1.ApplicationNumber: US-201615341998-A
1.PublishNumber: US-2017121742-A1
2.Date Publish: 20170504
3.Inventor: AJJAWI IMAD
SORIAGA LEAH
AQUI MOENA
MOELLERING ERIC R.

4.Inventor Harmonized: AJJAWI IMAD(US)
SORIAGA LEAH(US)
AQUI MOENA(US)
MOELLERING ERIC R(US)

5.Country: US
6.Claims:
(en)The present invention provides mutant microorganism that have higher lipid productivity than the wild type microorganisms from which they are derived while producing biomass at levels that are at least 45% of wild type biomass productivity under nitrogen replete conditions. Particular mutants produce at least 50% as much FAME lipid as wild type while producing at least the amount of biomass produced by wild type cells under nitrogen replete conditions. Also provided are methods of producing lipid using the mutant strains.

7.Description:
(en)CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims benefit of priority under 35 U.S.C. §119(e) of U.S. Ser. No. 62/249,834 filed Nov. 2, 2015, the entire contents of which is incorporated herein by reference in its entirety.


INCORPORATION OF SEQUENCE LISTING
The material in the accompanying sequence listing is hereby incorporated by reference into this application. The accompanying sequence listing text file, name SGII1980_1_Sequence_Listing.txt, was created on Nov. 1, 2016, and is 425 kb. The file can be assessed using Microsoft Word on a computer that uses Windows OS.
BACKGROUND OF THE INVENTION
The invention relates to mutant microorganisms, such as algae and heterokonts, having increased lipid productivity and methods of their use in producing lipids.
Many microorganisms such as algae, labyrinthulomycetes (“chytrids”), and oleaginous yeast induce lipid biosynthesis in response to nutrient stress, such as nitrogen starvation. Under conditions of nitrogen depletion, such microorganisms redirect compound biosynthesis from protein to storage lipids, typically triacylglyceride lipids (“TAG”). Because nitrogen depletion simultaneously decreases cell growth, optimal lipid biosynthesis is limited to a relatively short window before the cells become too metabolically impaired to maintain high levels of production.
Various attempts to improve lipid productivity by increasing lipid biosynthesis during nutrient replete growth have focused on manipulating genes encoding enzymes for nitrogen assimilation or lipid metabolism as well as genes encoding polypeptides involved in lipid storage. For example, US2014/0162330 discloses a Phaeodactylum tricornutum strain in which the nitrate reductase (NR) gene has been attenuated by RNAi-based knockdown; Trentacoste et al. ((2013) Proc. Natl. Acad. Sci. USA 110: 19748-19753) disclose diatoms transformed with an RNAi construct targeting the Thaps3_264297 gene predicted to be involved in lipid catabolism; and WO2011127118 discloses transformation of Chlamydomonas with genes encoding oleosins (lipid storage protein) as well as with genes encoding diacylglycerol transferase (DGAT) genes. Although in each case increased lipid production was asserted based on microscopy or staining with lipophilic dyes, no quantitation of lipid production by the manipulated cells was provided, nor was the relationship between biomass and lipid productivities over time determined.
Daboussi et al. 2014 ( Nature Comm. 5:3881) report that disruption of the UGPase gene in Phaeodactylum triconornutum , which is believed to provide precursors to laminarin (a storage carbohydrate) synthesis, results in increased lipid accumulation. However, no biochemical data was shown to indicate that laminarin content was affected (or even present) and lipid and biomass productivities were not reported. Similarly, several groups have reported increases in lipid accumulation in Chlamydomonas starchless mutants (Wang et al. 2009 Eukaryotic Cell 8:1856-1868; Li et al. 2010 Metab Eng. 12:387-391) however, successive reports that actually measured lipid productivity concluded that these strains were impaired in growth when grown in phototrophic conditions (Siaut et al. 2011 BMC Biotechnol. 11:7; Davey et al. 2014 Eukaryot Cell 13:392-400). These reports concluded that the highest lipid productivities (measured as TAG per liter per day) were actually achieved by the wild-type parental strain.
WO 2011/097261 and US20120322157 report that a gene denoted “SN03” encoding an arrestin protein has a role in increasing lipid production under nutrient replete conditions when overexpressed in Chlamydomonas . However, overexpression of the SN03 gene was observed to result in the appearance of unidentified polar lipids, which were not quantified, and did not result in an increase in triglycerides (TAG). Another polypeptide identified as potentially regulating stress-induced lipid biosynthesis has been described by Boyle et al. ((2012) J. Biol. Chem. 287:15811-15825). Knockout of the NRR1 gene in Chlamydomonas encoding a “SQUAMOUSA” domain polypeptide resulted in a reduction of lipid biosynthesis with respect to wild type cells under nitrogen depletion; however, no mutants were obtained demonstrating increased lipid production. US 2010/0255550 suggests the overexpression of putative transcription factors (TF1, TF2, TF3, TF4, and TF5) in algal cells to increase lipid production, but no such strains are disclosed.
WO 2015/130832 and Ngan et al. ( Nature Plants, 1:1507, 2015) report that overexpression of the PSR1 gene, a regulator of the phosphate starvation response, leads to cellular lipid accumulation in Chlamydomonas . Cells engineered to overexpress PSR1 are disclosed as having the large round phenotype of cells induced to produce lipid via nutrient starvation. Transformed cells were also observed to have higher amounts of cellular lipid with respect to control cells as visualized by electron microscopy and measured by staining with lipophilic fluorophores. However, increased lipid production by cultures on a volumetric basis was not demonstrated, nor was culture propagation, biomass accumulation, or overexpression of the PSR1 gene determined from the cultures in which lipid accumulation was observed. Conversely, growth rate of the PSR1-transformed cultures was demonstrated under conditions that were nonselective for the expression of the gene, and neither PSR1 gene expression level nor lipid production was assessed during the growth assay period.
Copending and commonly-owned U.S. Utility application Ser. No. 15/210,845 filed Jul. 14, 2016, entitled “Microorganisms Having Increased Lipid Productivity” discloses algal mutants having attenuated expression of a lipid regulator gene “ZnCys-2845” referred to herein as “LION1” mutants and their use in lipid production.
SUMMARY OF THE DISCLOSURE
Disclosed herein are mutant microorganisms that produce more lipid than a control microorganism (such as a wild type microorganisms from which the mutants are derived) while producing biomass at levels that are at least 45% of the level of biomass produced by the control microorganism cultured under the same conditions, which are conditions under which the control microorganism produces biomass, for example, nitrogen replete or nutrient replete conditions with respect to the control microorganism. In various embodiments, a mutant microorganism as provided herein can produce at least 20% more lipid than a control microorganism while experiencing a decrease of no more than 20%, no more than 15%, no more than 10%, no more than 5%, or no more than 3% in biomass accumulation with respect to the control microorganism. For example, a mutant microorganism can produce at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% more lipid than a control microorganism while producting at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, or approximately 100% of the biomass produced by the control microorganism cultured under the same conditions as the mutant microorganism, which are conditions in which the control microorganism produces biomass. The control microorganism can be, in some examples, a wild type microorganism, for example, a wild type microorganism from which the mutant microorganism is directly or indirectly derived. The mutant microorganisms may be generated, for example, by classical mutagenesis or by genetic engineering techniques, and can have a mutation in or attenuated expression of a gene encoding a polypeptide having a TAZ zinc finger domain and/or a Bromo domain, and/or a gene encoding a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% to any of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, or SEQ ID NO:46.
Provided herein, in a first aspect, is a mutant microorganism that produces at least 20% more lipid than is produced by a control microorganism while producing not less than 45% of the biomass produced by the control microorganism cultured under the same conditions, in which the culture conditions support production of biomass by the control microorganism. For example, a mutant microorganism as provided herein can produce at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 105%, at least 110%, or at least 115% more lipid than is produced by a control microorganism while producing at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% of the amount of biomass produced by the control microorganism, when the control microorganism is cultured under the same conditions as the mutant microorganism, which are conditions in which the control microorganism accumulates biomass. In some examples, a mutant microorganism as provided herein produces at least 20% more lipid, for example, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 105%, at least 110%, or at least 115% more lipid than is produced by a control microorganism while producing not less than 45% of the biomass produced by the control microorganism cultured under the same conditions, in which the culture conditions are nitrogen replete, and are preferably nutrient replete, with respect to the control microorganism and the culture conditions support production of biomass by both the control microorganism and the mutant microorganism. The lipid can be fatty acid methyl ester-derivatizable lipid, i.e., “FAME lipid” or “FAME”. Biomass accumulation can be, for example, dry weight, ash free dry weight (AFDW), or total organic carbon (TOC) accumulation. The control microorganism can be, in some examples, a wild type microorganism, for example, a wild type microorganism from which the mutant microorganism is directly or indirectly derived.
The increased productivity can be increased average daily productivity, where the amount produced is averaged over multiple days. The mutant microorganism can produce, in some embodiments, at least 45%, at least about 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least about 100% of the amount of biomass produced by a control microorganism and at least about 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, or at least 120% more lipid than is produced by a control microorganism cultured under the same conditions over a culture period of at least three, at least five, at least seven, at least eight, at least ten, at least twelve, at least fourteen, at least fifteen, at least twenty, at least twenty-five, at least thirty, at least sixty, or at least ninety days, where the control microorganism and the mutant microorganism both produce biomass during the culture period. For example, the mutant can produce an average daily amount of lipid that is at least 25% greater than the average daily amount of a control microorganism while producing an average daily amount of biomass that is at least 45% of the daily amount produced by the control cell, for a period of from three to ninety days, such as from five to sixty days, from five to thirty days, or from five to fourteen days. In various embodiments a mutant microorganism as provided herein produces at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the biomass and at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or at least 55% of the amount of lipid produced by a control microorganism for at least three, at least five, at least seven, at least eight, at least ten, at least twelve, at least fourteen, at least twenty, at least thirty, at least sixty, or at least ninety days, where the mutant and control microorganisms are cultured under the same conditions in which both the control microorganism and the mutant microorganism accumulate biomass. For example, the culture conditions can be nitrogen-replete with respect to the control microorganism and are in various examples nutrient-replete with respect to the control microorganism. In some embodiments in which a mutant microorganism as provided herein produces at least 45% of the biomass and at least 20% more lipid than a control microorganism (e.g., a wild type microorganism), the culture medium includes nitrate as substantially the sole nitrogen source for growth and propagation of the mutant and control microorganisms.
For example, a mutant microorganism as provided herein can produce more FAME-derivatizable lipids (“FAME lipids” or “FAME”), for example, at least 20% more FAME lipids, than a control microorganism while producing not less than 45% of the biomass produced by the control microorganism, when the mutant microorganism and control microorganism are cultured under the same culture conditions under which the control microorganism produces biomass. For example, the FAME productivity of a mutant as provided herein, which can be, for example, the average daily FAME productivity of a mutant as provided herein, can be at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or at least 55% greater than that of a control or wild type microorganism while the average daily biomass (e.g., AFDW or TOC) productivity can be at least 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% of the average daily biomass productivity of the control or wild type microorganism over a culture period of at least three days, at least five days, at least seven days, at least ten days, at least twelve days, at least fifteen days, at least twenty days, at least thirty days, or at least sixty days during which both the mutant microorganism and the control microorganism are producing biomass. In particular embodiments, a mutant microorganism as provided herein can produce at least 25% more FAME lipids than a control or wild type microorganism while producing at least 45% as much biomass as the control microorganism over a culture period of at least ten days, where the where the daily amount of FAME produced by the mutant is not lower than the daily amount of FAME produced by the control or wild type microorganism on any day during the at least ten day culture period, during which both the mutant and control microorganism accumulate biomass.
In some examples, the culture conditions under which the mutant produces more lipid than a control or wild type microorganism can be culture conditions in which the concentration of ammonium in the culture medium is less than about 2.5 mM, for example, the concentration of ammonium in the culture medium can be about 2 mM or less than about 2 mM, less than about 1.5 mM, less than about 1 mM, or less than or equal to about 0.5 mM. In some examples the culture medium can include no added ammonium or includes substantially no ammonium. In some examples, the culture medium can include no added source of reduced nitrogen for the microorganism, e.g., no added ammonium, urea, or amino acids that can support growth and/or propagation of the culture. The culture medium can in some examples include a nitrogen source such as nitrate. For example, nitrate can be present at a concentration at greater than about 1 mM, or of at least about 2 mM, at least about 2.5 mM, at least about 3 mM, at least about 4 mM, or at least about 5 mM. In some examples the culture medium includes nitrate as substantially the sole nitrogen source for growth and/or propagation of the culture. For example, in some embodiments a mutant microorganism as disclosed herein can produce more lipid than a control microorganism while producing biomass at levels that are at least 45% of the level of biomass produced by the control microorganism when cultured under nutrient replete conditions in which nitrate is substantially the sole source of nitrogen for growth and propagation of the culture. The culture medium can be, in various embodiments, nutrient replete with respect to a wild type microorganism of the species from which the mutant microorganism is derived.
A mutant microorganism as provided herein can produce more lipid, for example more FAME lipids, than a control or wild type microorganism while producing at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% the amount of biomass produced by a control or wild type microorganism in any of batch, continuous, or semi-continuous culture conditions. In some embodiments, the mutant microorganism is a mutant photosynthetic microorganism, such as an alga, and the culture is exposed to light for at least a portion of the culture period. In some examples the culture conditions under which the mutant photosynthetic microorganism produces at least 20% more lipid while producing at least 45% as much biomass as a control photosynthetic microorganism are photoautotrophic conditions, for example, conditions in which inorganic carbon is substantially the sole source of carbon available to the microorganism. In some examples, the mutant microorganism is a mutant photosynthetic microorganism, such as an alga, and the mutant demonstrates increased productivity under photoautotrophic conditions that include a diel cycle, where the light period can be between about two hours and about twenty-two hours, for example, between about four hours and about twenty hours per twenty-four hour cycle, such as between about six and about sixteen hours, between about eight and about eighteen hours, or between about twelve and about sixteen hours, per twenty-four hour diel cycle.
In some embodiments, a mutant microorganism as provided herein can produce at least 20% more lipid while producing at least 75% of the amount of biomass produced by a wild type or control microorganism during a culture period of at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, or at least thirteen days of culturing, for example, at least five, at least ten, at least fifteen, at least twenty, or at least thirty days where the mutant and control microorganism are cultured under the same conditions in which both the control and mutant microorganism cultures produce biomass. For example, the average daily FAME productivity of a mutant as provided herein can be at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or at least 55% more than that of a wild type or control microorganism under conditions in which both the control and mutant microorganism cultures produce biomass and the average daily biomass productivity (e.g., TOC productivity) of the mutant is at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the daily biomass productivity (e.g., TOC productivity) of a control microorganism, for example, under conditions in which the mutant has substantially the same biomass productivity as a control microorganism. In some examples, a mutant microorganism can produce at least 30% more, at least 35% more, at least 40% more, at least 45% more, or at least 50% more FAME lipids than a wild type or control microorganism while producing at least about 90% or at least about 95% of the biomass produced by a wild type microorganism cultured under identical conditions, which are nutrient replete with respect to the wild type microorganism. In other examples a mutant microorganism can produce at least 50% more FAME lipids than a wild type or control microorganism while producing approximately as much biomass as is produced by a wild type microorganism cultured under identical conditions under which the wild type or control microorganism produces biomass (e.g., within 5% of the amount of biomass produced by the control microorganism). In various examples, the average daily FAME productivity for at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, or at least thirteen days of culturing, for example, at least five, at least ten, at least fifteen, at least twenty, or at least thirty days of culturing can be at least 50% greater than the average daily FAME productivity of a wild type or control microorganism while the mutant microorganism exhibits approximately as much biomass productivity as a wild type microorganism (e.g., within about 5% of the amount of biomass productivity of the control microorganism) cultured under identical conditions, which are conditions in which the wild type or control microorganism produces biomass.
A mutant microorganism such as any provided herein that produces at least 25% more lipid than is produced by a control microorganism while producing not less than 45% or not less than about 50% of the biomass produced by the control microorganism when the mutant microorganism and control microorganism are cultured under the same culture conditions can have FAME lipids (FAME)/total organic carbon (TOC) ratios at least 25% higher than the FAME/TOC ratio of a wild type microorganism. The FAME/TOC ratio of a mutant microorganism as provided herein can be, for example, at least 30% higher, at least 40% higher, at least 50% higher, at least 60% higher, or at least 70% higher than the FAME/TOC ratio of a control microorganism cultured under identical conditions under which both the control microorganism and the mutant microorganism produce biomass. The FAME/TOC ratio of a mutant microorganism as provided herein can be, for example, at least 25% higher, at least 30% higher, at least 35% higher, at least 40% higher, at least 45% higher, at least 50% higher, at least 55% higher, at least 60% higher, at least 65% higher, or at least 70% higher than the FAME/TOC ratio of a control microorganism cultured under identical conditions under which both the control microorganism and the mutant microorganism produce biomass, and which are nutrient replete with respect to the wild type microorganism.
A mutant microorganism as provided herein having greater lipid productivity that a control microorganism while producing at least 45% of the biomass of the control microorganism can have attenuated expression of a gene encoding a protein whose attenuated expression affects the expression of other genes, e.g., at least ten, at least twenty, at least thirty, at least forty, at least fifty, at least sixty, at least seventy, at least eighty, at least ninety, or at least 100 additional genes. For example, a mutant as provided herein can have at least ten genes that are upregulated with respect to a wild type microorganism and at least ten genes that are downregulated with respect to a wild type microorganism under conditions in which the mutant phenotype (e.g., greater lipid production) is expressed. A mutant as provided herein can have at least twenty, at least thirty, at least forty, at least fifty, at least sixty, at least seventy, at least eighty, at least ninety, or at least 100 genes that are upregulated with respect to a wild type microorganism and at least twenty, at least thirty, at least forty, at least fifty, at least sixty, at least seventy, at least eighty, at least ninety, or at least 100 genes that are downregulated with respect to a wild type microorganism under conditions in which the mutant phenotype (e.g., greater lipid production with respect to the wild type microorganism) is expressed.
In various examples, mutants as disclosed herein that produces at least 20% more lipid than a control microorganism while producing at least 45% as much biomass as the control microorganism can have attenuated expression of a gene encoding a polypeptide that has a TAZ Zinc Finger domain, e.g., has an amino acid sequence encoding a TAZ Zinc Finger domain characterized as conserved domain c102660 or SMART domain smart00551 or a TAZ Zinc Finger domain belonging to pfam PF02135. In some embodiments, a mutant microorganism as provided herein can have attenuated expression of a gene encoding a polypeptide that includes an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to SEQ ID NO:9. Alternatively or in addition, a mutant microorganism such as any disclosed herein that produces at least 20% more lipid than a control microorganism while producing at least 45% as much biomass as the control microorganism can have attenuated expression of a gene encoding a polypeptide that has a Bromo domain, e.g., can have an amino acid sequence encoding a Bromo domain characterized as conserved domain cd05506 or SMART domain smart00297 or a Bromo domain belonging to pfam PF00439. In some examples, the Bromo domain can include an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to SEQ ID NO:10.
Thus, provided herein in various embodiments are mutant microorganisms having attenuated expression of a gene encoding a polypeptide having a TAZ Zinc Finger domain, in which the mutant microorganism produces at least 20% more lipid and at least 45% as much biomass, and in various embodiments produces at least 90% as much biomass as is produced by a control microorganism that does not have attenuated expression of the gene encoding a polypeptide having a TAZ Zinc Finger domain. For example, a mutant microorganism as provided herein having attenuated expression of a polypeptide having a TAZ Zinc Finger domain can produce at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or at least 55% more lipid (e.g., FAME) than is produced by a wild type or control microorganism under culture conditions in which both the control and mutant microorganism cultures produce biomass, and the mutant produces at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the biomass produced by a control microorganism when the mutant microorganism and control microorganism are cultured under identical conditions under which the control microorganism experiences an increase in TOC. The polypeptide that includes a TAZ Zinc Finger domain in some embodiments can further include a Bromo domain.
Also provided herein is a mutant microorganism having attenuated expression of a gene encoding a polypeptide having a Bromo domain, in which the mutant microorganism produces at least 20% more lipid and at least 45% as much biomass and in various examples at least 90% biomass, as a control microorganism that does not have attenuated expression of the gene encoding a polypeptide having a Bromo domain. For example, a mutant microorganism as provided herein having attenuated expression of a polypeptide having a Bromo domain can produce at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or at least 55% more lipid (e.g., FAME) than is produced by a wild type or control microorganism under conditions in which both the control and mutant microorganism cultures produce biomass, and the mutant produces at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the biomass produced by a control microorganism when the mutant microorganism and control microorganism are cultured under identical conditions under which the control microorganism culture experiences an increase in TOC. The polypeptide that includes a Bromo domain in some embodiments can further include a TAZ Zinc Finger domain.
Alternatively or in addition, a mutant microorganism as provided herein that produces at least 20% more lipid and at least 45% as much biomass as a control microorganism can have attenuated expression of a gene encoding a polypeptide including an amino acid sequence with at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to any of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, or SEQ ID NO:46. The encoded polypeptide can have at least one TAZ Zinc Finger domain and/or can have at least one Bromo domain. In exemplary embodiments a mutant microorganism as provided herein can have attenuated expression of a gene encoding a polypeptide that includes an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to any of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12.
An attenuated gene encoding a polypeptide having a TAZ Zinc Finger domain and/or a Bromo domain, or that has 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to any of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, or SEQ ID NO:46 can be a gene that has an insertion, deletion, and/or one or more base changes with respect to the wild type gene. The insertion, deletion, or one or more base changes can be in a coding region, intron, 3′ untranslated region, or 5′ untranslated region of the gene, or can be upstream of the 5′ untranslated region of the gene, e.g., in the promoter region of a gene. Alternatively or in addition, a mutant microorganism as provided herein can include an antisense molecule or construct, an RNAi molecule or construct, a guide RNA (gRNA) as part of a CRISPRi system or a construct for expressing a guide RNA, a ribozyme, or construct for producing a ribozyme that targets the gene encoding the polypeptide having a TAZ Zinc Finger domain and/or a Bromo domain or having at least 50% identity to any of the recited sequences, wherein the presence or expression of the construct results in reduced expression of the gene.
A mutant microorganism as provided herein can be any eukaryotic microorganism, and in some examples is a heterokont or alga. For example, the mutant microorganism can be a Labyrinthulomycte species, such as, for example, a species of Labryinthula, Labryinthuloides, Thraustochytrium, Schizochytrium, Aplanochytrium, Aurantiochytrium, Oblongichytrium, Japonochytrium, Diplophrys , or Ulkenia . Alternatively a mutant microorganism can be an algal species such as for example, a species belonging to any of the genera Achnanthes, Amphiprora, Amphora, Ankistrodesmus, Asteromonas, Boekelovia, Bolidomonas, Borodinella, Botrydium, Botryococcus, Bracteococcus, Chaetoceros, Carteria, Chlamydomonas, Chlorococcum, Chlorogonium, Chlorella, Chroomonas, Chrysosphaera, Cricosphaera, Crypthecodinium, Cryptomonas, Cyclotella, Desmodesmus, Dunaliella, Elipsoidon, Emiliania, Eremosphaera, Ernodesmius, Euglena, Eustigmatos, Franceia, Fragilaria, Fragilaropsis, Gloeothamnion, Haematococcus, Hantzschia, Heterosigma, Hymenomonas, Isochrysis, Lepocinclis, Micractinium, Monodus, Monoraphidium, Nannochloris, Nannochloropsis, Navicula, Neochloris, Nephrochloris, Nephroselmis, Nitzschia, Ochromonas, Oedogonium, Oocystis, Ostreococcus, Parachlorella, Parietochloris, Pascheria, Pavlova, Pelagomonas, Phwodactylum, Phagus, Picochlorum, Platymonas, Pleurochrysis, Pleurococcus, Prototheca, Pseudochlorella, Pseudoneochloris, Pseudostaurastrum, Pyramimonas, Pyrobotrys, Scenedesmus, Schizochlamydella, Skeletonema, Spyrogyra, Stichococcus, Tetrachlorella, Tetraselmis, Thalassiosira, Tribonema, Vaucheria, Viridiella, Vischeria , and Volvox In some examples, the mutant alga is a heterokont alga, and may belong to the diatoms (bacillariophytes), eustigmatophytes , xanthophytes, phaeophytes, chrysophytes, or raphidophytes. In some examples, the mutant alga is a diatom and belongs to a genus such as but not limited to Amphiprora, Amphora, Chaetoceros, Cyclotella, Fragilaria, Fragilaropsis, Hantzschia, Navicula, Nitzschia, Phæodactylum, Phæodactylum, Skeletonema , and Thalassiosira . In some examples, the mutant alga is a Eustigmatophyte and belongs to a genus selected from the group consisting of Chloridella, Chlorobptrys, Ellipsoidion, Eustigmatos, Goniochloris, Monodopsis, Monodus, Nannochloropsis, Pseudocharaciopsis, Pseudostaruastrum, Pseudotetraedriella , and Vischeria . In some examples, the mutant alga cell is a Nannochloropsis species.
A further aspect is a method of producing lipid, comprising culturing a mutant microorganism as provided herein and isolating lipid from the microorganism, the culture medium, or both. The culture medium can be nutrient replete with respect to a control (e.g. wild type) microorganism. In some embodiments of the method the mutant microorganism can be cultured in a medium that comprises less than about 5 mM ammonium, for example, less than about 2.5 mM ammonium, less than or equal to about 1.5 mM ammonium, less than or equal to about 2 mM ammonium, less than or equal to about 1.5 mM ammonium, less than or equal to about 1 mM ammonium, or less than or equal to about 0.5 mM. The culture medium can include, for example, from about 0 to about 5 mM ammonium, from about 0 to about 4 mM ammonium, from about 0 to about 3 mM ammonium, from about 0 to about 2.5 mM ammonium, from about 0.1 to about 2.5 mM ammonium, from about 0.5 to about 2.5 mM ammonium, from about 0 to about 2 mM ammonium, from about 0.1 to about 2 mM ammonium, from about 0.2 to about 2 mM ammonium, from about 0.5 to about 1.5 mM ammonium, from about 0.1 to about 1.5 mM ammonium, from about 0.2 to about 1.5 mM ammonium, from about 0.5 to about 1.5 mM ammonium, from about 1 mM to about 1.5 mM ammonium, from about 0.1 to about 1.5 mM ammonium, or from about 0.2 to about 1 mM ammonium. The microorganism can be cultured in a medium that includes nitrate, which in some examples may be substantially the sole nitrogen source in the culture medium or may be present in addition to ammonium that may be present at a concentration of less than 5 mM, less than 2.5 mM, less than 2 mM, less than 1.5 mM, or less than 1 mM. In some examples the mutant microorganism is a mutant alga and the microorganism is exposed to light during at least a portion of the culturing period. In some examples the mutant microorganism is a mutant alga and the microorganism is cultured under photoautotrophic conditions e.g., conditions in which inorganic carbon is substantially the sole carbon source in the culture medium. The method can further include extracting lipid from the microorganism, the culture medium, or both.
Yet another aspect of the disclosure is a nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide including an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, or SEQ ID NO:46. For example, the nucleic acid molecule can have at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, or SEQ ID NO:45.
The encoded polypeptide encoded by a nucleic acid molecule as provided herein can include an amino acid sequence encoding a TAZ Zinc Finger domain and/or can include an amino acid sequence encoding a Bromo domain. The nucleic acid molecule in various examples can be or comprise a cDNA and/or can lack one or more introns present in the naturally-occurring gene, or, alternatively or in addition, the nucleic acid molecule can include one or more introns not present in the naturally-occurring gene. The nucleic acid molecule in various examples can have a sequence that is not 100% identical to a naturally-occurring gene. The nucleic acid molecule in various examples can comprise a heterologous promoter operably linked to the sequence encoding a polypeptide that includes an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, or SEQ ID NO:46 and/or can comprise a vector that includes a sequence encoding a polypeptide that includes an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, or SEQ ID NO:46. For example, the nucleic acid molecule can be a cDNA, expression cassette, or vector comprising a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, or SEQ ID NO:45.
A further aspect is a construct designed for attenuating expression of a gene encoding a polypeptide containing a TAZ Zinc Finger domain and/or a Bromo domain. The construct can be or comprise, in various examples, a sequence encoding a guide RNA of a CRISPR system, an RNAi construct, an antisense construct, a ribozyme construct, or a construct for homologous recombination, e.g., a construct having one or more nucleotide sequences having homology to a naturally-occurring TAZ Zinc Finger domain-encoding and/or Bromo domain-encoding gene as disclosed herein and/or sequences adjacent thereto in the native genome from which the gene is derived. For example, the construct can include at least a portion of a gene that encodes a polypeptide having a TAZ Zinc Finger domain and/or Bromo domain or a sequence homologous to at least a portion of an gene that encodes a polypeptide that includes an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, or SEQ ID NO:46, including a nucleic acid sequence complementary to the coding strand of a gene or portion thereof. Alternatively or in addition, the construct can include a sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, least 65%, at least 70%, at least 75%, at least 80%, at least 85% at least 90%, or at least 95% identity to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, or SEQ ID NO:45, or a portion thereof, including a sequence complementary to the coding strand of any of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, or SEQ ID NO:45, a sequence having at least least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, least 65%, at least 70%, at least 75%, at least 80%, at least 85% at least 90%, or at least 95% identity to any thereof or to a portion of any thereof.
Also included is a guide RNA targeting a gene encoding a polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, or SEQ ID NO:46 or guide RNA targeting a gene having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, least 65%, at least 70%, at least 75%, at least 80%, at least 85% at least 90%, or at least 95% identity to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, or SEQ ID NO:45, or a portion thereof. In particular embodiments the invention provides a guide RNA that includes a sequence corresponding to the target sequence of any of SEQ ID NO:61, SEQ ID NO:68, SEQ ID NO:70, and SEQ ID NO:72, and in exemplary embodiments may be a chimeric guide such as any of SEQ ID NO:60, SEQ ID NO:67, SEQ ID NO:69, and SEQ ID NO:71.
These and other objects and features of the invention will become more fully apparent when the following detailed description of the invention is read in conjunction with the accompanying drawings.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A is a graph showing the FAME/TOC ratio of wild type Nannochloropsis gaditana strain WT-3730 grown in batch assay on nitrate-containing culture medium alongside the FAME to TOC rations of eighteen mutant strains, each of which knocked out (has a gene disrupting insertion) in a transcription factor gene whose genome locus is provided under each column along the x axis of the graph. Of the eighteen strains assayed, only the strain knocked out in the gene identified as the Naga_100104 g18 has a higher FAME/TOC ratio than wild type.
FIG. 1B is a schematic depiction of the N. gaditana Bromo-1091 gene. Boxes denote the positions of the TAZ Zn finger domain (TAZ) and the Bromo domain (BR). The approximate location of the putative nuclear localization signal is also shown (NLS), as well as three glutamine-rich regions, denoted by the letter “Q”. Arrows 1-5 point to positions upstream of the coding region that were targeted by CRISPR guide sequences. The arrow labeled GE-8563 demonstrates the region of the gene just downstream of the TAZ Zn finger domain where the Hygromycin resistance marker was inserted using CRISPR/Cas9 to generated knockout strain GE-8563. Primer sites for qRT-PCR to determine transcript levels of knockdowns are also denoted. The figure is not to scale.
FIGS. 2A-2C are an alignment of the four Bromo-1091 isoforms encoded by four Bromo-1091 transcripts of the N. gaditana Bromo-1091 gene. FIG. 2A ) provides the N-terminal portions of the polypeptides; FIG. 2B ) provides the invariant central portion of the polypeptides; and C) provides the C terminal regions of the polypeptides.
FIG. 3 is a schematic map of vector pSGE-6206 used to introduce Cas9 into the N. gaditana wild type strain WT-3730 to generate the Cas9 Editor line.
FIGS. 4A-4C are graphs depicting FAME/TOC values. FIG. 4A ) is a graph depicting FAME productivity of wild-type and Bromo-1091 knockout N. gaditana cells cultured in batch mode in nitrate-only medium as determined from samples taken on odd days (3, 5, and 7) of the culture; FIG. 4B ) is a graph depicting TOC values for days 3-7 of the batch productivity assay. FIG. 4C ) is a graph depicting FAME/TOC ratios calculated from samples taken on odd days of the culture. Error bars in graphs represent the standard deviation of the three independent cultures (biological replicates). Symbols used in graphs: asterisks represent wild type WT-3730 pre-cultured in nitrate-plus-ammonium medium PM124, black diamonds represent knockout mutant GE-8563 pre-cultured in nitrate-plus-ammonium medium PM124, X's represent wild type WT-3730 pre-cultured in nitrate-only medium PM074, and black circles represent knockout mutant GE-8563 pre-cultured in nitrate-only medium PM074.
FIGS. 5A-5C provide graphs depicting productivities of the N. gaditana wild type strain and the GE-8563 Bromo-1091 knockout strain in a semi-continuous assay in which the culture medium includes nitrate as the sole nitrogen source. FIG. 5A ) shows daily FAME productivity over seven days of the assay; FIG. 5B ) shows daily TOC productivity over the same seven days of the assay; and FIG. 5C ) provides the FAME/TOC ratios for the same seven days of the assay. Error bars in graphs represent the standard deviation of the three independent cultures (biological replicates). Symbols used in graphs: circles represent wild type WT-3730 and Xs represent knockout mutant GE-8563.
FIG. 6A ) is a schematic depiction of the Bromo-1091 gene with the positions of the nuclear localization signal (NLS), TAZ Zn finger domain (TAZ) and Bromo domain (BR) shown as boxes, and arrows depicting the sites of CRISPR-targeted mutations, FIG. 6B ) shows the relative transcript levels of the corresponding CRISPR-targeted mutants (position of primers used for transcript assessment shown in FIG. 6A ), where “BASH-1” is strain GE-13027, “BASH-4” is GE-13030, and “BASH-5” is GE-13032, and the Bromo-1091 knockout strain is GE-8563. Normalized expression levels are relative to the average wild type level which was set to 1.0.
FIG. 7A ) is a graph depicting FAME productivity of wild-type and Bromo-1091 knockdown N. gaditana cells cultured in batch mode in nitrate-only medium; FIG. 7B ) is a graph depicting TOC values for the odd days of the batch screen in nitrate-only medium (days 3, 5, and 7); and FIG. 7C ) is a graph providing FAME/TOC ratios of the cultures calculated on days 3, 5, and 7. Symbols used in graphs: open circles represent wild type WT-3730, an asterisk represents “BASH-1” knockdown mutant GE-13027; a plus sign represents “BASH-4” knockdown mutant GE-13130; and X's represent “BASH-5” knockdown mutant GE-13132; open triangles represent Bromo-1091 knockout mutant GE-8563.
FIGS. 8A-8F provides tables and graphs depicting productivities of the N. gaditana wild type strain and Bromo-1091 knockdown strains in a semi-continuous assay in which the culture medium used for daily dilution includes nitrate as the sole nitrogen source. FIG. 8A ) is a table of average FAME productivity from three cultures of each Bromo-1091 knockdown strain as well as wild type strain WT-3730 on each day of the ten day semi-continuous assay, standard deviations for the three culture averages are in parentheses; FIG. 8B ) is a graph showing average daily FAME productivities (mg/L culture) over the ten days of the assay; FIG. 8C ) is a table of average TOC productivity from three cultures of each Bromo-1091 knockdown strain as well as wild type strain WT-3730 on each day of the ten day semi-continuous assay, standard deviations for the three culture averages are in parentheses; FIG. 8D ) is a graph showing the daily TOC productivities of the cultures in g/m 2 /day over ten days of the assay; FIG. 8E ) is a table providing the FAME/TOC ratios for each day of the assay; and FIG. 8F ) is a graph providing the daily FAME/TOC ratios of the cultures over ten days of the assay. Symbols used in graphs: circles represent wild type WT-3730; triangles represent “BASH-1” knockdown mutant GE-13127; Xs represent “BASH-4” knockdown mutant GE-13130; and diamonds represent “BASH-5” knockdown mutant GE-13132. Error bars in graphs represent the standard deviation of the three independent cultures (biological replicates).
FIG. 9 is a bar graph of the biomolecular composition of Bromo knockdown mutants as compared to wild type cells.
FIG. 10 is a bar chart showing chlorophyll content of two Bromo-1091 knockdown strains, GE-13027 (BASH-1) and GE-13132 (BASH-5) compared to wild type strain WT-3730 (WT).
FIGS. 11A-11B depicts a transcriptomics “heat map” in which the darkness of the bands, each of which represents a gene, is correlated with the level of expression of the gene (assessed by FPKM value). FIG. 11A ) depicts relative transcript abundance in the presence of ammonium. Left to right: Bromo 1091 knockdown strain GE-13032 sample 1, Bromo 1091 knockdown strain GE-13032 sample 2, ZnCys-2845 knockout strain GE-8564 sample 1, ZnCys-2845 knockout strain GE-8564 sample 2, Cas9 parental strain sample 1, and Cas9 parental strain sample 2; FIG. 11B ) depicts relative transcript abundance in the presence of nitrate. Left to right: Bromo 1091 knockdown strain GE-13032 sample 1, Bromo 1091 knockdown strain GE-13032 sample 2, ZnCys-2845 knockout strain GE-8564 sample 1, ZnCys-2845 knockout strain GE-8564 sample 2, NR knockout strain sample 1, NR knockout sample 2, Cas9 parental strain sample 1, and Cas9 parental strain sample 2.



DETAILED DESCRIPTION
Definitions
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present application including the definitions will control. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. All ranges provided within the application are inclusive of the values of the upper and lower ends of the range unless specifically indicated otherwise.
All publications, patents and other references mentioned herein are incorporated by reference in their entireties for all purposes as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.
The term “and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B”, “A or B”, “A”, and “B”.
“About” means either within 10% of the stated value, or within 5% of the stated value, or in some cases within 2.5% of the stated value, or, “about” can mean rounded to the nearest significant digit.
The term “gene” is used broadly to refer to any segment of a nucleic acid molecule (typically DNA, but optionally RNA) encoding a polypeptide or expressed RNA. Thus, genes include sequences encoding expressed RNA (which can include polypeptide coding sequences or, for example, functional RNAs, such as ribosomal RNAs, tRNAs, antisense RNAs, microRNAs, short hairpin RNAs, ribozymes, etc.). Genes may further comprise regulatory sequences required for or affecting their expression, as well as sequences associated with the protein or RNA-encoding sequence in its natural state, such as, for example, intron sequences, 5′ or 3′ untranslated sequences, etc. In some examples, “gene” may only refer to a protein-encoding portion of a DNA or RNA molecule, which may or may not include introns. A gene is preferably greater than 50 nucleotides in length, more preferably greater than 100 nucleotide in length, and can be, for example, between 50 nucleotides and 500,000 nucleotides in length, such as between 100 nucleotides and 100,000 nucleotides in length or between about 200 nucleotides and about 50,000 nucleotides in length, or about 200 nucleotides and about 20,000 nucleotides in length. Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information.
The term “nucleic acid” or “nucleic acid molecule” refers to, a segment of DNA or RNA (e.g., mRNA), and also includes nucleic acids having modified backbones (e.g., peptide nucleic acids, locked nucleic acids and other modified nucleic acids or nucleic acid analogs (e.g., Efimov and Chakhmakhcheva (2005) Methods Mol Biol. 288: 147-163)) or modified or non-naturally-occurring nucleobases. The nucleic acid molecules can be double-stranded or single-stranded; a single stranded nucleic acid molecule that comprises a gene or a portion thereof can be a coding (sense) strand or a non-coding (antisense) strand.
A nucleic acid molecule may be “derived from” an indicated source, which includes the isolation (in whole or in part) of a nucleic acid segment from an indicated source. A nucleic acid molecule may also be derived from an indicated source by, for example, direct cloning, PCR amplification, or artificial synthesis from the indicated polynucleotide source or based on a sequence associated with the indicated polynucleotide source, which may be, for example, a species of organism. Genes or nucleic acid molecules derived from a particular source or species also include genes or nucleic acid molecules having sequence modifications with respect to the source nucleic acid molecules. For example, a gene or nucleic acid molecule derived from a source (e.g., a particular referenced gene) can include one or more mutations with respect to the source gene or nucleic acid molecule that are unintended or that are deliberately introduced, and if one or more mutations, including substitutions, deletions, or insertions, are deliberately introduced the sequence alterations can be introduced by random or targeted mutation of cells or nucleic acids, by amplification or other gene synthesis or molecular biology techniques, or by chemical synthesis, or any combination thereof. A gene or nucleic acid molecule that is derived from a referenced gene or nucleic acid molecule that encodes a functional RNA or polypeptide can encode a functional RNA or polypeptide having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, sequence identity with the referenced or source functional RNA or polypeptide, or to a functional fragment thereof. For example, a gene or nucleic acid molecule that is derived from a referenced gene or nucleic acid molecule that encodes a functional RNA or polypeptide can encode a functional RNA or polypeptide having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the referenced or source functional RNA or polypeptide, or to a functional fragment thereof.
As used herein, an “isolated” nucleic acid or protein is removed from its natural milieu or the context in which the nucleic acid or protein exists in nature. For example, an isolated protein or nucleic acid molecule is removed from the cell or organism with which it is associated in its native or natural environment. An isolated nucleic acid or protein can be, in some instances, partially or substantially purified, but no particular level of purification is required for isolation. Thus, for example, an isolated nucleic acid molecule can be a nucleic acid sequence that has been excised from the chromosome, genome, or episome that it is integrated into in nature.
A “purified” nucleic acid molecule or nucleotide sequence, or protein or polypeptide sequence, is substantially free of cellular material and cellular components. The purified nucleic acid molecule or protein may be substantially free of chemicals beyond buffer or solvent, for example “Substantially free” is not intended to mean that other components beyond the novel nucleic acid molecules are undetectable.
The terms “naturally-occurring” and “wild type” refer to a form found in nature. For example, a naturally occurring or wild type nucleic acid molecule, nucleotide sequence or protein may be present in and isolated from a natural source, and is not intentionally modified by human manipulation.
As used herein “attenuated” means reduced in amount, degree, intensity, or strength. Attenuated gene expression may refer to a significantly reduced amount and/or rate of transcription of the gene in question, or of translation, folding, or assembly of the encoded protein. As nonlimiting examples, an attenuated gene may be a mutated or disrupted gene (e.g., a gene disrupted by partial or total deletion, truncation, frameshifting, or insertional mutation) that does not encode a complete functional open reading frame or that has decreased expression due to alteration or disruption of gene regulatory sequences. An attenuated gene may also be a gene targeted by a construct that reduces expression of the gene, such as, for example, an antisense RNA, microRNA, RNAi molecule, or ribozyme. Attenuated gene expression can be gene expression that is eliminated, for example, reduced to an amount that is insignificant or undetectable. Attenuated gene expression can also be gene expression that results in an RNA or protein that is not fully functional or nonfunctional, for example, attenuated gene expression can be gene expression that results in a truncated RNA and/or polypeptide.
“Exogenous nucleic acid molecule” or “exogenous gene” refers to a nucleic acid molecule or gene that has been introduced (“transformed”) into a cell. A transformed cell may be referred to as a recombinant cell, into which additional exogenous gene(s) may be introduced. A descendent of a cell transformed with a nucleic acid molecule is also referred to as “transformed” if it has inherited the exogenous nucleic acid molecule. The exogenous gene or nucleic acid molecule may be derived from a different species (and so “heterologous”), or from the same species (and so “homologous”), relative to the cell being transformed. An “endogenous” nucleic acid molecule, gene or protein is a native nucleic acid molecule, gene, or protein as it occurs in, or is naturally produced by, the host.
The term “native” is used herein to refer to nucleic acid sequences or amino acid sequences as they naturally occur in the host. The term “non-native” is used herein to refer to nucleic acid sequences or amino acid sequences that do not occur naturally in the host. Thus, a “non-native” nucleic acid molecule is a nucleic molecule that is not naturally present in the host cell, for example, the non-native nucleic acid molecule is exogenous to the host cell or microorganism into which it is introduced, and may be heterologous with respect to the host cell or microorganism. Additionally, a nucleic acid sequence or amino acid sequence that has been removed from a cell, subjected to laboratory manipulation, and introduced or reintroduced into a host cell such that it differs in sequence or location in the genome with respect to its position in a non-manipulated organism (i.e., is juxtaposed with or operably linked to sequences it is not juxtaposed with or operably linked to in a non-transformed organism) is considered “non-native”. Non-native genes also include genes endogenous to the host microorganism operably linked to one or more heterologous regulatory sequences that have been recombined into the host genome.
A “recombinant” or “engineered” nucleic acid molecule is a nucleic acid molecule that has been altered through human manipulation. As non-limiting examples, a recombinant nucleic acid molecule includes any nucleic acid molecule that: 1) has been partially or fully synthesized or modified in vitro, for example, using chemical or enzymatic techniques (e.g., by use of chemical nucleic acid synthesis, or by use of enzymes for the replication, polymerization, digestion (exonucleolytic or endonucleolytic), ligation, reverse transcription, transcription, base modification (including, e.g., methylation), integration or recombination (including homologous and site-specific recombination) of nucleic acid molecules); 2) includes conjoined nucleotide sequences that are not conjoined in nature; 3) has been engineered using molecular cloning techniques such that it lacks one or more nucleotides with respect to the naturally occurring nucleic acid molecule sequence; and/or 4) has been manipulated using molecular cloning techniques such that it has one or more sequence changes or rearrangements with respect to the naturally occurring nucleic acid sequence. As non-limiting examples, a cDNA is a recombinant DNA molecule, as is any nucleic acid molecule that has been generated by in vitro polymerase reaction(s), or to which linkers have been attached, or that has been integrated into a vector, such as a cloning vector or expression vector.
The term “recombinant protein” as used herein refers to a protein produced by genetic engineering regardless of whether the amino acid varies from that of a wild-type protein.
When applied to organisms, the term recombinant, engineered, or genetically engineered refers to organisms that have been manipulated by introduction of a heterologous or exogenous recombinant nucleic acid sequence into the organism (e.g., a non-native nucleic acid sequence), and includes gene knockouts, targeted mutations, gene replacement, and promoter replacement, deletion, disruption, or insertion, as well as introduction of transgenes or synthetic genes or nucleic acid sequences into the organism. That is, recombinant, engineered, or genetically engineered refers to organisms that have been altered by human intervention. Recombinant or genetically engineered organisms can also be organisms into which constructs for gene “knockdown” have been introduced. Such constructs include, but are not limited to, RNAi, microRNA, shRNA, siRNA, antisense, and ribozyme constructs. Also included are organisms whose genomes have been altered by the activity of meganucleases, zinc finger nucleases, TALENs, or cas/CRISPR systems. An exogenous or recombinant nucleic acid molecule can be integrated into the recombinant/genetically engineered organism's genome or in other instances may not be integrated into the host genome. As used herein, “recombinant microorganism” or “recombinant host cell” includes progeny or derivatives of the recombinant microorganisms of the invention. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny or derivatives may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
The term “promoter” refers to a nucleic acid sequence capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3′ direction) coding sequence. A promoter includes the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. A promoter can include a transcription initiation site as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eukaryotic promoters often, but not always, contain “TATA” boxes and “CAT” boxes. Prokaryotic promoters may contain −10 and −35 prokaryotic promoter consensus sequences. A large number of promoters, including constitutive, inducible and repressible promoters, from a variety of different sources are well known in the art. Representative sources include for example, algal, viral, mammalian, insect, plant, yeast, and bacterial cell types, and suitable promoters from these sources are readily available, or can be made synthetically, based on sequences publicly available on line or, for example, from depositories such as the ATCC as well as other commercial or individual sources. Promoters can be unidirectional (initiate transcription in one direction) or bi-directional (initiate transcription in either direction). A promoter may be a constitutive promoter, a repressible promoter, or an inducible promoter. A promoter region can include, in addition to the gene-proximal promoter where RNA polymerase binds to initiate transcription, additional sequences upstream of the gene that can be within 1 kb, 2 kb, 3 kb, 4 kb, 5 kb or more of the transcriptional start site of a gene, where the additional sequences can influence the rate of transcription of the downstream gene and optionally the responsiveness of the promoter to developmental, environmental, or biochemical (e.g., metabolic) conditions.
The term “heterologous” when used in reference to a polynucleotide, gene, nucleic acid, polypeptide, or enzyme refers to a polynucleotide, gene, nucleic acid, polypeptide, or enzyme that is from a source or derived from a source other than the host organism species. In contrast a “homologous” polynucleotide, gene, nucleic acid, polypeptide, or enzyme is used herein to denote a polynucleotide, gene, nucleic acid, polypeptide, or enzyme that is derived from the host organism species. When referring to a gene regulatory sequence or to an auxiliary nucleic acid sequence used for maintaining or manipulating a gene sequence (e.g. a promoter, a 5′ untranslated region, 3′ untranslated region, poly A addition sequence, intron sequence, splice site, ribosome binding site, internal ribosome entry sequence, genome homology region, recombination site, etc.), “heterologous” means that the regulatory sequence or auxiliary sequence is not naturally associated with the gene with which the regulatory or auxiliary nucleic acid sequence is juxtaposed in a construct, genome, chromosome, or episome. Thus, a promoter operably linked to a gene to which it is not operably linked to in its natural state (i.e. in the genome of a non-genetically engineered organism) is referred to herein as a “heterologous promoter,” even though the promoter may be derived from the same species (or, in some cases, the same organism) as the gene to which it is linked.
As used herein, the term “protein” or “polypeptide” is intended to encompass a singular “polypeptide” as well as plural “polypeptides,” and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term “polypeptide” refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, “protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids, are included within the definition of “polypeptide,” and the term “polypeptide” can be used instead of, or interchangeably with any of these terms.
Gene and protein Accession numbers, commonly provided in parenthesis after a gene or species name, are unique identifiers for a sequence record publicly available at the National Center for Biotechnology Information (NCBI) website (ncbi.nlm.nih.gov) maintained by the United States National Institutes of Health. The “GenInfo Identifier” (GI) sequence identification number is specific to a nucleotide or amino acid sequence. If a sequence changes in any way, a new GI number is assigned. A Sequence Revision History tool is available to track the various GI numbers, version numbers, and update dates for sequences that appear in a specific GenBank record. Searching and obtaining nucleic acid or gene sequences or protein sequences based on Accession numbers and GI numbers is well known in the arts of, e.g., cell biology, biochemistry, molecular biology, and molecular genetics. Gene loci identifiers refer to the published genome described in Corteggiani Carpinelli et al. (2014) Mol Plant 7:323-335 and available online at nannochloropsis.org.
As used herein, the terms “percent identity” or “homology” with respect to nucleic acid or polypeptide sequences are defined as the percentage of nucleotide or amino acid residues in the candidate sequence that are identical with the known polypeptides, after aligning the sequences for maximum percent identity and introducing gaps, if necessary, to achieve the maximum percent homology. For polypeptide sequences, N-terminal or C-terminal insertions or deletions shall not be construed as affecting homology, and internal deletions and/or insertions into the polypeptide sequence of less than about 65, less than about 60, less than about 50, less than about 40, less than about 30, less than about 20, or less than about 10 amino acid residues shall not be construed as affecting homology of compared amino acid (protein) sequences. For nucleic acid sequences, 5′ end or 3′ end insertions or deletions shall not be construed as affecting homology, and internal deletions and/or insertions into the polypeptide sequence of less than about 200, less than about 180, less than about 150, less than about 120, less than about 100, less than about 90, less than about 80, less than about 70, less than about 60, less than about 50, less than aobut 40, or less than about 30 nucleotides shall not be construed as affecting homology of compared nucleic acid sequences. Homology or identity at the nucleotide or amino acid sequence level can be determined by BLAST (Basic Local Alignment Search Tool) analysis using the algorithm employed by the programs blastp, blastn, blastx, tblastn, and tblastx (Altschul (1997), Nucleic Acids Res. 25, 3389-3402, and Karlin (1990), Proc. Natl. Acad. Sci. USA 87, 2264-2268), which are tailored for sequence similarity searching. The approach used by the BLAST program is to first consider similar segments, with and without gaps, between a query sequence and a database sequence, then to evaluate the statistical significance of all matches that are identified, and finally to summarize only those matches which satisfy a preselected threshold of significance. For a discussion of basic issues in similarity searching of sequence databases, see Altschul (1994), Nature Genetics 6, 119-129. The search parameters for histogram, descriptions, alignments, expect (i.e., the statistical significance threshold for reporting matches against database sequences), cutoff, matrix, and filter (low complexity) can be at the default settings. The default scoring matrix used by blastp, blastx, tblastn, and tblastx is the BLOSUM62 matrix (Henikoff (1992), Proc. Natl. Acad. Sci. USA 89, 10915-10919), recommended for query sequences over 85 in length (nucleotide bases or amino acids).
For blastn, designed for comparing nucleotide sequences, the scoring matrix is set by the ratios of M (i.e., the reward score for a pair of matching residues) to N (i.e., the penalty score for mismatching residues), wherein the default values for M and N can be +5 and −4, respectively. Four blastn parameters can be adjusted as follows: Q=10 (gap creation penalty); R=10 (gap extension penalty); wink=1 (generates word hits at every winkth position along the query); and gapw=16 (sets the window width within which gapped alignments are generated). The equivalent Blastp parameter settings for comparison of amino acid sequences can be: Q=9; R=2; wink=1; and gapw=32. A Bestfit comparison between sequences, available in the GCG package version 10.0, can use DNA parameters GAP=50 (gap creation penalty) and LEN=3 (gap extension penalty), and the equivalent settings in protein comparisons can be GAP=8 and LEN=2.
Thus, when referring to the polypeptide or nucleic acid sequences of the present invention, included are sequence identities of at least 40%, at least 45%, at least 50%, at least 55%, of at least 70%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85%, for example at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity with the full-length polypeptide or nucleic acid sequence, or to fragments thereof comprising a consecutive sequence of at least 100, at least 125, at least 150 or more amino acid residues of the entire protein; variants of such sequences, e.g., wherein at least one amino acid residue has been inserted N- and/or C-terminal to, and/or within, the disclosed sequence(s) which contain(s) the insertion and substitution. Contemplated variants can additionally or alternately include those containing predetermined mutations by, e.g., homologous recombination or site-directed or PCR mutagenesis, and the corresponding polypeptides or nucleic acids of other species, including, but not limited to, those described herein, the alleles or other naturally occurring variants of the family of polypeptides or nucleic acids which contain an insertion and substitution; and/or derivatives wherein the polypeptide has been covalently modified by substitution, chemical, enzymatic, or other appropriate means with a moiety other than a naturally occurring amino acid which contains the insertion and substitution (for example, a detectable moiety such as an enzyme).
As used herein, the phrase “conservative amino acid substitution” or “conservative mutation” refers to the replacement of one amino acid by another amino acid with a common property. A functional way to define common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz (1979) Principles of Protein Structure, Springer-Verlag). According to such analyses, groups of amino acids can be defined where amino acids within a group exchange preferentially with each other, and therefore resemble each other most in their impact on the overall protein structure (Schulz (1979) Principles of Protein Structure, Springer-Verlag). Examples of amino acid groups defined in this manner can include: a “charged/polar group” including Glu, Asp, Asn, Gln, Lys, Arg, and His; an “aromatic or cyclic group” including Pro, Phe, Tyr, and Trp; and an “aliphatic group” including Gly, Ala, Val, Leu, Ile, Met, Ser, Thr, and Cys. Within each group, subgroups can also be identified. For example, the group of charged/polar amino acids can be sub-divided into sub-groups including: the “positively-charged sub-group” comprising Lys, Arg and His; the “negatively-charged sub-group” comprising Glu and Asp; and the “polar sub-group” comprising Asn and Gln. In another example, the aromatic or cyclic group can be sub-divided into sub-groups including: the “nitrogen ring sub-group” comprising Pro, His, and Trp; and the “phenyl sub-group” comprising Phe and Tyr. In another further example, the aliphatic group can be sub-divided into sub-groups including: the “large aliphatic non-polar sub-group” comprising Val, Leu, and Ile; the “aliphatic slightly-polar sub-group” comprising Met, Ser, Thr, and Cys; and the “small-residue sub-group” comprising Gly and Ala. Examples of conservative mutations include amino acid substitutions of amino acids within the sub-groups above, such as, but not limited to: Lys for Arg or vice versa, such that a positive charge can be maintained; Glu for Asp or vice versa, such that a negative charge can be maintained; Ser for Thr or vice versa, such that a free —OH can be maintained; and Gln for Asn or vice versa, such that a free —NH2 can be maintained. A “conservative variant” is a polypeptide that includes one or more amino acids that have been substituted to replace one or more amino acids of the reference polypeptide (for example, a polypeptide whose sequence is disclosed in a publication or sequence database, or whose sequence has been determined by nucleic acid sequencing) with an amino acid having common properties, e.g., belonging to the same amino acid group or sub-group as delineated above.
As used herein, “expression” includes the expression of a gene at least at the level of RNA production, and an “expression product” includes the resultant product, e.g., a polypeptide or functional RNA (e.g., a ribosomal RNA, a tRNA, an antisense RNA, a micro RNA, an shRNA, a ribozyme, etc.), of an expressed gene. The term “increased expression” includes an alteration in gene expression to facilitate increased mRNA production and/or increased polypeptide expression. “Increased production” [of a gene product] includes an increase in the amount of polypeptide expression, in the level of the enzymatic activity of a polypeptide, or a combination of both, as compared to the native production or enzymatic activity of the polypeptide.
Some aspects of the present invention include the partial, substantial, or complete deletion, silencing, inactivation, or down-regulation of expression of particular polynucleotide sequences. The genes may be partially, substantially, or completely deleted, silenced, inactivated, or their expression may be down-regulated in order to affect the activity performed by the polypeptide they encode, such as the activity of an enzyme. Genes can be partially, substantially, or completely deleted, silenced, inactivated, or down-regulated by insertion of nucleic acid sequences that disrupt the function and/or expression of the gene (e.g., viral insertion, transposon mutagenesis, meganuclease engineering, homologous recombination, or other methods known in the art). The terms “eliminate,” “elimination,” and “knockout” can be used interchangeably with the terms “deletion,” “partial deletion,” “substantial deletion,” or “complete deletion.” In certain embodiments, a microorganism of interest may be engineered by site directed homologous recombination or targeted integration or mutation using a cas/CRISPR system to knockout a particular gene of interest. In still other embodiments, targeted insertion into or mutation of a gene regulatory region using a cas/CRISPR system, RNAi, or antisense DNA (asDNA) constructs may be used to partially, substantially, or completely silence, inactivate, or down-regulate a particular gene of interest.
These insertions, deletions, or other modifications of certain nucleic acid molecules or particular polynucleotide sequences may be understood to encompass “genetic modification(s)” or “transformation(s)” such that the resulting strains of the microorganisms or host cells may be understood to be “genetically modified”, “genetically engineered” or “transformed.”
As used herein, “up-regulated” or “up-regulation” includes an increase in expression of a gene or nucleic acid molecule of interest or the activity of an enzyme, e.g., an increase in gene expression or enzymatic activity as compared to the expression or activity in an otherwise identical gene or enzyme that has not been up-regulated.
As used herein, “down-regulated” or “down-regulation” includes a decrease in expression of a gene or nucleic acid molecule of interest or the activity of an enzyme, e.g., a decrease in gene expression or enzymatic activity as compared to the expression or activity in an otherwise identical gene or enzyme that has not been down-regulated.
As used herein, “mutant” refers to an organism that has a mutation in a gene that is the result of classical mutagenesis, for example, using gamma irradiation, UV, or chemical mutagens. “Mutant” as used herein also refers to a recombinant cell that has altered structure or expression of a gene as a result of genetic engineering that many include, as non-limiting examples, overexpression, including expression of a gene under different temporal, biological, or environmental regulation and/or to a different degree than occurs naturally and/or expression of a gene that is not naturally expressed in the recombinant cell; homologous recombination, including knock-outs and knock-ins (for example, gene replacement with genes encoding polypeptides having greater or lesser activity than the wild type polypeptide, and/or dominant negative polypeptides); gene attenuation via RNAi, antisense RNA, or ribozymes, or the like; and genome engineering using meganucleases, TALENs, and/or CRISPR technologies, and the like. A mutant is therefore not a naturally-occurring organism. A mutant organism of interest will typically have a phenotype different than that of the corresponding wild type or progenitor strain that lacks the mutation, where the phenotype can be assessed by growth assays, product analysis, photosynthetic properties, biochemical assays, etc. When referring to a gene “mutant” means the gene has at least one base (nucleotide) change, deletion, or insertion with respect to a native or wild type gene. The mutation (change, deletion, and/or insertion of one or more nucleotides) can be in the coding region of the gene or can be in an intron, 3′ UTR, 5′ UTR, or promoter region, e.g., within 2 kb of the transcriptional start site or within 3 kb or the translational start site. For example, a mutant having attenuated expression of a gene as disclosed herein can have a mutation, which can be one or more nucleobase changes and/or one or more nucleobase deletions and/or one or more nucleobase insertions, into the region of a gene 5′ of the transcriptional start site, such as, in non-limiting examples, within 2 kb, within 1.5 kb, within 1 kb, or within 0.5 kb of the known or putative transcriptional start site, or within 3 kb, within 2.5 kb, within 2 kb, within 1.5 kb, within 1 kb, or within 0.5 kb of the translational start site. As nonlimiting examples, a mutant gene can be a gene that has a mutation, insertion, or deletion within the promoter region that can either increase or decrease expression of the gene; can be a gene that has a deletion that results in production of a nonfunctional protein, truncated protein, dominant negative protein, or no protein; can be a gene that has one or more point mutations leading to a change in the amino acid of the encoded protein or results in aberrant splicing of the gene transcript, etc.
Conserved domains of polypeptides include those identified in the “cd” (conserved domain) database, the COG database, the SMART database, the PRK database, the TIGRFAM database, or others known the art. The National Center for Biotechnology Information website (ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) sponsored by the U.S. National Institutes of Health includes a conserved domain database (CDD) which it describes as “a protein annotation resource that consists of a collection of well-annotated multiple sequence alignment models for ancient domains and full-length proteins. These are available as position-specific score matrices (PSSMs) for fast identification of conserved domains in protein sequences via RPS-BLAST. CDD content includes NCBI-curated domains, which use 3D-structure information to explicitly define domain boundaries and provide insights into sequence/structure/function relationships, as well as domain models imported from a number of external source databases (Pfam, SMART, COG, PRK, TIGRFAM).” Sequences can be searched for conserved domains at the cdd database of NCBI (ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). See, Marchler-Bauer et al. (2015) Nucleic Acids Res. 43(D) 222-226.
The term “Pfam” refers to a large collection of protein domains and protein families maintained by the Pfam Consortium and available at several sponsored world wide web sites, including: pfam.sanger.ac.uk/ (Welcome Trust, Sanger Institute); pfam.sbc.su.se (Stockholm Bioinformatics Center); pfam.janelia.org/ (Janelia Farm, Howard Hughes Medical Institute); pfam.jouy.inra.fr/ (Institut national de la Recherche Agronomique); and pfam.ccbb.re.kr. The latest release of Pfam is Pfam 30.0 (June 2016) based on the UniProt protein database release 2012_06. Pfam domains and families are identified using multiple sequence alignments and hidden Markov models (HMMs). Pfam-A family or domain assignments, are high quality assignments generated by a curated seed alignment using representative members of a protein family and profile hidden Markov models based on the seed alignment. (Unless otherwise specified, matches of a queried protein to a Pfam domain or family are Pfam-A matches.) All identified sequences belonging to the family are then used to automatically generate a full alignment for the family (Sonnhammer (1998) Nucleic Acids Research 26, 320-322; Bateman (2000) Nucleic Acids Research 26, 263-266; Bateman (2004) Nucleic Acids Research 32, Database Issue, D138-D141; Finn (2006) Nucleic Acids Research Database Issue 34, D247-251; Finn (2010) Nucleic Acids Research Database Issue 38, D211-222). By accessing the Pfam database, for example, using any of the above-reference websites, protein sequences can be queried against the HMMs using HMMER homology search software (e.g., HMMER2, HMMER3, or a higher version, hmmer.janelia.org/). Significant matches that identify a queried protein as being in a pfam family (or as having a particular Pfam domain) are those in which the bit score is greater than or equal to the gathering threshold for the Pfam domain Expectation values (e values) can also be used as a criterion for inclusion of a queried protein in a Pfam or for determining whether a queried protein has a particular Pfam domain, where low e values (much less than 1.0, for example less than 0.1, or less than or equal to 0.01) represent low probabilities that a match is due to chance.
A “cDNA” is a DNA molecule that comprises at least a portion the nucleotide sequence of an mRNA molecule, with the exception that the DNA molecule substitutes the nucleobase thymine, or T, in place of uridine, or U, occurring in the mRNA sequence. A cDNA can be double stranded or single stranded and can be, for example, the complement of the mRNA sequence. In preferred examples, a cDNA does not include one or more intron sequences that occur in the naturally-occurring gene that the cDNA corresponds to (i.e., the gene as it occurs in the genome of an organism). For example, a cDNA can have sequences from upstream of an intron of a naturally-occurring gene juxtaposed to sequences downstream of the intron of the naturally-occurring gene, where the upstream and downstream sequences are not juxtaposed in a DNA molecule in nature (i.e., the sequences are not juxtaposed in the naturally occurring gene). A cDNA can be produced by reverse transcription of mRNA molecules, or can be synthesized, for example, by chemical synthesis and/or by using one or more restriction enzymes, one or more ligases, one or more polymerases (including, but not limited to, high temperature tolerant polymerases that can be used in polymerase chain reactions (PCRs)), one or more recombinases, etc., based on knowledge of the cDNA sequence, where the knowledge of the cDNA sequence can optionally be based on the identification of coding regions from genome sequences or compiled from the sequences multiple partial cDNAs.
Reference to properties that are “substantially the same” or “substantially identical” without further explanation of the intended meaning, is intended to mean the properties are within 10%, and preferably within 5%, and may be within 2.5%, of the reference value. Where the intended meaning of “substantially” in a particular context is not set forth, the term is used to include minor and irrelevant deviations that are not material to the characteristics considered important in the context of the subject matter of the invention.
A “control cell” or “control microorganism” is either a wild type cell or microorganism from which the mutant microorganism (genetically engineered or mutagenized microorganism) is directly or indirectly derived, or is a cell or microorganism that is substantially identical to the mutant cell or microorganism referred to (i.e., of the same genus and species, preferably of the same strain) with the exception that the control cell or microorganism does not have the mutation resulting in increased lipid production that the subject microroganism has. For example, where the mutant microorganism has attenuated expression of a gene encoding a polypeptide that includes a TAZ zinc finger domain and/or a Bromo domain or a gene encoding a polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to any of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, or SEQ ID NO:46, a control cell can be substantially identical to the mutant microorganism with the exception that the control microorganism does not have attenuated expression of a gene encoding a polypeptide that includes a TAZ zinc finger domain and/or a Bromo domain or a gene encoding a polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to any of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, or SEQ ID NO:46.
“The same conditions” or “the same culture conditions”, as used herein, means substantially the same conditions, that is, any differences between the referenced conditions that may be present are minor and not relevant to the function or properties of the microorganism that are material to the invention, including lipid production or biomass production.
As used herein “lipid” or “lipids” refers to fats, waxes, fatty acids, fatty acid derivatives such as fatty alcohols, wax esters, alkanes, and alkenes, sterols, monoglycerides, diglycerides, triglycerides, phospholipids, sphingolipids, saccharolipids, and glycerolipids. “FAME lipids” or “FAME” refers to lipids having acyl moieties that can be derivatized to fatty acid methyl esters, such as, for example, monoacylglycerides, diacylglycerides, triacylglycerides, wax esters, and membrane lipids such as phospholipids, galactolipids, etc. Lipid productivity can be assessed as FAME productivity in milligrams per liter (mg/L) and for algae, may be reported as grams per meter 2 per day (g/m 2 /day). In the semi-continuous assays provided herein, mg/L values are converted to g/m2/day by taking into account the area of incident irradiance (the SCPA flask rack aperture of 1½″×33/8″, or 0.003145 m 2 ) and the volume of the culture (550 ml). To obtain productivity values in g/m 2 /day, mg/L values are multiplied by the daily dilution rate (30%) and a conversion factor of 0.175. Where lipid or subcategories thereof (for example, TAG or FAME) are referred to as a percentage, the percentage is a weight percent unless indicated otherwise.
“Biomass” refers to cellular mass, whether of living or dead cells, and can be assessed, for example, as aspirated pellet weight, but is more preferably dry weight (e.g., lyophilate of a culture sample or pelleted cells), ash-free dry weight (AFDW), or total organic carbon (TOC), using methods known in the art. Biomass increases during the growth of a culture under growth permissive conditions and may be referred to as “biomass accumulation” in batch cultures, for example. In continuous or semi-continuous cultures that undergo steady or regular dilution, biomass that is produced that would otherwise accumulate in the culture is removed during culture dilution. Thus, daily biomass productivity (increases in biomass) by these cultures can also be referred to as “biomass accumulation”. Biomass productivity can be assessed as TOC productivity in milligrams per liter (mg/L) and for algae, may be reported as grams per meter 2 per day (g/m 2 /day). In the semi-continuous assays provided herein, mg/L values are converted to g/m2/day by taking into account the area of incident irradiance (the SCPA flask rack aperture of 1½″×33/8″, or 0.003145 m 2 ) and the volume of the culture (550 ml). To obtain productivity values in g/m 2 /day, mg/L values are multiplied by the daily dilution rate (30%) and a conversion factor of 0.175. Where biomass is expressed as a percentage, the percentage is a weight percent unless indicated otherwise.
In the context of the invention, a “nitrogen source” is a source of nitrogen that can be taken up and metabolized by the subject microorganism and incorporated into biomolecules for growth and propagation. For example, compounds including nitrogen that cannot be taken up and/or metabolized by the microorganism for growth (e.g., nitrogen-containing biological buffers such as Hepes, Tris, etc.) are not considered nitrogen sources in the context of the invention.
“Reduced nitrogen”, as used herein, is nitrogen in the chemical form of ammonium, ammonia, urea, or an amino acid (e.g., an amino acid that can be taken up and metabolized by the microorganism being cultured to provide a source of nitrogen for incorporation into biomolecules, thereby supporting growth). Examples of amino acids that may be nitrogen sources can include, without limitation, glutamate, glutamine, histidine, proline, lysine, arginine, asparagine, alanine, and glycine. “Non-reduced nitrogen” in the context of a nitrogen source that can be present in a culture medium for microorganisms refers to nitrate or nitrite that must be reduced prior to assimilation into organic compounds by the microorganism.
“The sole source of nitrogen [in the culture medium]” is used interchangeably with “substantially the sole source of nitrogen” and indicates that no other nitrogen source that can be metabolized by the microorganism (i.e., the nitrogen source provides nitrogen that can be taken up by the microorganism and incorporated by the microorganism into biomolecules such as proteins and nucleic acids) is intentionally added to the culture medium, or that no other nitrogen source is present in an amount sufficient to significantly increase the growth of the microorganisms or cells cultured in the referenced medium. Throughout this application, for brevity, the terms “nitrate-only” is used to characterize culture media in which nitrate is the only source of nitrogen that is available to the microorganisms for supporting growth.
Similarly, “the sole source of carbon [in the culture medium]” is used interchangeably with “substantially the sole source of carbon” and indicates that no other carbon source that can be metabolized by the microorganism (i.e., used for energy or for as a carbon source for the production of biomolecules) is present in an amount sufficient to increase the productivity, growth, or propagation of the microorganisms or cells cultured in the referenced medium or that can become incorporated into biomolecules such as lipids produced by the microorganisms or cells at a percentage of greater than 5% of the carbon incorporated into the biomolecule.
“Nitrogen replete” conditions refer to media conditions in which no further growth or propagation benefit is conferred by adding additional nitrogen (in a form that can be used by the microorganism) to the medium. Similarly, “nutrient replete” conditions refer to media conditions in which no nutrient is limiting to growth or propagation, that is, when a medium is nutrient replete, adding additional nutrient(s) to the medium does not result in an improved growth or propagation rate. In the context of “nutrient replete”, “nutrients” includes, as nonlimiting examples, phosphate, sulfur, iron, and optionally silica, but excludes carbon sources such as sugars or organic acids that may be used by the organism as an energy source.
Disclosed herein are methods for manipulating, assaying, culturing, and analyzing microorganisms. The invention set forth herein also makes use of standard methods, techniques, and reagents for cell culture, transformation of microorganisms, genetic engineering, and biochemical analysis that are known in the art. Although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present invention, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and are not intended to be limiting. Other features and advantages of the invention will be apparent from the detailed description and from the claims.
All references cited herein are incorporated by reference in their entireties. All headings are for the convenience of the reader and do not limit the invention in any way. References to aspects or embodiments of the invention do not necessarily indicate that the described aspects may not be combined with other described aspects of the invention or features of other aspects of the invention.
Mutant Microorganisms Having Increased Lipid Productivity
The invention provides mutant microorganisms (for example, microorganisms obtained by classical mutagenesis or genetic engineering) having at least 20% higher lipid productivity with respect to a control microorganism while producing at least 45% of the biomass produced by the control microorganism when both the mutant microorganism and control microorganism are cultured under substantially identical conditions in which the control microorganism culture produces biomass, for example, under culture conditions in which both the mutant and control microorganism are producing biomass. The culture conditions under which a mutant microorganism as provided herein produces at least 20% more lipid and at least 45% as much biomass as a control microorganism can be nitrogen replete, and can be nutrient replete, with respect to the control microorganism. In some embodiments the control microorganism is a wild type microorganism of the same species from which the mutant is directly or indirectly derived, and the culture conditions under which a mutant microorganism as provided herein produces at least 20% more lipid and at least 45% as much biomass as a wild type microorganism are nitrogen replete, and can be nutrient replete, with respect to the wild type microorganism.
A mutant microorganism as provided herein can demonstrate greater lipid productivity than a control microorganism and at least 45% of the biomass productivity (e.g., average daily biomass productivity) of the control microorganism over a culture period of at least three days, for example, over a culture period of at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least twenty, at least thirty, or at least sixty days when the mutant microorganism and the control microorganism are cultured under substantially identical conditions that support growth and propagation of the control microorganism, i.e., under conditions in which the control microorganism culture produces biomass. In some examples the culture period in which a mutant microorganism as provided herein produces at least 45% of the biomass and produces at least 20% more lipid with respect to a control microorganism can be less than 180 days, less than 120 days, or less than 90 days, where the mutant can have a higher average daily lipid productivity over the time period. For example, a mutant microorganism as provided herein can produce at least 45% of the biomass and at least 20% more lipid than a control microorganism during a culture period of from three to 90 days, from three to 60 days, from three to thirty days, or from three to fifteen days. For example, a mutant microorganism as provided herein can produce at least 45% of the biomass and at least 20% more lipid than a control microorganism during a culture period ranging from five to 90 days, from five to 60 days, from five to thirty days, or from five to fifteen days, or from seven to 90 days, from seven to 60 days, from seven to thirty days, from seven to twenty days, or from seven to at least fifteen days.
Productivity can be volumetric productivity, for example, the productivity of a culture can be expressed as weight per milliliter or liter of culture, and can be a daily productivity (e.g., mg/liter/day or g/liter/day), for example, an average daily productivity over multiple days of the culture (for example, at least three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen fifteen, or more days), or can be a total amount produced per unit volume for a defined period of time in culture. Productivity is preferably measured multiple times during the culture period, for example, at least twice or at least three times, and may be assessed every day, every other day, every third day, etc.
Biomass productivity can be assessed, for example, by measuring total organic carbon (TOC) or by other methods, such as measuring dry weight, ash-free dry weight (AFDW). Methods for measuring TOC are known in the art (e.g., U.S. Pat. No. 8,835,149) and are provided herein. Methods of measuring AFDW are also well-known and can be found, for example, in U.S. Pat. No. 8,940,508, incorporated herein by reference in its entirety.
Methods of measuring the amount of lipid produced by microorganisms are also well-known in the art and provided in the examples herein. For example, total extractable lipid can be determined according to Folch et al. (1957) J. Biol. Chem. 226: 497-509; Bligh & Dyer (1959) Can. J. Biochem. Physiol. 37: 911-917; or Matyash et al. (2008) J. Lipid Res. 49:1137-1146, for example, and the percentage of biomass present as lipid can also be assessed using Fourier transform infrared spectroscopy (FT-IR) (Pistorius et al. (2008) Biotechnol & Bioengin. 103:123-129). Additional references for gravimetric analysis of FAME and TAGs are provided in U.S. Pat. No. 8,207,363 and WO 2011127118 for example, each incorporated herein by reference in its entirety.
A mutant as provided herein can produce, in various embodiments, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 80%, at least 90%, or at least 100% more lipid with respect to a control microorganism under culture conditions in which both the mutant and control microorganism are producing biomass and the mutant produces at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 105%, at least 110%, or at least 115% of the biomass produced by a wild type microorganism per day averaged over at least three, at least five, at least seven, at least nine, at least ten, at least twelve, at least thirteen, or at least fourteen days. In some embodiments, a mutant microorganism as provided herein produces higher amounts of lipid with respect to a control microorganism and at least 45% of the biomass but less than 150%, less than 200%, less than 250%, or less than 300% of the biomass produced by the control microorganism. In some embodiments, a mutant microorganism as provided herein produces at least 45% of the biomass with respect to a control microorganism and at least 20% more lipid but not more than 100%, not more than than 150%, not more than 200% more lipid than is produced by the control microorganism.
In various examples, a mutant microorganism as provided herein produces an average of at least 25% more FAME lipids per day, for example, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 80%, at least 90%, or at least 100% more lipid per day with respect to a control microorganism while producing not less than an average of about 50% of the biomass produced by the control microorganism per day, and can produce at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 105%, at least 110%, or at least 115% of the biomass produced by a wild type microorganism per day, when the mutant microorganism and control microorganism are cultured under the same culture conditions under which the culture of the control microorganism produces biomass, where the culture conditions are nitrogen-replete, and are preferably nutrient replete culture conditions with respect to the control microorganism, over a period of at least three days, at least four days, at least five days, at least seven days, at least ten days, at least twelve days, or at least fourteen days. The culture conditions can include culturing in a culture medium that includes less than about 3 mM, less than about 2.5 mM, less than about 2 mM, or less than about 1.5 mM of a reduced nitrogen source such as ammonium. Alternatively or in addition, the culture conditions can include culturing in a culture medium that includes nitrate as substantially the sole source of nitrogen. The control microorganism in some examples is a wild type microorganism, e.g., a wild type microorganism from which the mutant microorganism is directly or indirectly derived.
In some embodiments, a mutant microorganism as disclosed herein can be an algal or heterokont cell that produces at least 25% more FAME while producing at least 45% of the amount of TOC as a control microorganism in a culture medium that includes less than about 5 mM, less than about 4.5 mM, less than about 4 mM, less than about 3.5 mM, less than about 3 mM, about 2.5 mM ammonium or less, about 2.0 mM ammonium or less, about 1.5 mM ammonium or less, about 1.0 mM ammonium or less, about 0.5 mM ammonium or less, or substantially no ammonium, and includes, for example, at least 1.0 mM, at least 2.0 mM, at least 3.0 mM, at least 4.0 mM, at least 5.0 mM, at least 6.0 mM, at least 7.0 mM, at least 8.0 mM, or at least 10.0 mM nitrate. For example, the ammonium concentration may be at a concentration ranging from about 0 to about 5 mM, from about 0 to about 4.0 mM, from about 0 to about 3 mM, from about 0 to about 2.5 mM, from about 0 to about 2.0 mM, from about 0 to about 1.5 mM, from about 0 to about 1.0 mM, or from about 0 to about 0.5 mM. The ammonium concentration may be at a concentration ranging from about 0.2 to about 3 mM, 0.2 to about 2.5 mM, from about 0.2 to about 2.0 mM, from about 0.2 to about 1.5 mM, about 0.2 to about 1 mM, or from about 0.3 to about 2.5 mM, or from about 0.3 to about 1 mM, from about 0.3 to about 1.5 mM, or from about 0.3 to about 2 mM. In further examples, the ammonium concentration may be at a concentration ranging from about 0.5 mM to about 2.5 mM, from about 0.4 to about 2 mM, or from about 0.4 to about 1.5 mM.
Alternatively or in addition, a mutant as provided herein can produce at least 45% of the biomass produced by a control cell and at least 25% more lipid than the control cell over the same time period in a culture that includes nitrate, for example, at least about 1 mM, at least about 1.5 mM, at least about 2 mM, at least about 2.5 mM, at least about 3 mM, at least about 3.5 mM, at least about 4 mM, at least about 4.5 mM, or at least about 5 mM nitrate and in some examples can further optionally include less than about 5 mM ammonium, such as less than about 2.5 mM, ammonium, less than about 2 mM, ammonium, less than about 1 mM ammonium, or less than about 0.5 mM ammonium.
The mutant microorganism can be, in some embodiments, a photosynthetic microorganism and can produce more lipid and at least 45% of the lipid of a control or wild type cell under photoautotrophic conditions, which may be under a diel cycle. The light period of the diel cycle may be of any length and can be, for example, from about four hours to about twenty-two hours, and can be, for example, from about six hours to about twenty hours, e.g., from about eight hours to about eighteen hours per twenty four hour cycle. The microorganism can be exposed to natural or artificial light or a combination thereof. The available light can vary in intensity throughout the light period.
Mutant microorganisms provided herein can have greater partitioning of carbon to lipid with respect to a control microorganism cultured under identical conditions in which both the control microorganism and the mutant microorganism are producing biomass. A mutant having increased partitioning of carbon to lipid with respect to a control microorganism can have increased partitioning of carbon to total extractable lipid, to total neutral lipids, to triglycerides, and/or to FAME-derivatizable lipids. For example, a mutant microorganism as provided herein can have a ratio of the amount of FAME-derivatizable lipids (“FAME”) produced to biomass (TOC or ash-free dry weight (AFDW), for example) produced that is at least 25% higher than that of a control microorganism. Lipid and biomass production and/or production can be assessed, for example, by gravimetric analysis as known in the art and demonstrated in the examples herein. For example, a mutant microorganism as provided herein can have a ratio of FAME to TOC that is at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% higher than the FAME/TOC ratio of a control microorganism when both the mutant microorganism and the control microorganism are cultured under conditions in which both the culture of the mutant microorganism and the culture of the control microorganism produce biomass. In some embodiments, the FAME/TOC ratio of a mutant microorganism as provided herein can be increased with respect to the FAME/TOC ratio of a control microorganism cultured under identical conditions by less than about 200% or less than about 150%.
In various examples a mutant microorganism as provided herein can have a ratio of the amount of FAME produced to TOC produced that is at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% higher than the FAME/TOC ratio of a control microorganism when both the mutant microorganism and the control microorganism are cultured under conditions in which the control culture produces biomass (e.g., TOC) and the mutant culture produces at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the amount of biomass that is produced by the control culture. In various examples, the FAME/TOC ratio of a mutant as provided herein can be at least 0.27, at least 0.30, at least 0.35, at least 0.40, at least 0.45, at least 0.50, or at least 0.55 when cultured under conditions in which the mutant microorganism culture produces at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85, at least 90%, or at least 95% as much biomass (e.g., TOC) as a control microorganism culture, under conditions where both the control and mutant cultures produce biomass. In various examples, the FAME/TOC ratio of a mutant as provided herein can be at least 0.27, at least 0.30, at least 0.35, at least 0.40, at least 0.45, at least 0.50, or at least 0.55 when cultured under conditions in which the mutant culture produces at least about 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85, at least 90%, at least 95%, or at least 100% as much biomass (e.g., TOC) as a control microorganism produces when both the mutant and the control microorganism are cultured under conditions that are nitrogen replete, for example, nutrient replete, with respect to the control microorganism.
In some examples, a mutant microorganism as provided herein can produce at least 50% more FAME while producing at least 80%, at least 85%, or at least 90% of the TOC produced by a control cell (such as a wild type cell) when cultured under conditions in which both the control and mutant microorganism produce biomass, and the FAME/TOC ratio of the mutant microorganism is at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% higher than the FAME/TOC ratio of a control microorganism. The FAME/TOC ratio of the mutant microorganism can be, for example, at least 0.30, at least 0.35, at least 0.40, at least 0.45, or at least 0.50. The culture conditions can include, for example, a culture medium that includes less than 2.5 mM, less than 2 mM, less than 1.5 mM, less than 1.0 mM, or less than 0.5 mM ammonium and in some examples can include at least 2 mM, at least 4 mM, or at least 6 mM nitrate. The culture conditions can in some examples include substantially no ammonium, and in some examples can include substantially no reduced nitrogen as a nitrogen source. The culture in some examples includes nitrate as a nitrogen source, which can optionally be substantially the sole nitrogen source in the culture medium.
The properties of a mutant as provided herein having increased lipid production are compared to the same properties of a control microorganism that may be a wild type organism of the same species as the mutant, preferably the progenitor strain of the lipid-overproducing mutant. Alternatively, a control microorganism can be a microorganism that is substantially identical to the mutant microorganism with the exception that the control microorganism does not have the mutation that leads to higher lipid productivity. For example, a control microorganism can be a genetically engineered microorganism or classically mutated organism that has been further mutated or engineered to generate a mutant having increased lipid productivity and/or increased lipid partitioning as disclosed herein.
In some examples, a control microorganism can be a microorganism that is substantially identical to the mutant microorganism, with the exception that the control microorganism does not have a mutation in a gene that regulates lipid induction (i.e., the gene whose mutation results in increased lipid production under conditions in which the mutant microorganism has at least about half the biomass productivity of the progenitor strain). The properties of a lipid-overproducing mutant having a disrupted, attenuated, or otherwise directly or indirectly genetically manipulated gene (resulting in altered structure or expression of the lipid induction regulator gene) are also be compared with the same properties of a control cell that does not have a disrupted, attenuated, or otherwise directly or indirectly genetically manipulated lipid induction regulator gene resulting in altered structure or expression of the lipid induction regulator gene (regardless of whether the cell is “wild type”). For example, a control cell may be a recombinant cell that includes one or more non-native genes or a cell mutated in a gene other than the lipid induction regulator gene whose effects are being assessed, etc.
Heterokont species considered for use in the invention include, but are not limited to, Bacillariophytes (diatoms), Eustigmatophytes , Labrinthulids, and Thraustochytrids, such as, for example, species of Labryinthula, Labryinthuloides, Thraustochytrium, Schizochytrium, Aplanochytrium, Aurantiochytrium, Oblongichytrium, Japonochytrium, Diplophrys , or Ulkenia.
Mutant microorganisms having the properties disclosed herein, such as mutant microorganisms having attenuated expression of a gene that regulates lipid biosynthesis, such as the Bromo-1091 gene of N. gaditana and orthologs thereof can be, in various examples, of any eukaryotic microalgal strain such as, for example, any species of any of the genera Achnanthes, Amphiprora, Amphora, Ankistrodesmus, Asteromonas, Boekelovia, Bolidomonas, Borodinella, Botrydium, Botryococcus, Bracteococcus, Chaetoceros, Carteria, Chlamydomonas, Chlorococcum, Chlorogonium, Chlorella, Chroomonas, Chrysosphaera, Cricosphaera, Crypthecodinium, Cryptomonas, Cyclotella, Desmodesmus, Dunaliella, Elipsoidon, Emiliania, Eremosphaera, Ernodesmius, Euglena, Eustigmatos, Franceia, Fragilaria, Fragilaropsis, Gloeothamnion, Haematococcus, Hantzschia, Heterosigma, Hymenomonas, Isochrysis, Lepocinclis, Micractinium, Monodus, Monoraphidium, Nannochloris, Nannochloropsis, Navicula, Neochloris, Nephrochloris, Nephroselmis, Nitzschia, Ochromonas, Oedogonium, Oocystis, Ostreococcus, Parachlorella, Parietochloris, Pascheria, Pavlova, Pelagomonas, Phwodactylum, Phagus, Picochlorum, Platymonas, Pleurochrysis, Pleurococcus, Prototheca, Pseudochlorella, Pseudoneochloris, Pseudostaurastrum, Pyramimonas, Pyrobotrys, Scenedesmus, Schizochlamydella, Skeletonema, Spyrogyra, Stichococcus, Tetrachlorella, Tetraselmis, Thalassiosira, Tribonema, Vaucheria, Viridiella, Vischeria , and Volvox . Non-limiting examples of particularly suitable species include, for instance, diatoms such as, for example, a species of any of the genera Amphora, Chaetoceros, Cyclotella, Fragilaria, Fragilaropsis, Hantzschia, Monodus, Navicula, Nitzschia, Phæodactylum , or Thalassiosira , or Eustigmatophytes , e.g., Eustigmatos, Nannochloropsis, Pseudostaurastrum , or Vischeria.
The mutants can be spontaneous mutants, classically-derived mutants, or engineered mutants having attenuated expression of a regulator gene, for example, a gene whose expression affects the the expression of many other genes such as a gene encoding a transcription factor or a transcriptional activator. For example, a mutant microorganism as disclosed herein that produces at least 25% more lipid and at least 45% as much biomass as a control microorganism under conditions in which both the control microorganism and the mutant microorganism are accumulating biomass (e.g., under conditions that are nutrient replete with respect to the control microorganism, which can be a wild type microorganism) can have attenuated expression of a gene encoding a polypeptide that has a TAZ Zinc Finger domain, e.g., has an amino acid sequence encoding a TAZ Zinc Finger domain characterized as belonging to conserved domain family cd15614 or SMART domain 00551 or a TAZ Zinc Finger domain belonging to pfam PF02135. TAZ zinc finger (Transcription Adaptor putative Zinc finger) domains are zinc binding domains found in the transcriptional co-activators CREB-binding protein (CBP) and P300. Transcriptional coactivators are proteins that are recruited to DNA-binding transcription factors through their activation domains and increase transcription. CBP and P300 are histone acetyltransferases (EC) that catalyse the reversible acetylation of all four histones in nucleosomes, acting to regulate transcription via chromatin remodelling (De Guzman et al. (2004) J. Biol. Chem. 279:3042-3049). Such domains can be identified in a polypeptide encoded by a gene as provided herein with an e value of less than about 0.01, less than about 0.001, or less than about 10 −6 , for example. In some embodiments, a mutant microorganism as provided herein can have attenuated expression of a gene encoding a polypeptide that recruits to pfam PF02135 with a bit score of greater than 20.0 (the gathering cutoff of PF02135) and an e valure of less than 0.01, less than about 0.001, or less than about 10 −6 , for example. In some embodiments, a mutant microorganism as provided herein can have attenuated expression of a gene encoding a polypeptide that has an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to SEQ ID NO:9.
Alternatively or in addition, a mutant microorganism such as any disclosed herein that produces at least 20% more lipid than a control microorganism while producing at least 45% as much biomass as the control microorganism can have attenuated expression of a gene encoding a polypeptide that has a Bromo domain, e.g., can have an amino acid sequence encoding a Bromo domain characterized as conserved domain cd05506, SMART domain 00297, COG domain 5076, or a Bromo domain belonging to pfam PF00439. A bromodomain is an approximately 110 amino acid protein domain found on some chromatin associated proteins. The bromodomain recognizes acetylatedlysine residues, such as those on the N-terminal tails of histones. Changes in histone acetylation has been found to accompany chromatin remodelling that occurs with changes in transcriptional activity (Dhalluin et al. Nature 399:491-496). In some embodiments, a mutant microorganism as provided herein can have attenuated expression of a gene encoding a polypeptide that recruits to pfam PF00439 with a bit score of greater than 21.0 (the gathering cutoff of PF00439) and an e valure of less than 0.01, less than about 0.001, or less than about 10 −6 , for example. In some embodiments, a mutant microorganism as provided herein can have attenuated expression of a gene encoding a polypeptide that has an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to SEQ ID NO:10.
For example, a mutant microorganism as provided herein that produces at least 20% more lipid than a control microorganism while producing at least 45% as much biomass as the control microorganism can have attenuated expression of a gene encoding a polypeptide that includes a TAZ Zinc Finger domain and/or includes a Bromo domain. In some embodiments the mutant microorganism has attenuated expression of a gene encoding a polypeptide having a TAZ domain characterized as pfam PF02135 with a bit score of greater than 20.0 and an e value of less than 0.01, and a Bromo domain characterized as pfam PF00439 with a bit score of greater than 21.0 and an e value of less than 0.01. Alternatively or in addition, a mutant microorgnaism can include an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to SEQ ID NO:9 and/or an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to SEQ ID NO:10.
Alternatively or in addition, in various embodiments, a mutant microorganism as disclosed herein that produces at least 25% more lipid and at least 45% as much biomass as a control microorganism under conditions in which both the control microorganism and the mutant microorganism are accumulating biomass (e.g., under conditions that are nutrient replete with respect to the control microorganism, which can be a wild type microorganism) can have attenuated expression of a gene encoding a polypeptide that has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, or SEQ ID NO:46 and/or has a coding region having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, or SEQ ID NO:45.
The mutant microorganism having attenuated expression of a gene that regulates lipid production can be a “knockout” mutant, for example, in which the reading frame of the polypeptide is disrupted such that the functional protein is not produced. For example, the gene can include an insertion, deletion, or mutation in the reading frame that results in no functional protein being made. In various examples, a knockout mutation can be generated by insertion of a sequence, often but not necessarily including a selectable marker gene, into the gene, for example, into the coding region of the gene. Such an insertion can be by use is a cas/CRISPR system that inegrates a donor fragment into a targeted locus, or can be by homologous recombination, for example Such an insertion can disrupt an open reading frame and/or splicing signals, or generate nonfunctional fusion proteins or truncated proteins. In other examples, the mutant microorganism can be a “knockdown” mutant in which expression of the gene is reduced but not eliminated, for example, reduced from 5% or less to 95% or more, for example, from 5% to 95% or 10% to 90%, with respect to expression levels of a wild type cell. Knockdowns can be mutants in which a mutation, insertion, or deletion occurs in a non-coding region of the gene, for example, the 5′ or 3′ region of a gene, or can be effected by expressing constructs in the cells that reduce expression of the targeted gene, such as RNAi, ribozyme, or antisense constructs. In addition to CRISPR systems, homologous recombination can be used to generate insertion mutants (either knockdown or knockout).
A mutant microorganism as provided herein can be designed by targeting an endogenous gene of a microorganism of interest that encodes a polypeptide that includes a TAZ Zinc Finger domain as disclosed herein and/or a Bromo domain as disclosed herein. Such genes can be identified in a microorgnaism of interest using bioinformatics methods, molecular biology techniques and combinations thereof. For example, a gene encoding a polypeptide that includes a TAZ Zinc Finger domain and/or a Bromo domain can be identified using Southern hybridization, screening of cDNA libraries by hybridization, or PCR, for example, using degenerate probes and/or primers. Genome sequences available in public or proprietary databases can be searched by any of a number of programs that perform sequence matching (e.g., blast programs such as blastp, blastn, and tblastn (protein sequence queried against translated nucleotide sequence)) or analyze domain structures of encoded amino acid sequences. For example, hmmer.org provides software for analyzing structural and functional domains encoded by genes that can be used to scan genome sequences, including, for example, hmmsearch and hmmscan. Such searches can be done online. Programs such as MUSCLE and hmmalign can also be used to search for orthologs of proteins such as the proteins disclosed herein (e.g., TAZ Zinc Finger domain-containing polypeptides) by constructing phylogenetic trees to determine relationships among proteins. Gene targeting can make use of sequences identified in the genome of the microorganism of interest. It is not necessary to resolve the complete structure of a gene to target the gene for attenuation. For example, using methods disclosed herein, including, without limitation, cas/CRISPR genome editing, RNAi constructs, antisense constructs, homologous recombination constructs, and ribozyme constructs, only a portion of a gene sequence can be employed in gene attenuation constructs and techniques.
Gene Attenuation
A mutant microorganism as provided herein having attenuated expression of a gene that regulates lipid biosynthesis is a mutant generated by human intervention, for example, by classical mutagenesis or genetic engineering. For example, a mutant microorganism as provided herein can be a mutant generated by any feasible mutagenesis method, including but not limited to UV irradiation, gamma irradiation, or chemical mutagenesis, and screening for mutants having increased lipid production, for example by staining with lipophilic dyes such as Nile Red or BODIPY (e.g., Cabanelas et al. (2015) Bioresource Technology 184:47-52). Methods for generating mutants of microbial strains are well-known.
A mutant as provided herein that produces at least 25% more lipid while producing at least 50% of the biomass as the progenitor cell can also be a genetically engineered mutant, for example, a mutant in which a regulatory gene such as Bromo-1091 or an ortholog thereof (e.g., a gene encoding a polypeptide having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, or SEQ ID NO:46) has been targeted by homologous recombination for knock-out or gene replacement (for example with mutated form of the gene that may encode a polypeptide having reduced activity with respect to the wild type polypeptide). For example, a microbial strain of interest may be engineered by site directed homologous recombination to insert a sequence into a genomic locus and thereby alter a gene and/or its expression, or to insert a promoter into a genetic locus of the host microorganism to affect the expression of a particular gene or set of genes at the locus.
For example, gene knockout or replacement by homologous recombination can be by transformation of a nucleic acid (e.g., DNA) fragment that includes a sequence homologous to the region of the genome to be altered, where the homologous sequence is interrupted by a foreign sequence, typically a selectable marker gene that allows selection for the integrated construct. The genome-homologous flanking sequences on either side of the foreign sequence or mutated gene sequence can be for example, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1,000, at least 1,200, at least 1,500, at least 1,750, or at least 2,000 nucleotides in length. A gene knockout or gene “knock in” construct in which a foreign sequence is flanked by target gene sequences, can be provided in a vector that can optionally be linearized, for example, outside of the region that is to undergo homologous recombination, or can be provided as a linear fragment that is not in the context of a vector, for example, the knock-out or knock-in construct can be an isolated or synthesized fragment, including but not limited to a PCR product. In some instances, a split marker system can be used to generate gene knock-outs by homologous recombination, where two DNA fragments can be introduced that can regenerate a selectable marker and disrupt the gene locus of interest via three crossover events (Jeong et al. (2007) FEMS Microbiol Lett 273: 157-163).
In one aspect the invention provides genetically modified organisms, e.g. microorganisms having one or more genetic modifications for attenuating expression of a lipid regulator gene such as a gene encoding a polypeptide having at least 55% identity to any of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, or SEQ ID NO:46, or a gene having a coding region with at least 55% identity to any of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, or SEQ ID NO:45. As used herein “attenuating expression of a lipid regulator gene” means reducing or eliminating expression of the gene in any manner that reduces production of the fully functional protein. Means for attenuating a lipid regulator gene include, for example, homologous recombination constructs; CRISPR systems, including guide RNAs, Cas9 or other cas enzymes, and optionally, donor fragments for insertion into the targeted site; RNAi constructs, including shRNAs, antisense RNA constructs; ribozyme constructs; TALENS, Zinc Finger nucleases; and meganucleases.
For example, a recombinant microorganism engineered to have attenuated expression of a lipid regulator gene can have a disrupted lipid regulator gene that includes as least one insertion, mutation, or deletion that reduces or abolishes expression of the gene such that a fully functional lipid regulator gene is not produced or is produced in lower amounts than is produced by a control microorganism that does not include a disrupted lipid regulator gene.
The mutation (change, deletion, and/or insertion of one or more nucleotides) can be in the coding region of the gene or can be in an intron, 3′ UTR, 5′ UTR, or promoter region, e.g., within 2 kb of the transcriptional start site or within 3 kb or the translational start site. For example, a mutant having attenuated expression of a gene as disclosed herein can have a mutation, which can be one or more nucleobase changes and/or one or more nucleobase deletions and/or one or more nucleobase insertions, into the region of a gene 5′ of the transcriptional start site, such as, in non-limiting examples, within 2 kb, within 1.5 kb, within 1 kb, or within 0.5 kb of the known or putative transcriptional start site, or within 3 kb, within 2.5 kb, within 2 kb, within 1.5 kb, within 1 kb, or within 0.5 kb of the translational start site. As nonlimiting examples, a mutant gene can be a gene that has a mutation, insertion, or deletion within the promoter region that can either increase or decrease expression of the gene; can be a gene that has a deletion that results in production of a nonfunctional protein, truncated protein, dominant negative protein, or no protein; can be a gene that has one or more point mutations leading to a change in the amino acid of the encoded protein or results in aberrant splicing of the gene transcript, etc.
The disrupted lipid regulator gene can be disrupted by, for example, an insertion or gene replacement mediated by homologous recombination and/or by the activity of a meganuclease, zinc finger nuclease (Perez-Pinera et al. (2012) Curr. Opin. Chem. Biol. 16: 268-277), TALEN (WO 2014/207043; WO 2014/076571), or a cas protein (e.g., a Cas9 protein) of a CRISPR system.
CRISPR systems, reviewed recently by Hsu et al. ( Cell 157:1262-1278, 2014) include, in addition to the cas nuclease polypeptide or complex, a targeting RNA, often denoted “crRNA”, that interacts with the genome target site by complementarity with a target site sequence, a trans-activating (“tracr”) RNA that complexes with the cas polypeptide and also includes a region that binds (by complementarity) the targeting crRNA.
The invention contemplates the use of two RNA molecules (a “crRNA” and a “tracrRNA”) that can be co-transformed into a host strain (or expressed in a host strain) that expresses or is transfected with a cas protein for genome editing, or the use of a single guide RNA that includes a sequence complementary to a target sequence as well as a sequence that interacts with a cas protein. That is, in some strategies a CRISPR system as used herein can comprise two separate RNA molecules (RNA polynucleotides: a “tracr-RNA” and a “targeter-RNA” or “crRNA”, see below) and referred to herein as a “double-molecule DNA-targeting RNA” or a “two-molecule DNA-targeting RNA.” Alternatively, as illustrated in the examples, the DNA-targeting RNA can also include the trans-activating sequence for interaction with the cas protein (in addition to the target-homologous (“cr”) sequences), that is, the DNA-targeting RNA can be a single RNA molecule (single RNA polynucleotide) and is referred to herein as a “chimeric guide RNA,” a “single-guide RNA,” or an “sgRNA.” The terms “DNA-targeting RNA” and “gRNA” are inclusive, referring both to double-molecule DNA-targeting RNAs and to single-molecule DNA-targeting RNAs (i.e., sgRNAs). Both single-molecule guide RNAs and two RNA systems have been described in detail in the literature and for example, in U.S. Patent Application Publication No. US 2014/0068797, incorporated by reference herein in its entirety.
Any cas protein can be used in the methods herein, e.g., Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Cbf1, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Cpf1, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, C2c2, C2c3, and homologs thereof, or modified versions thereof. The cas protein can be a Cas9 protein, such as a Cas9 protein of Staphylococcus pyogenes, S. thermophilus, S. pneumonia, S. aureus , or Neisseria meningitidis , as nonlimiting examples. Also considered are the Cas9 proteins provided as SEQ ID NOs:1-256 and 795-1346 in U.S. Patent Application Publication No. US 2014/0068797, incorporated herein by reference in its entirety, and chimeric Cas9 proteins that may combine domains from more than one Cas9 protein, as well variants and mutants of identified Cas9 proteins.
Cas nuclease activity cleaves target DNA to produce double strand breaks. These breaks are then repaired by the cell in one of two ways: non-homologous end joining or homology-directed repair. In non-homologous end joining (NHEJ), the double-strand breaks are repaired by direct ligation of the break ends to one another. In this case, no new nucleic acid material is inserted into the site, although some nucleic acid material may be lost, resulting in a deletion, or altered, often resulting in mutation. In homology-directed repair, a donor polynucleotide (sometimes referred to as a “donor DNA” or “editing DNA”) which may have homology to the cleaved target DNA sequence is used as a template for repair of the cleaved target DNA sequence, resulting in the transfer of genetic information from the donor polynucleotide to the target DNA. As such, new nucleic acid material may be inserted/copied into the site. The modifications of the target DNA due to NHEJ and/or homology-directed repair (for example using a donor DNA molecule) can lead to, for example, gene correction, gene replacement, gene tagging, transgene insertion, nucleotide deletion, gene disruption, gene mutation, etc.
In some instances, cleavage of DNA by a site-directed modifying polypeptide (e.g., a cas nuclease, zinc finger nuclease, meganuclease, or TALEN) may be used to delete nucleic acid material from a target DNA sequence by cleaving the target DNA sequence and allowing the cell to repair the sequence in the absence of an exogenously provided donor polynucleotide. Such NHEJ events can result in mutations (“mis-repair”) at the site of rejoining of the cleaved ends that can resulting in gene disruption.
Alternatively, if a DNA-targeting RNA is co-administered to cells that express a cas nuclease along with a donor DNA, the subject methods may be used to add, i.e. insert or replace, nucleic acid material to a target DNA sequence (e.g. “knock out” by insertional mutagenesis, or “knock in” a nucleic acid that encodes a protein (e.g., a selectable marker and/or any protein of interest), an siRNA, a miRNA, etc., to modify a nucleic acid sequence (e.g., introduce a mutation), and the like.
A donor DNA can in particular embodiments include a gene regulatory sequence (e.g., a promoter) that can, using CRISPR targeting, be inserted upstream of the coding regions of the gene and upstream of the presumed proximal promoter region of the gene, for example, at least 50 bp, at least 100 bp, at least 120 bp, at least 150 bp, at least 200 bp, at least 250 bp, at least 300 bp, at least 350 bp, at least 400 bp, at least 450 bp, or at least 500 bp upstream of the initiating ATG of the coding region of the lipid regulator gene. The donor DNA can include a sequence, such as for example a selectable marker or any convenient sequence, that may be interfere with the native promoter. The additional sequence inserted upstream of the initiating ATG of the lipid regulator open reading frame (e.g., in the 5′UTR or upstream of the transcriptional start site of the lipid regulator gene) can decrease or even eliminate expression of the endogenous lipid regulator gene. Alternatively or in addition, the native lipid regulator gene can have its endogenous promoter wholly or partially replaced by a weaker or differently regulated promoter, or a non-promoter sequence.
In some examples, a nucleic acid molecule introduced into a host cell for generating a high efficiency genome editing cell line encodes a Cas9 enzyme that is mutated to with respect to the corresponding wild-type enzyme such that the mutated Cas9 enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence. For example, an aspartate-to-alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (an enzyme that cleaves a single strand). Other examples of mutations that render Cas9 a nickase include, without limitation, H840A, N854A, and N863A. In some embodiments, a Cas9 nickase may be used in combination with guide sequence(s), e.g., two guide sequences, which target respectively sense and antisense strands of the DNA target. This combination allows both strands to be nicked and used to induce NHEJ. Two nickase targets (within close proximity but targeting different strands of the DNA) can be used to inducing mutagenic NHEJ. Such targeting of a locus using enzymes that cleave opposite strains at staggered positions can also reduce nontarget cleavage, as both strands must be accurately and specifically cleaved to achieve genome mutation.
In additional examples, a mutant Cas9 enzyme that is impaired in its ability to cleave DNA can be expressed in the cell, where one or more guide RNAs that target a sequence upstream of the transcriptional or translational start site of the targeted gene are also introduced. In this case, the cas enzyme may bind the target sequence and block transcription of the targeted gene (Qi et al. (2013) Cell 152:1173-1183). This CRISPR interference of gene expression can be referred to as RNAi and is also described in detail in Larson et al. (2013) Nat. Protoc. 8: 2180-2196.
In some cases, a cas polypeptide such as a Cas9 polypeptide is a fusion polypeptide, comprising, e.g.: i) a Cas9 polypeptide (which can optionally be variant Cas9 polypeptide as described above); and b) a covalently linked heterologous polypeptide (also referred to as a “fusion partner”). A heterologous nucleic acid sequence may be linked to another nucleic acid sequence (e.g., by genetic engineering) to generate a chimeric nucleotide sequence encoding a chimeric polypeptide. In some embodiments, a Cas9 fusion polypeptide is generated by fusing a Cas9 polypeptide with a heterologous sequence that provides for subcellular localization (i.e., the heterologous sequence is a subcellular localization sequence, e.g., a nuclear localization signal (NLS) for targeting to the nucleus; a mitochondrial localization signal for targeting to the mitochondria; a chloroplast localization signal for targeting to a chloroplast; an ER retention signal; and the like). In some embodiments, the heterologous sequence can provide a tag (i.e., the heterologous sequence is a detectable label) for ease of tracking and/or purification (e.g., a fluorescent protein, e.g., green fluorescent protein (GFP), YFP, RFP, CFP, mCherry, tdTomato, and the like; a hemagglutinin (HA) tag; a FLAG tag; a Myc tag; and the like).
Host cells can be genetically engineered (e.g. transduced or transformed or transfected) with, for example, a vector construct that can be, for example, a vector for homologous recombination that includes nucleic acid sequences homologous to a portion of a lipid regulator gene locus of the host cell or to regions adjacent thereto, or can be an expression vector for the expression of any or a combination of: a cas protein (e.g., a Cas9 protein), a CRISPR chimeric guide RNA, a crRNA, and/or a tracrRNA, an RNAi construct (e.g., a shRNA), an antisense RNA, or a ribozyme. The vector can be, for example, in the form of a plasmid, a viral particle, a phage, etc. A vector for expression of a polypeptide or RNA for genome editing can also be designed for integration into the host, e.g., by homologous recombination. A vector containing a polynucleotide sequence as described herein, e.g., sequences having homology to host lipid regulator gene sequences (including sequences that are upstream and downstream of the lipid regulator-encoding sequences), as well as, optionally, a selectable marker or reporter gene, can be employed to transform an appropriate host to cause attenuation of a lipid regulator gene.
The recombinant microorganism in some examples can have reduced but not abolished expression of the lipid regulator gene, and the recombinant microorganism can have an increase in lipid production of from about 25% to about 200% or more, for example. A genetically modified microorganism as provided herein can in some examples include a nucleic acid construct for attenuating the expression of a lipid regulator gene, such as, for example, a gene encoding a polypeptide having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, or SEQ ID NO:46. For example, a host microorganism can include a construct for expressing an RNAi molecule, ribozyme, or antisense molecule that reduces expression of a lipid regulator gene encoding a polypeptide having at least 55% identity to any of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, or SEQ ID NO:46. In some examples, a recombinant microorganism as provided herein can include at least one introduced (exogenous or non-native) construct for reducing expression of a lipid regulator gene.
In some examples, engineered strains can be selected for expression of a lipid regulator gene that is decreased with respect to a control cell that does not include a genetic modification for attenuating lipid regulator gene expression, but not eliminated, using methods known in the art, such as, for example, RNA-Seq or reverse transcription-PCR (RT-PCR).
A genetically engineered strain as provided herein can be engineered to include a construct for attenuating gene expression by reducing the amount, stability, or translatability of mRNA of a gene encoding a lipid regulator. For example, a microorganism such as an algal or heterokont strain can be transformed with an antisense RNA, RNAi, or ribozyme construct targeting an mRNA of a lipid regulator gene using methods known in the art. For example, an antisense RNA construct that includes all or a portion of the transcribed region of a gene can be introduced into a microorganism to decrease gene expression (Shroda et al. (1999) The Plant Cell 11:1165-78; Ngiam et al. (2000) Appl. Environ. Microbiol. 66: 775-782; Ohnuma et al. (2009) Protoplasma 236: 107-112; Lavaud et al. (2012) PLoS One 7:e36806). Alternatively or in addition, an RNAi construct (for example, a construct encoding a short hairpin RNA) targeting a gene having a TAZ zinc finger domain and/or a Bromo domain can be introduced into a microorganism such as an alga or heterokont for reducing expression of the lipid regulator gene (see, for example, Cerruti et al. (2011) Eukaryotic Cell (2011) 10: 1164-1172; Shroda et al. (2006) Curr. Genet. 49:69-84).
Ribozymes are RNA-protein complexes that cleave nucleic acids in a site-specific fashion. Ribozymes have specific catalytic domains that possess endonuclease activity. For example, U.S. Pat. No. 5,354,855 (incorporated herein by reference) reports that certain ribozymes can act as endonucleases with a sequence specificity greater than that of known ribonucleases and approaching that of the DNA restriction enzymes. Catalytic RNA constructs (ribozymes) can be designed to base pair with an mRNA encoding a gene as provided herein to cleave the mRNA target. In some examples, ribozyme sequences can be integrated within an antisense RNA construct to mediate cleavage of the target. Various types of ribozymes can be considered, their design and use is known in the art and described, for example, in Haseloff et al. (1988) Nature 334:585-591.
Ribozymes are targeted to a given sequence by virtue of annealing to a site by complimentary base pair interactions. Two stretches of homology are required for this targeting. These stretches of homologous sequences flank the catalytic ribozyme structure defined above. Each stretch of homologous sequence can vary in length from 7 to 15 nucleotides. The only requirement for defining the homologous sequences is that, on the target RNA, they are separated by a specific sequence which is the cleavage site. For hammerhead ribozyme, the cleavage site is a dinucleotide sequence on the target RNA is a uracil (U) followed by either an adenine, cytosine or uracil (A, C or U) (Thompson et al., (1995) Nucl Acids Res 23:2250-68). The frequency of this dinucleotide occurring in any given RNA is statistically 3 out of 16. Therefore, for a given target messenger RNA of 1,000 bases, 187 dinucleotide cleavage sites are statistically possible.
The general design and optimization of ribozyme directed RNA cleavage activity has been discussed in detail (Haseloff and Gerlach (1988) Nature 334:585-591; Symons (1992) Ann Rev Biochem 61: 641-71; Chowrira et al. (1994) J Biol Chem 269:25856-64; Thompson et al. (1995) supra), all incorporated by reference in their entireties. Designing and testing ribozymes for efficient cleavage of a target RNA is a process well known to those skilled in the art. Examples of scientific methods for designing and testing ribozymes are described by Chowrira et al., (1994) supra and Lieber and Strauss (1995) Mol Cell Biol. 15: 540-51, each incorporated by reference. The identification of operative and preferred sequences for use in down regulating a given gene is a matter of preparing and testing a given sequence, and is a routinely practiced “screening” method known to those of skill in the art.
The use of RNAi constructs is described in literature cited above as well as in US2005/0166289 and WO 2013/016267 (both of which are incorporated herein by reference), for example A double stranded RNA with homology to the target gene is delivered to the cell or produced in the cell by expression of an RNAi construct, for example, an RNAi short hairpin (sh) construct. The construct can include a sequence that is identical to the target gene, or at least 70%, 80%, 90%, 95%, or between 95% and 100% identical to a sequence of the target gene. The construct can have at least 20, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1 kb of sequence homologous to the target gene. Expression vectors can be engineered using promoters selected for continuous or inducible expression of an RNAi construct, such as a construct that produces an shRNA.
A nucleic acid construct for gene attenuation, e.g., a ribozyme, RNAi, or antisense construct can include at least fifteen, at least twenty, at least thirty, at least forty, at least fifty, or at least sixty nucleotides having at least 80% identity, such as at least 85%, at least 90%, at least 95%, or at least 99% or complementarity to at least a portion of the sequence of an endogenous lipid regulator gene of the microorganism to be engineered. A nucleic acid construct for gene attenuation, e.g., a ribozyme, RNAi, or antisense construct can include at least fifteen, at least twenty, at least thirty, at least forty, at least fifty, or at least sixty nucleotides having at least 80%, such as at least 95% or about 100%, identity or complementarity to the sequence of a naturally-occurring gene, such as a gene having encoding a polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80% or at least 85%, at least 90%, or at least 95% sequence identity to an endogenous lipid regulator gene. For example, a nucleic acid construct for gene attenuation, e.g., a ribozyme, RNAi, or antisense construct can include at least fifteen, at least twenty, at least thirty, at least forty, at least fifty, or at least sixty nucleotides having at least 80% identity or complementarity to the sequence of a naturally-occurring lipid regulator gene, such as any provided herein. The nucleotide sequence can be, for example, from about 30 nucleotides to about 3 kilobases or greater, for example, from 30-50 nucleotides in length, from 50 to 100 nucleotides in length, from 100 to 500 nucleotides in length, from 500 nucleotides to 1 kb in length, from 1 kb to 2 kb in length, or from 2 to 5 kb. For example, an antisense sequence can be from about 100 nucleotides to about 1 kb in length. For example, a nucleic acid construct for gene attenuation, e.g., a ribozyme, RNAi, or antisense construct can include at least fifteen, at least twenty, at least thirty, at least forty, at least fifty, at least sixty, or at least 100 nucleotides having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85%, for example at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95% identity or complementarity to an endogenous lipid regulator gene or a portion thereof.
Promoters used in antisense, RNAi, or ribozyme constructs can be any that are functional in the host organism and that are suitable for the levels of expression required for reducing expression of the target gene to a desired amount. Promoters functional in algae and heterokonts are known in the art and disclosed herein. The construct can be transformed into algae using any feasible method, include any disclosed herein. A recombinant organism or microorganism transformed with a nucleic acid molecule for attenuating lipid regulator gene expression, such as but not limited to an antisense, RNAi, or ribozyme construct, can have the properties of a lipid regulator mutant as described herein, including, for example, reduced chlorophyll, increased photosynthetic efficiency, and increased productivity in culture, with respect to a host organism or microorganism that does not include the exogenous nucleic acid molecule that results in attenuated gene expression.
Nucleic Acid Molecules and Constructs
Also provided herein are nucleic acid molecules encoding polypeptides that include amino acid sequences having at least 60%, at least 65%, at least 70%, or at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, or SEQ ID NO:46. Alternatively or in addition, a nucleic acid molecule as provided herein can include a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, or SEQ ID NO:45. The polypeptide having at least 60% identity to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, and SEQ ID NO:46, and/or encoded by a nucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, and SEQ ID NO:45 can include an amino acid sequence encoding a TAZ zinc finger domain, e g, a domain belonging to pfam PF02135. For example, the polypeptide encoded by the nucleic acid molecule can include a TAZ zinc finger domain having an amino acid sequence with at least 60%, at least 65%, at least 70%, or at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to SEQ ID NO:9. Alternatively or in addition, a polypeptide encoded by a nucleic acid molecule as provided herein can optionally further include a Bromo domain, e.g., a domain belonging to pfam PF00439. For example a polypeptide encoded by a nucleic acid molecule as provided herein can include a Bromo domain having at least 60%, at least 65%, at least 70%, or at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to SEQ ID NO:10.
The nucleic acid molecule in various examples can be or comprise a cDNA that lacks one or more introns present in the naturally-occurring gene, or, alternatively, can include one or more introns not present in the naturally-occurring gene. The nucleic acid molecule in various examples can have a sequence that is not 100% identical to a naturally-occurring gene. For example, the nucleic acid molecule can include a mutation with respect to a naturally-occurring gene that reduces the activity of the encoded polypeptide or reduces expression of the mRNA or protein encoded by the gene.
The nucleic acid molecule in various examples can comprise a heterologous promoter operably linked to the sequence encoding a polypeptide that includes an amino acid sequence having at least 60%, at least 65%, at least 70%, or at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, or SEQ ID NO:46 and/or having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, or SEQ ID NO:45. Alternatively or in addition, a nucleic acid molecule can comprise a vector that includes a sequence encoding a polypeptide that includes an amino acid sequence having at least 60%, at least 65%, at least 70%, or at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, or SEQ ID NO:46 and/or has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, or SEQ ID NO:45.
A further aspect of the invention is a construct designed for attenuating expression of a gene encoding a TAZ zinc finger domain and/or a Bromo domain. The construct can be or comprise, in various examples, a sequence encoding a guide RNA of a CRISPR system, an RNAi construct, an antisense construct, a ribozyme construct, or a construct for homologous recombination, e.g., a construct having one or more nucleotide sequences having homology to a naturally-occurring TAZ zinc finger and/or Bromo domain-encoding gene as disclosed herein and/or sequences adjacent thereto in the native genome from which the gene is derived. For example, the construct can include at least a portion of a gene encoding a polypeptide having a TAZ zinc finger domain and/or a Bromo domain, e.g., a sequence homologous to at least a portion of an gene that encodes a polypeptide that includes an amino acid sequence having at least 60%, at least 65%, at least 70%, or at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to SEQ ID NO:9 or SEQ ID NO:10.
The construct for gene attenuation can include, for example, at least a portion of the coding region, intron, 5′UTR, promoter region, or 3′ UTR of a gene encoding a polypeptide having a TAZ zinc finger domain and/or a Bromo domain or a polypeptide having at least 60% identity to any of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, or SEQ ID NO:46, or at least a portion of a gene having at least 50% identity to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, or SEQ ID NO:45, in either sense or antisense orientation.
In further examples a construct can be designed for the in vitro or in vivo expression of a guide RNA (e.g., of a CRISPR system) designed to target a gene having a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to at least a portion of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, or SEQ ID NO:45, or coding a polypeptide having a TAZ Zinc Finger domain and/or a Bromo domain or a polypeptide having at least 60% identity to any of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, or SEQ ID NO:46, and/or can include a sequence homologous to a portion of a gene encoding a polypeptide having a TAZ Zinc Finger domain and/or a Bromo domain, including, for example, an intron, a 5′UTR, a promoter region, and/or a 3′ UTR.
In yet further examples, a construct for attenuating expression of a gene encoding a TAZ Zinc Finger domain and/or a Bromo domain-containing polypeptide can be a guide RNA or antisense oligonucleotide, where the sequence having homology to a transcribed region of a gene encoding a polypeptide having a TAZ Zinc Finger domain and/or a Bromo domain in antisense orientation.
Nucleic acid constructs for attenuating expression of a TAZ Zinc Finger domain and/or a Bromo domain-encoding gene or a gene encoding a polypeptide having at least 60% identity to any of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, or SEQ ID NO:46 can include, for example at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 nucleotides of sequence of a naturally occurring TAZ Zinc Finger domain and/or a Bromo domain-encoding gene or a gene encoding a polypeptide having at least 60% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, or SEQ ID NO:46 and/or a gene having at least 50% identity to a portion of any of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, or SEQ ID NO:45.
In one example, provided herein is a nucleic acid molecule having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to at least a portion of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, or SEQ ID NO:45, where the nucleic acid molecule encodes a guide RNA of a CRISPR system. The nucleic acid molecule can include, for example at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 nucleotides of sequence of a naturally occurring TAZ Zinc Finger domain and/or a Bromo domain gene, such as but not limited to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, or SEQ ID NO:45.
In addition, provided herein are antisense, ribozyme, or RNAi constructs that include at least a portion of a gene having encoding a TAZ zinc finger domain and/or a Bromo domain or a polypeptide having at least 60% identity to any of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, or SEQ ID NO:46 and/or a gene having at least 50% identity to a portion of any of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, or SEQ ID NO:45, in which a promoter, such as a heterologous promoter, is operably linked to the TAZ zinc finger domain and/or a Bromo domain gene sequence and the TAZ zinc finger domain and/or a Bromo domain gene sequence is in antisense orientation.
Further, provided herein are constructs for homologous recombination that include at least one sequence from a TAZ zinc finger domain and/or a Bromo domain-encoding gene locus of the genome of an alga juxtaposed with a heterologous nucleic acid sequence that can be, in nonlimiting examples, a selectable marker or detectable marker gene. In some examples a construct for homologous recombination includes two nucleic acid sequences from a TAZ zinc finger domain and/or Bromo domain-encoding gene locus of the genome of an alga where the two sequences flank a heterologous sequence for insertion into the TAZ zinc finger domain and/or Bromo domain gene locus.
One skilled in the art will appreciate that a number of transformation methods can be used for genetic transformation of microorganisms and, therefore, can be deployed for the methods of the present invention. “Stable transformation” is intended to mean that the nucleic acid construct introduced into an organism integrates into the genome of the organism or is part of a stable episomal construct and is capable of being inherited by the progeny thereof. “Transient transformation” is intended to mean that a polynucleotide is introduced into the organism and does not integrate into the genome or otherwise become established and stably inherited by successive generations.
Genetic transformation can result in stable insertion and/or expression of transgenes, constructs from either the nucleus or the plastid, and in some cases can result in transient expression of transgenes. The transformation methods can also be used for the introduction of guide RNAs or editing DNAs. Genetic transformation of microalgae has been reported successful for more than 30 different strains of microalgae, which belong to at least ˜22 species of green, red, and brown algae, diatoms, euglenids, and dianoflagellates (see, e.g., Radakovits et al., Eukaryotic Cell, 2010; and Gong et al., J. Ind. Microbiol. Biotechnol., 2011). Non-limiting examples of such useful transformation methods include agitation of cells in the presence of glass beads or silicon carbide whiskers as reported by, for example, Dunahay, Biotechniques, 15(3):452-460, 1993; Kindle, Proc. Natl. Acad. Sci. U.S.A., 1990; Michael and Miller, Plant J., 13, 427-435, 1998. Electroporation techniques have been successfully used for genetic transformation of several microalgal species including Nannochloropsis sp. (see, e.g., Chen et al., J. Phycol., 44:768-76, 2008), Chlorella sp. (see, e.g., Chen et al., Curr. Genet., 39:365-370, 2001; Chow and Tung, Plant Cell Rep . Vol. 18, No. 9, 778-780, 1999), Chlamydomonas (Shimogawara et al., Genetics, 148: 1821-1828, 1998), Dunaliella (Sun et al., Mol. Biotechnol., 30(3): 185-192, 2005). Micro-projectile bombardment, also referred to as microparticle bombardment, gene gun transformation, or biolistic bombardment, has been used successfully for several algal species including, for example, diatoms species such as Phaeodactylum (Apt et al., Mol. Gen. Genet., 252:572-579, 1996), Cyclotella and Navicula (Dunahay et al., J. Phycol., 31:1004-1012, 1995), Cylindrotheca (Fischer et al., J. Phycol., 35:113-120, 1999), and Chaetoceros sp. (Miyagawa-Yamaguchi et al., Phycol. Res. 59: 113-119, 2011), as well as green algal species such as Chlorella (El-Sheekh, Biologia Plantarum , Vol. 42, No. 2: 209-216, 1999), and Volvox species (Jakobiak et al., Protist, 155:381-93, 2004). Additionally, Agrobacterium -mediated gene transfer techniques can also be useful for genetic transformation of microalgae, as has been reported by, for example, Kumar, Plant Sci., 166(3):731-738, 2004, and Cheney et al., J. Phycol ., Vol. 37, Suppl. 11, 2001.
A transformation vector or construct as described herein will typically comprise a marker gene that confers a selectable or scorable phenotype on target host cells, e.g., algal cells or may be co-transformed with a construct that includes a marker. A number of selectable markers have been successfully developed for efficient isolation of genetic transformants of algae. Common selectable markers include antibiotic resistance, fluorescent markers, and biochemical markers. Several different antibiotic resistance genes have been used successfully for selection of microalgal transformants, including blastocidin, bleomycin (see, for example, Apt et al., 1996, supra; Fischer et al., 1999, supra; Fuhrmann et al., Plant J., 19, 353-61, 1999, Lumbreras et al., Plant J., 14(4):441-447, 1998; Zaslayskaia et al., J. Phycol., 36:379-386, 2000), spectinomycin (Cerutti et al., Genetics, 145: 97-110, 1997; Doetsch et al., Curr. Genet., 39, 49-60, 2001; Fargo, Mol. Cell. Biol., 19:6980-90, 1999), streptomycin (Berthold et al., Protist, 153:401-412, 2002), paromomycin (Jakobiak et al., Protist , supra.; Sizova et al., Gene, 277:221-229, 2001), nourseothricin (Zaslayskaia et al., 2000, supra), G418 (Dunahay et al., 1995, supra; Poulsen and Kroger, FEBS Lett., 272:3413-3423, 2005, Zaslayskaia et al., 2000, supra), hygromycin (Berthold et al., 2002, supra), chloramphenicol (Poulsen and Kroger, 2005, supra), and many others. Additional selectable markers for use in microalgae such as Chlamydomonas can be markers that provide resistance to kanamycin and amikacin resistance (Bateman, Mol. Gen. Genet. 263:404-10, 2000), zeomycin and phleomycin (e.g., ZEOCIN™ pheomycin D1) resistance (Stevens, Mol. Gen. Genet. 251:23-30, 1996), and paramomycin and neomycin resistance (Sizova et al., 2001, supra). Other fluorescent or chromogenic markers that have been used include luciferase (Falciatore et al., J. Mar. Biotechnol., 1: 239-251, 1999; Fuhrmann et al., Plant Mol. Biol., 2004; Jarvis and Brown, Curr. Genet., 19: 317-322, 1991), β-glucuronidase (Chen et al., 2001, supra; Cheney et al., 2001, supra; Chow and Tung, 1999, supra; El-Sheekh, 1999, supra; Falciatore et al., 1999, supra; Kubler et al., J. Mar. Biotechnol., 1:165-169, 1994), β-galactosidase (Gan et al., J. Appl. Phycol., 15:345-349, 2003; Jiang et al., Plant Cell Rep., 21:1211-1216, 2003; Qin et al., High Technol. Lett., 13:87-89, 2003), and green fluorescent protein (GFP) (Cheney et al., 2001, supra; Ender et al., Plant Cell, 2002, Franklin et al., Plant J., 2002; 56, 148, 210).
One skilled in the art will readily appreciate that a variety of known promoter sequences can be usefully deployed for transformation systems of microalgal species in accordance with the present invention. For example, the promoters commonly used to drive transgene expression in microalgae include various versions of the of cauliflower mosaic virus promoter 35S (CaMV35S), which has been used in both dinoflagellates and chlorophyta (Chow et al, Plant Cell Rep., 18:778-780, 1999; Jarvis and Brown, Curr. Genet., 317-321, 1991; Lohuis and Miller, Plant J., 13:427-435, 1998). The SV40 promoter from simian virus has also reported to be active in several algae (Gan et al., J. Appl. Phycol., 151 345-349, 2003; Qin et al., Hydrobiologia 398-399, 469-472, 1999). The promoters of RBCS2 (ribulose bisphosphate carboxylase, small subunit) (Fuhrmann et al., Plant J., 19:353-361, 1999) and PsaD (abundant protein of photosystem I complex; Fischer and Rochaix, FEBS Lett. 581:5555-5560, 2001) from Chlamydomonas can also be useful. The fusion promoters of HSP70A/RBCS2 and HSP70A/β2TUB (tubulin) (Schroda et al., Plant J., 21:121-131, 2000) can also be useful for an improved expression of transgenes, in which HSP70A promoter may serve as a transcriptional activator when placed upstream of other promoters. High-level expression of a gene of interest can also be achieved in, for example diatoms species, under the control of a promoter of an fcp gene encoding a diatom fucoxanthin-chlorophyll a/b binding protein (Falciatore et al., Mar. Biotechnol., 1:239-251, 1999; Zaslayskaia et al., J. Phycol. 36:379-386, 2000) or the vcp gene encoding a eustigmatophyte violaxanthin-chlorophyll a/b binding protein (see U.S. Pat. No. 8,318,482, incorporated by reference herein). If so desired, inducible promoters can provide rapid and tightly controlled expression of genes in transgenic microalgae. For example, promoter regions of the NR genes encoding nitrate reductase can be used as such inducible promoters. The NR promoter activity is typically suppressed by ammonium and induced when ammonium is replaced by nitrate (Poulsen and Kroger, FEBS Lett 272:3413-3423, 2005), thus gene expression can be switched off or on when microalgal cells are grown in the presence of ammonium/nitrate. Additional algal promoters that can find use in the constructs and transformation systems provided herein include those disclosed in U.S. Pat. No. 8,883,993; U.S. Patent Appl. Pub. No. US 2013/0023035; U.S. Patent Application Pub. No. US 2013/0323780; and U.S. Patent Application Pub. No. US 2014/0363892, all incorporated herein by reference in their entireties.
Host cells can be either untransformed cells or cells that are already transfected with at least one nucleic acid molecule. For example, an algal host cell that is engineered to have attenuated expression of a lipid regulator gene can further include one or more genes that may confer any desirable trait, such as, but not limited to, increased production of biomolecules of interest, such as one or more proteins, pigments, alcohols, or lipids.
Methods of Producing Lipids
Also provided herein are methods of producing lipid by culturing a mutant microorganism as provided herein that has increased lipid productivity with respect to a control cell while producing at least 45% of the biomass produced by a control cell under the same culture conditions. The methods include culturing a mutant microorganism as provided herein in a suitable medium to produce lipid and recovering biomass or at least one lipid from the culture. The microorganism can in some examples be an alga, and the culture can be a photoautotrophic culture. Culturing can be in batch, semi-continuous, or continuous mode.
The mutant microorganism in some examples can be cultured in a medium that comprises less than about 5 mM ammonium, for example, less than about 2.5 mM ammonium, less than about 2 mM ammonium, less than about 1.5 mM ammonium, less than or equal to about 1 mM ammonium, or less than or equal to about 0.5 mM. The culture medium can include, for example, from about 0 to about 2.5 mM ammonium, from about 0.1 to about 2.5 mM ammonium, from about 0.5 to about 2.5 mM ammonium, from about 0 to about 1.5 mM ammonium, from about 0.1 to about 1 mM ammonium, or from about 0.2 to about 1 mM ammonium. The microorganism can be cultured in a medium that includes nitrate, which in some examples may be substantially the sole nitrogen source in the culture medium or may be present in addition to less than 5 mM ammonium, less than 2.5 mM ammonium, less than 1.0 mM ammonium, or less than or equal to about 0.5 mM ammonium. Alternatively or in addition, the culture medium can comprises urea, which in some examples can be substantially the sole source of nitrogen in the culture medium.
The lipid producing microorganisms may be cultured in any suitable vessel(s), including flasks or bioreactors. In some examples, the mutant microorganism is an alga and is exposed to light for at least a portion of the culture period, in which the algae may be exposed to artificial or natural light (or natural light supplemented with artificial light). The culture comprising mutant algae that are deregulated in their response to low light may be cultured on a light/dark cycle that may be, for example, a natural or programmed light/dark cycle, and as illustrative examples, may provide twelve hours of light to twelve hours of darkness, fourteen hours of light to ten hours of darkness, sixteen hours of light to eight hours of darkness, etc. Alternatively, an algal mutant can be cultured in continuous light.
Culturing refers to the intentional fostering of growth (e.g., increases in cell size, cellular contents, and/or cellular activity) and/or propagation (e.g., increases in cell numbers via mitosis) of one or more cells by use of selected and/or controlled conditions. The combination of both growth and propagation may be termed proliferation. A microorganism as provided herein may be cultured for at least five, at least six, at least seven at least eight, at least nine, at least ten, at least eleven at least twelve, at least thirteen, at least fourteen, or at least fifteen days, or at least one, two three, four, five, six, seven, eight, nine, or ten weeks, or longer. The culturing can be in a culture medium that is nutrient replete with respect to a control alga.
Non-limiting examples of selected and/or controlled conditions that can be used for culturing the recombinant microorganism can include the use of a defined medium (with known characteristics such as pH, ionic strength, and/or carbon source), specified temperature, oxygen tension, carbon dioxide levels, growth in a bioreactor, or the like, or combinations thereof. In some embodiments, the microorganism or host cell can be grown mixotrophically, using both light and a reduced carbon source. Alternatively, the microorganism or host cell can be cultured phototrophically. When growing phototrophically, the algal strain can advantageously use light as an energy source. An inorganic carbon source, such as CO 2 or bicarbonate can be used for synthesis of biomolecules by the microorganism. “Inorganic carbon”, as used herein, includes carbon-containing compounds or molecules that cannot be used as a sustainable energy source by an organism. Typically “inorganic carbon” can be in the form of CO 2 (carbon dioxide), carbonic acid, bicarbonate salts, carbonate salts, hydrogen carbonate salts, or the like, or combinations thereof, which cannot be further oxidized for sustainable energy nor used as a source of reducing power by organisms. A microorganism grown photoautotrophically can be grown on a culture medium in which inorganic carbon is substantially the sole source of carbon. For example, in a culture in which inorganic carbon is substantially the sole source of carbon, any organic (reduced) carbon molecule or organic carbon compound that may be provided in the culture medium either cannot be taken up and/or metabolized by the cell for energy and/or is not present in an amount sufficient to provide sustainable energy for the growth and proliferation of the cell culture.
Microorganisms and host cells that can be useful in accordance with the methods of the present invention can be found in various locations and environments throughout the world. The particular growth medium for optimal propagation and generation of lipid and/or other products can vary and may be optimized to promote growth, propagation, or production of a product such as a lipid, protein, pigment, antioxidant, etc. In some cases, certain strains of microorganisms may be unable to grow in a particular growth medium because of the presence of some inhibitory component or the absence of some essential nutritional requirement of the particular strain of microorganism or host cell.
Solid and liquid growth media are generally available from a wide variety of sources, as are instructions for the preparation of particular media suitable for a wide variety of strains of microorganisms. For example, various fresh water and salt water media can include those described in Barsanti (2005) Algae: Anatomy, Biochemistry & Biotechnology, CRC Press for media and methods for culturing algae. Algal media recipes can also be found at the websites of various algal culture collections, including, as nonlimiting examples, the UTEX Culture Collection of Algae (www.sbs.utexas.edu/utex/media.aspx); Culture Collection of Algae and Protozoa (www.ccap.ac.uk); and Katedra Botaniky (botany.natur.cuni.cz/algo/caup-media.html).
The culture methods can optionally include inducing expression of one or more genes and/or regulating a metabolic pathway in the microorganism. Inducing expression can include adding a nutrient or compound to the culture, removing one or more components from the culture medium, increasing or decreasing light and/or temperature, and/or other manipulations that promote expression of the gene of interest. Such manipulations can largely depend on the nature of the (heterologous) promoter operably linked to the gene of interest.
In some embodiments of the present invention, the microorganisms having increased lipid productivity can be cultured in a photobioreactor equipped with an artificial light source, and/or having one or more walls that is transparent enough to light, including sunlight, to enable, facilitate, and/or maintain acceptable microorganism growth and proliferation. For production of fatty acid products or triglycerides, photosynthetic microorganisms or host cells can additionally or alternately be cultured in shake flasks, test tubes, vials, microtiter dishes, petri dishes, or the like, or combinations thereof.
Additionally or alternately, mutant or recombinant photosynthetic microorganisms or host cells may be grown in ponds, canals, sea-based growth containers, trenches, raceways, channels, or the like, or combinations thereof. In such systems, the temperature may be unregulated, or various heating or cooling method or devices may be employed As with standard bioreactors, a source of inorganic carbon (such as, but not limited to, CO 2 , bicarbonate, carbonate salts, and the like), including, but not limited to, air, CO 2 -enriched air, flue gas, or the like, or combinations thereof, can be supplied to the culture. When supplying flue gas and/or other sources of inorganic that may contain CO in addition to CO 2 , it may be necessary to pre-treat such sources such that the CO level introduced into the (photo)bioreactor do not constitute a dangerous and/or lethal dose with respect to the growth, proliferation, and/or survival of the microorganisms.
The mutant microorganisms can optionally include one or more non-native genes encoding a polypeptide for the production of a product, such as but not limited to a lipid.
The methods include culturing a mutant microorganism as provided herein, such as a mutant microorganism as provided herein that has increased lipid productivity with respect to a control cell while producing at least 50% of the biomass produced by a control cell under the same culture conditions to produce biomass or lipid. Lipids can be recovered from culture by recovery means known to those of ordinary skill in the art, such as by whole culture extraction, for example, using organic solvents or by first isolating biomass from which lipids are extracted (see, for example, Hussein et al. Appl. Biochem. Biotechnol. 175:3048-3057; Grima et al. (2003) Biotechnol. Advances 20:491-515). In some cases, recovery of fatty acid products can be enhanced by homogenization of the cells (Gunerken et al. (2015) Biotechnol. Advances 33:243-260). For example, lipids such as fatty acids, fatty acid derivatives, and/or triglycerides can be isolated from algae by extraction of the algae with a solvent at elevated temperature and/or pressure, as described in the co-pending, commonly-assigned U.S. patent publication No. US 2013/0225846 entitled “Solvent Extraction of Products from Algae”, filed on Feb. 29, 2012, which is incorporated herein by reference in its entirety.
Biomass can be harvested, for example, by centrifugation or filtering. The biomass may be dried and/or frozen. Further products may be isolated from biomass, such as, for example, various lipids or one or more proteins. Also included in the invention is an algal biomass comprising biomass of lipid regulator mutant, such as any disclosed herein, such as but not limited to a lipid regulator mutant that includes a mutation in a gene encoding a polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, and SEQ ID NO:46.
Alternatively or in addition to any of the forgoing embodiments, the invention provides the following embodiments:
Embodiment 1 is a mutant microorganism that produces at least 25% more lipid and at least 45% more biomass than is produced by a control microorganism cultured under substantially identical conditions under which the control microorganism and the mutant microorganism produce biomass, optionally wherein any one or more of the following are fulfilled:
(a) the control microorganism is a wild type microorganism;
(b) the mutant microorganism produces at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 105%, at least 110%, at least 120%, or at least 150% as much biomass as the control microorganism, which can be assessed as average biomass (e.g., TOC) productivity per day, during a culture period of at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen days, at least fourteen, at least fifteen, at least twenty, at least thirty, or at least sixty days;
(c) the mutant microorganism produces at least 25%, at least 30%, at least 55%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 115%, at least 120%, or at least 150% more lipid, which can be assessed as average lipid (e.g., FAME) productivity per day, than is produced by a control microorganism during a culture period of at least at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen days, at least fourteen, at least fifteen, at least twenty, at least thirty, or at least sixty days;
(d) the culture medium comprises less than about 5 mM, less than about 4 mM, less than about 3 mM, less than 2.5 mM ammonium, less than or equal to about 2 mM ammonium, less than or equal to about 1.5 mM ammonium, less than or equal to about 1 mM ammonium, or less than or equal to about 0.5 mM ammonium;
(e) the culture medium includes nitrate, optionally wherein nitrate is substantially the sole nitrogen source in the culture medium; and/or
(f) the microorganism is a heterokont or alga.
Embodiment 2 is a mutant microorganism according to embodiment 1 in which the mutant has attenuated expression of a gene encoding a polypeptide that:
(a) includes a TAZ zinc finger domain and/or a Bromo domain; and/or
(b) has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, or SEQ ID NO:46.
Embodiment 3 is a mutant microorganism according to embodiment 1 or embodiment 2, wherein the mutant a classically-derived mutant or an engineered mutant, optionally wherein the mutant is an engineered mutant that:
(a) has a disrupted gene encoding a regulator of lipid biosynthesis, wherein the gene is disrupted in a coding region or in a noncoding region;
(b) is deleted in all or a portion of a gene encoding a regulator of lipid biosynthesis;
(c) includes an antisense construct, an RNAi construct, a guide RNA construct, or a ribozyme construct that targets a gene encoding a regulator of lipid biosynthesis;
(d) includes an insertion into a gene encoding a regulator of lipid biosynthesis, optionally wherein the insertion is generated by CRISPR/cas genome editing, further optionally wherein the insertion is in the non-coding portion of the gene, such as, for example, the 5′ UTR, the promoter region, the 3′ UTR, or an intron of the gene; and/or
(e) includes a mutation in a gene encoding a regulator of lipid biosynthesis optionally generated by CRISPR/cas genome editing, optionally wherein the mutation is in the non-coding portion of the gene, further optionally wherein the mutation is in the 5′ UTR, the promoter region, the 3′ UTR, or an intron of the gene.
Embodiment 4 is a mutant microorganism according to any of embodiments 1-3, wherein:
(a) the mutant produces at least 50% more FAME (e.g., average productivity per day) while producing at least 85% or at least 90% of the TOC produced by a control cell, e.g., TOC productivity on a per day basis, when cultured under conditions in which both the control and mutant microorganism produce biomass; and/or
(b) wherein the FAME/TOC ratio of the mutant microorganism is at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% higher than the FAME/TOC of the control microorganism while producing at least 85% or at least 90% of the TOC produced by a control cell (such as a wild type cell) when cultured under conditions in which both the control and mutant microorganism produce biomass; and/or
(c) the FAME/TOC ratio of the mutant microorganism is at least 0.30, at least 0.35 at least 0.40, at least 0.5, or between about 0.3 and about 0.8 when cultured under conditions in which both the control and mutant microorganism produce biomass and/or
(d) wherein the FAME/TOC ratio is maintained between about 0.3 and about 0.7 for a culture period of at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, or at least thirteen days during which the mutant microorganism produces at least 50%, at least 60%, at least 70%, or at least 75%, at least 80% or at least 85% of the biomass produced by a control microorganism cultured under the same conditions in which the control microorganism accumulates biomass.
Embodiment 6 is a mutant microorganism according to any of embodiments 1-3, wherein:
(a) the mutant produces at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% more FAME (e.g., on an average per day basis) while producing at least 90%, at least 95%, at least 100% of the TOC produced (e.g., on an average per day basis) by a control microorganism (such as a wild type cell) when cultured under conditions in which both wild type and mutant microorganism are producing biomass; and/or
(b) the FAME/TOC ratio of the mutant microorganism is at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% greater than the FAME/TOC ratio of a control microorganism when cultured under conditions in which both wild type and mutant microorganism are producing biomass; and/or
(c) the FAME/TOC ratio of the mutant microorganism is at least 0.30, at least 0.35, at least 0.40, at least 0.45, or at least 0.50 and the mutant microorganism produces at least 80%, at least 85%, at least 90%, or at least 95% of the TOC produced by a control microorganism when cultured under conditions in which the wild type accumulates biomass.
Embodiment 7 is a mutant microorganism according to any of embodiments 1-6, wherein:
(a) the culture conditions under which the mutant microorganism produces more lipid is batch, semi-continuous, or continuous culture; and/or
(b) the daily lipid productivity of the mutant is greater than the daily lipid productivity of the control microorganism throughout the culture period, e.g., every day throughout a culture period of at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen days, at least fourteen, at least fifteen, at least twenty, at least thirty, or at least sixty days.
Embodiment 8 is a mutant microorganism according to any of embodiments 1-7 in which the mutant microorganism comprises a mutation in a non-coding region of a gene that reduces expression of the gene, optionally wherein the mutation is an insertion.
Embodiment 9 is a mutant microorganism according to any of embodiments 1-7 in which the mutant microorganism comprises a construct that reduces expression of a gene, wherein the construct encodes an RNAi, a guide RNA, an antisense transcript, or a ribozyme.
Embodiment 10 is a mutant microorganism according to any of embodiments 1-9, wherein the mutant microorganism is a labyrinthulomycte species,
optionally wherein the mutant microorganism is a species belonging to any of the genera Labryinthula, Labryinthuloides, Thraustochytrium, Schizochytrium, Aplanochytrium, Aurantiochytrium, Oblongichytrium, Japonochytrium, Diplophrys , or Ulkenia ; or wherein the mutant microorganism is an algal species,
optionally a species belonging to any of the genera Achnanthes, Amphiprora, Amphora, Ankistrodesmus, Asteromonas, Boekelovia, Bolidomonas, Borodinella, Botrydium, Botryococcus, Bracteococcus, Chaetoceros, Carteria, Chlamydomonas, Chlorococcum, Chlorogonium, Chlorella, Chroomonas, Chrysosphaera, Cricosphaera, Crypthecodinium, Cryptomonas, Cyclotella, Desmodesmus, Dunaliella, Elipsoidon, Emiliania, Eremosphaera, Ernodesmius, Euglena, Eustigmatos, Franceia, Fragilaria, Fragilaropsis, Gloeothamnion, Haematococcus, Hantzschia, Heterosigma, Hymenomonas, Isochrysis, Lepocinclis, Micractinium, Monodus, Monoraphidium, Nannochloris, Nannochloropsis, Navicula, Neochloris, Nephrochloris, Nephroselmis, Nitzschia, Ochromonas, Oedogonium, Oocystis, Ostreococcus, Parachlorella, Parietochloris, Pascheria, Pavlova, Pelagomonas, Phæodactylum, Phagus, Picochlorum, Platymonas, Pleurochrysis, Pleurococcus, Prototheca, Pseudochlorella, Pseudoneochloris, Pseudostaurastrum, Pyramimonas, Pyrobotrys, Scenedesmus, Schizochlamydella, Skeletonema, Spyrogyra, Stichococcus, Tetrachlorella, Tetraselmis, Thalassiosira, Tribonema, Vaucheria, Viridiella, Vischeria , and Volvox.
Embodiment 11 is biomass comprising any of the mutant microorganisms of any of embodiments 1-10.
Embodiment 12 is a nucleic acid molecule comprising a sequence encoding a polypeptide having at least 60%, at least 65%, at least 70%, or at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, or SEQ ID NO:46, wherein any one or more of the following are satisfied:
(a) the polypeptide includes an amino acid sequence encoding a TAZ Zinc Finger domain, optionally wherein the TAZ Zinc Finger domain has at least 60%, at least 65%, at least 70%, or at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to SEQ ID NO:9;
(b) the polypeptide includes an amino acid sequence encoding a Bromo domain, optionally wherein the Bromo domain has at least 60%, at least 65%, at least 70%, or at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to SEQ ID NO:10;
(c) the nucleic acid molecule in various examples comprises a cDNA that lacks one or more introns present in the naturally-occurring gene or is a gene construct that includes one or more introns not present in the naturally-occurring gene;
(d) the nucleic acid molecule in various examples can have a sequence that is not 100% identical to a naturally-occurring gene;
(e) the nucleic acid molecule has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, or a portion of any thereof;
(f) the nucleic acid molecule comprises a heterologous promoter operably linked to the sequence; and/or
(g) the nucleic acid molecule comprises a vector.
Embodiment 13 is a nucleic acid molecule construct for attenuating expression of a gene encoding a polypeptide according to having at least 60%, at least 65%, at least 70%, or at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, or SEQ ID NO:46, wherein the nucleic acid molecule construct comprises:
a sequence encoding a guide RNA of a CRISPR system, an RNAi construct, an antisense construct, a ribozyme construct, or a construct for homologous recombination;

further optionally wherein the nucleic acid molecule construct comprises one or more nucleotide sequences having homology to a naturally-occurring TAZ Zinc Finger domain-encoding gene as disclosed herein and/or sequences adjacent thereto in the native genome from which the gene is derived.

Embodiment 14 is method of engineering a cell for increased lipid production comprising attenuating expression of a gene encoding a polypeptide having at least 60%, at least 65%, at least 70%, or at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, or SEQ ID NO:46, optionally a gene having a coding sequence with at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to any of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, or SEQ ID NO:45, into a microorganism to produce a mutant microorganism having higher lipid productivity than the progenitor microorganism, optionally wherein attenuating expression of the gene comprises introducing a nucleic acid molecule according to embodiment 13 into the microorganism.
Embodiment 15 is method for producing lipid comprising culturing a mutant according to any of embodiments 1-10 to produce lipid, optionally wherein any one or more of the following are satisfied:
(a) the culture medium includes nitrate;
(b) the culture medium includes less than 5 mM, less than 4 mM, less than 3 mM, less than 2.5 mM ammonium, less than or equal to about 2 mM ammonium, less than or equal to about 1.5 mM ammonium, less than or equal to about 1 mM ammonium, or less than or equal to about 0.5 mM ammonium;
(c) the culture is a batch, semi-continuous, or continuous culture;
(d) the culture period is at least 5, 7, 8, 9, 10, 11, 12, 13 day, at least 15, 20, 30, 40, 50, or 60 days;
(e) the mutant is an algal mutant and the culture is photoautotrophic;
(f) the mutant produces at least 25% more lipid, preferably FAME lipid, and at least 45% of the biomass of a control microorganism during the culture period;
(g) the mutant produces more lipid, preferably FAME lipid, on each day of the culture period; and/or
(h) the mutant accumulates biomass on each day of the culture period.
Embodiment 16 is method for producing lipid comprising culturing a microorganism under conditions in which the FAME/TOC ratio is maintained at between about 0.3 and about 0.8 throughout the culture period, optionally wherein any one or more of the following are satisfied:
(a) the culture medium includes nitrate;

(b) the culture medium includes less than 5 mM, less than 4 mM, less than 3 mM, less than 2.5 mM ammonium, less than 2 mM ammonium, less than or equal to about 1.5 mM ammonium, less than or equal to about 1 mM ammonium, or less than or equal to about 0.5 mM ammonium;

(c) the culture is a batch, semi-continuous, or continuous culture;

(d) the culture period is at least 5, 7, 8, 9, 10, 11, 12, 13 day, at least 15, 20, 30, 40, 50, or 60 days;

(e) the microorganism is an algal microorganism and the culture is photoautotrophic;

(f) the microorganism accumulates biomass on each day of the culture period; and/or

(g) the microorganism is a mutant microorganism according to any of embodiments 1-10.

EXAMPLES
Media Used in Examples
PM066 medium (Example 1) includes 10 mM nitrate as the sole nitrogen source. PM066 medium included 10 mM nitrate (NO 3 ) and 0.417 mM phosphate (PO 4 ) along with trace metals and vitamins in Instant Ocean salts. PM066 media was made by adding 5.71 ml of a 1.75 M NaNO 3 stock solution (148.7 g/L), and 5.41 ml of a 77 mM K 2 HPO 4 .3H 2 O stock solution (17.57 g/L) to 981 mls of Instant Ocean salts solution (35 g/L) along with 4 ml of Chelated Metals Stock Solution and ml of 4 ml Vitamin Stock Solution. Chelated Metals Stock Solution was prepared by adding to 400 mls of water 2.18 g Na 2 EDTA.2H 2 O; 1.575 g FeCl3.6H 2 O; 500 μl of 39.2 mM stock solution (0.98 g/100 ml) CuSO 4 .5H 2 O; 500 μl of 77.5 mM stock solution (2.23 g/100 ml) ZnSO 4 .7H 2 O; 500 μl of 42.0 mM stock solution (1.00 g/100 ml) CoCl 2 .6H2O; 500 μl of 910.0 mM stock solution (18.0/100 ml) MnCl2.4H2O; 500 μl of 26.0 mM stock solution (0.63 g/100 ml) Na 2 MoO 4 .2H 2 O; bringing up to 500 ml final volume, and filter sterilizing. Vitamin Stock Solution was prepared by adding to 400 mls of water 0.05 g Thiamine HCl; 500 μl of 0.37 mM stock solution (0.05 g/100 ml) of cyanocobalamin; and 2.5 ml of 0.41 mM stock solution (0.01 g/100 ml) of biotin, bringing up to a final volume of 500 mls, and filter sterilizing.
PM067 medium included no nitrogen source (no nitrate or ammonium), and 0.417 mM phosphate (PO 4 ) along with trace metals and vitamins in Instant Ocean salts. PM067 media was made by adding 5.41 ml of a 77 mM K 2 HPO 4 .3H 2 O stock solution (17.57 g/L) to 987 mls of Instant Ocean salts solution (35 g/L) along with 4 ml of Chelated Metals Stock Solution and ml of 4 ml Vitamin Stock Solution. Chelated Metals Stock Solution was prepared by adding to 400 mls of water 2.18 g Na 2 EDTA.2H 2 O; 1.575 g FeCl3.6H 2 O; 500 μl of 39.2 mM stock solution (0.98 g/100 ml) CuSO 4 .5H 2 O; 500 μl of 77.5 mM stock solution (2.23 g/100 ml) ZnSO 4 .7H 2 O; 500 μl of 42.0 mM stock solution (1.00 g/100 ml) CoCl 2 .6H2O; 500 μl of 910.0 mM stock solution (18.0/100 ml) MnCl 2 .4H 2 O; 500 μl of 26.0 mM stock solution (0.63 g/100 ml) Na 2 MoO 4 .2H 2 O; bringing up to 500 ml final volume, and filter sterilizing. Vitamin Stock Solution was prepared by adding to 400 mls of water 0.05 g Thiamine HCl; 500 μl of 0.37 mM stock solution (0.05 g/100 ml) of cyanocobalamin; and 2.5 ml of 0.41 mM stock solution (0.01 g/100 ml) of biotin, bringing up to a final volume of 500 mls, and filter sterilizing.
PM074 is a nitrogen replete (“nitrate-only”) medium that is 10×F/2 made by adding 1.3 ml PROLINE® F/2 Algae Feed Part A (Aquatic Eco-Systems) and 1.3 ml PROLINE® F/2 Algae Feed Part B (Aquatic Eco-Systems) to a final volume of 1 liter of a solution of Instant Ocean salts (35 g/L) (Aquatic Eco Systems, Apopka, Fla.). Proline A and Proline B together include 8.8 mM NaNO 3 , 0.361 mM NaH 2 PO 4 .H 2 O, 10×F/2 Trace metals, and 10×F/2 Vitamins (Guillard (1975) Culture of phytoplankton for feeding marine invertebrates. in “Culture of Marine Invertebrate Animals.” (eds: Smith W. L. and Chanley M. H.) Plenum Press, New York, USA. pp 26-60).
PM123 medium is PM074 medium supplemented with additional Proline B so that the concentration of nitrate was increased from approximately 8.8 mM to approximately 15 mM. This is also a “nitrate only” medium.
PM124 medium is PM074 supplemented with 5 mM Ammonium and 10 mM HEPES pH 8.0. It is made by adding 10 mls of 1 M HEPES pH 8 and 5 mls of NH 4 Cl to the PM074 recipe (final volume of 1 L). Additional media with controlled ammonium levels was made by adjusting the ammonium concentration of PM074 and adding additional Hepes buffer.
PM066, PM074, PM123, and PM124 media are nitrogen replete and nutrient replete with respect to wild type Nannochloropsis.
Example 1. Identification of a Polypeptide Downregulated During Nitrogen Starvation
To identify genes that influence lipid biosynthesis, a comparative transcriptomics experiment was performed in which the RNA transcript levels of genes of Nannochloropsis gaditana cells under nitrogen starvation, under which Nannochloropsis induces storage lipid biosynthesis, were compared with the levels of RNA transcripts of the same strain of Nannochloropsis gaditana grown under identical conditions except that the amount of nitrogen in the growth medium was not limiting.
Wild type N. gaditana (WT-3730) cells were grown in nutrient replete medium under a 16 hour light (120 μE)/8 hour dark cycle to light limitation and at the beginning of the photoperiod were spun down and resuspended in either nitrogen replete medium PM074 or culture medium lacking a nitrogen source (“nitrogen deplete” medium PM067). RNA was isolated from each sample three hours after resuspension in nitrogen replete or nitrogen depete medium. RNA was isolated by spinning down 10 mLs of each algal cell culture (4000×g for 5 minutes) and decanting the supernatant. The pellets were resuspended in 1.8 mL Buffer A (5 mL TLE Grinding Buffer, 5 mL phenol, 1 mL 1-bromo-3-chloropropane and 20 mercaptoethanol, where TLE Grinding Buffer includes 9 mL of 1M Tris pH 8, 5 mL of 10% SDS, 0.6 mL of 7.5 M LiCl, and 0.45 M EDTA in a final volume of 50 mL) and transferred to 2 mL microcentrifuge tubes containing approximately 0.5 mL of 200 μm zirconium beads. The tubes were vortexed vigorously for 5 min at 4° C. and then centrifuged for 2 min at 11.8×g. The aqueous layers were then removed and pipetted into new 2 mL tubes, to which 1 mL 25:24:1 phenol extraction buffer (25 mL phenol pH 8 or 5.1; 24 mL 1-bromo-3-chloropropane, and 1 mL isoamyl alcohol) was added. The tubes were shaken vigorously and centrifuged for 2 min at 11.8×g. After centrifugation, the aqueous layer was removed and pipetted into new 2 mL centrifuge tubes, to which 1 ml 1-bromo-3-chloropropane was added. The tubes were shaken and again centrifuged for 2 min at 11.8×g. The aqueous layer was removed to a new tube and 0.356 volumes of 7.5 M LiCl were added. The tubes were inverted 10-12 times and stored at −20° C. overnight. The next day, samples were allowed to come to room temperature without mixing and were centrifuged at 16,000×g for 30 minutes. The supernatants were removed and the pellets were washed with 1 mL of ice cold 80% ethanol. The tubes were centrifuged for 30 min at 16,000×g and allowed to air dry after the supernatants had been removed. Finally, the RNA pellets were resuspended in 50 μl ultrapure water. The RNA quality was assessed by on-chip gel electrophoresis using an Agilent 2100 Bioanalyzer and RNA6000 LabChip according to manufacturer instructions.
Next-generation sequencing libraries were prepared from the isolated RNA utilizing the TruSeq Stranded mRNA Sample Prep Kit (Illumina, San Diego, Calif.) following manufacturer instructions. The TruSeq libraries were sequenced using sequencing-by-synthesis (Illumina MiSeq) to generate 100 bp paired-end reads using the mRNA-Seq procedure (described in Mortazavi et al. (2008) Nature Methods 5:621-628). Mappable reads were aligned to the N. gaditana reference genome sequence using TopHat (tophat.cbcb.umd.edu/). Expression levels were computed for every annotated gene using the Cuffdiff component of the Cufflinks software (cufflinks.cbcb.umd.edu). Differential expression analysis was performed using the R package edgeR (McCarthy et al. (2012) Nucl. Acids Res. 40:doi:10/1093/nar/gks042)). Expression levels in units of fragments per kilobase per million (FPKM) were reported for every gene in each sample using standard parameters. FPKM is a measure of relative transcriptional levels that normalizes for differences in transcript length.
From this analysis a list was compiled of differentially expressed genes under nitrogen-replete and nitrogen-deplete conditions. The list of differentially expressed genes was compared with a bioinformatically curated list of putative Nannochloropsis transcription factors previously generated in-house by mining the Nannochloropsis genome for proteins containing DNA binding domains and other conserved pfam domains typical of characterized transcription factors using the Plant Transcription Factor Database as a reference (Perez-Rodriguez et al. (2010) Nucl. Acids Res. 38:D822-D827; Jin et al. (2013) Nucl. Acids Res. 42: D1182-D1187).
As disclosed in commonly-owned, copending U.S. patent application Ser. No. 15/210,845, the ZnCys-2845 gene was identified as a transcription factor gene differentially regulated at the outset of nitrogen deprivation ( FIG. 1A ). A second gene that was not identified as a transcription factor was also identified as differentially expressed between the N-replete and N-deplete samples. This gene (cDNA sequence provided as SEQ ID NO:1) encoding a polypeptide (SEQ ID NO:2) encoded a polypeptide having domains found in transcriptional co-regulators. The polypeptide was observed to have a TAZ Zinc Finger domain (Pfam PF02135), a protein-protein interaction domain found in transcriptional regulators (see, for example, Guzman et al. (2004) J. Biol. Chem. 279: 3042-3049) and a Bromo domain (pfam PF00439) (SEQ ID NO:10), a domain known to bind acetylated histones, and was named “Bromo-1091”. A diagram of the polypeptide of SEQ ID NO:2 is provided in FIG. 1B .
Example 2. Bioinformatic Analysis of the Bromo-1091 Protein: Domains and Orthologs
In addition to the transcript encoding SEQ ID NO:2, three additional transcripts were identified by RNA Seq and 3′ RACE (rapid amplification of cDNA ends; Frohman, et al. (1988). Proc. of the Natl Acad Sci. USA 85: 8998-9002) that originate from the Bromo-1091 gene and extensively overlap with SEQ ID NO:1. RACE was performed using the MARATHON® cDNA amplification kit (Clontech, Mountain/View, Calif.) to obtain cDNAs from RNA isolated using the NUCLEO TRAP® mRNA mini RNA isolation kit (Clontech). The nucleotide sequences of these transcripts are provided as SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7, encoding the polypeptides of SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8, respectively. An alignment of the sequences of the polypeptide variants encoded by the four cDNAs is provided as FIG. 2A-C . Each of the polypeptides encoded by transcripts of the Bromo-1091 gene includes the same “TAZ” zinc finger domain (PF002135), amino acids 827-892 of SEQ ID NO:2 and amino acids 769-834 of SEQ ID NO:4 (SEQ ID NO:9). SEQ ID NO:2 and SEQ ID NO:4 also include the same “Bromo” domain (pfam PF00439), amino acids 1223 to 1264 of SEQ ID NO:2 and 1165-1206 of SEQ ID NO:4 (SEQ ID NO:10), that were identified as conserved domains in the polypeptide encoded by the Bromo-1091 cDNA SEQ ID NO:2. For simplicity, we refer herein to all four polypeptides as the “Bromo-1091 protein” or “Bromo-1091 polypeptide”, where the different isoforms (isoforms a, b, c, and d), which appear to result from alternative splicing (referred to as Bromo-1091 transcripts a, b, c, and d), differ primarily in the carboxy terminus (e.g., the terminal 25-35 amino acids of the polypeptides of SEQ ID NOs:2, 4, 6, and 8). SEQ ID NO:4 also lacks a stretch of amino acids present in the other isoforms extending from amino acid 226 to amino acid 289 of SEQ ID NO:2.
Co-pending and commonly-owned U.S. patent application Ser. No. 15/210,845, filed Jul. 14, 2016, discloses a regulator gene “ZnCys-2845” whose mutation results in the ability of the mutant strain to induce lipid synthesis in the presence of a nitrogen source such as nitrate even while continuing to propagate and accumulate biomass. The “Bromo-1091” gene newly disclosed herein that includes a TAZ Zinc Finger domain and a bromo domain is the second gene discovered whose mutation results in the ability of the mutant strain to induce lipid synthesis while continuing to propagate and accumulate biomass in the presence of a nitrogen source such as nitrate. The ZnCys-2845 gene and orthologs thereof in other species may be referred to herein as LION1 (“Lipid Induced On Nitrate 1”) genes and the Bromo-1091 gene and orthologs thereof are referred to herein as “LION2” (“Lipid Induced On Nitrate 2”) genes.
Several potential orthologs of Bromo-1091 were identified by bioinformatics. In particular, several genes were identified in stramenopiles (heterokonts) that had both a TAZ zinc finger domain and a bromo domain, including a partial sequence of an ortholog in Nannochloropsis oceanica (SEQ ID NO:11, encoding amino acid sequence SEQ ID NO:12), in which the amino acid sequence (SEQ ID NO:12), which does not appear to be the complete polypeptide, demonstrates 84% identity to a portion of SEQ ID NO:2, and includes a TAZ Zinc Finger domain that has 95% identity to the TAZ Zinc Finger domain of the Nannochloropsis gaditana Bromo-1091 (“LION2”) polypeptide (SEQ ID NO:9). Other putative orthologs that include both a bromo domain and a TAZ zinc finger domain include polypeptides from diatoms, e.g., Phaeodactylum, Thalassiosira, Navicula, Fragilariopsis , and Cyclotella , as well as Aureococcus, Ectocarpus , and the labyrinthulomycete Schizochytrium (see Table 1).





TABLE 1



Putative Bromo-1091 Orthologs of N. gaditana Bromo-1091 in Heterokonts








Nucleotide
Amino acid
TAZ
Bromo


sequence
sequence
domain(s)
domain

Species
(cDNA)
(polypeptide)
amino acids
amino acids




Nannochloropsis

SEQ ID NO: 11
SEQ ID NO: 12
65-127



oceanica


Cyclotella sp.
SEQ ID NO: 13
SEQ ID NO: 14
221-284;
631-715




1338-1399

Cyclotella sp.
SEQ ID NO: 15
SEQ ID NO: 16
1438-1515
470-564

Cyclotella sp.
SEQ ID NO: 17
SEQ ID NO: 18
704-765;
 911-1007




476-357


Fragilariopsis

SEQ ID NO: 19
SEQ ID NO: 20
350-411;
549-644


cylindrus



1328-1387


Fragilariopsis

SEQ ID NO: 21
SEQ ID NO: 22
1-42
183-273


cylindrus



Thalassiosira

SEQ ID NO: 23
SEQ ID NO: 24
761-822
 51-146


pseudonana



Thalassiosira

SEQ ID NO: 25
SEQ ID NO: 26
648-709;
833-929


pseudonana



441-502


Phaeodactylum

SEQ ID NO: 27
SEQ ID NO: 28
862-925;
1251-1341


tricornutum



2323-2384


Phaeodactylum

SEQ ID NO: 29
SEQ ID NO: 30
339-400;
582-670


tricornutum



1494-1571


Phaeodactylum

SEQ ID NO: 31
SEQ ID NO: 32
1-42;
179-271


tricornutum



957-1016

Navicula sp.
SEQ ID NO: 33
SEQ ID NO: 34
242-302;
671-762




460-521;




1438-1497

Navicula sp.
SEQ ID NO: 35
SEQ ID NO: 36
357-420;
764-860




2136-2197

Navicula sp.
SEQ ID NO: 37
SEQ ID NO: 38
277-338;
530-520




1444-1521


Ectocarpus

SEQ ID NO: 39
SEQ ID NO: 40
1244-1322
420-516


silicosus



Aureococcus

SEQ ID NO: 41
SEQ ID NO: 42
758-836
 2-78


anophagefferens



Schizochytrium

SEQ ID NO: 43
SEQ ID NO: 44
1-42;
244-330


limacinum



1533-1600


Schizochytrium

SEQ ID NO: 45
SEQ ID NO: 46
1-42;
171-261


limacinum



936-998


























































Example 3. Knockout of the Bromo-1091 Locus in Nannochloropsis
In order to determine whether there was any relationship between the Bromo-1091 polypeptide and lipid induction, the Bromo-1091 gene was knocked out using CRISPR technology as described in commonly owned co-pending U.S. patent application Ser. No. 14/986,492 and corresponding PCT application publication no. WO2016/109840, filed Dec. 31, 2015. As described in U.S. Ser. No. 14/986,492, a highly efficient Nannochloropsis Cas9 Editor line, N. gaditana strain pSGIE-6791, expressing a gene encoding the Streptococcus pyogenes Cas9 nuclease was used as a host for transformation with a chimeric guide RNA and donor DNA for insertional knockout.
To produce the high efficiency Nannochloropsis Cas9 Editor line, a Nannochloropsis strain was engineered and isolated that exhibited expression of the introduced Cas9 genes in close to 100% of the cell population of a growing culture. The vector pSGE-6206 (SEQ ID NO:47), used to transform wild type N. gaditana strain pSGIE-6791, included the following three elements: 1) a Cas9 expression cassette which contained a Cas9 gene from Streptococcus pyogenes codon optimized for Nannochloropsis gaditana (SEQ ID NO:48) that included sequences encoding an N-terminal FLAG tag (SEQ ID NO:49), nuclear localization signal (SEQ ID NO:50), and peptide linker (SEQ ID NO:51), driven by the N. gaditana RPL24 promoter (SEQ ID NO:52) and terminated by N. gaditana bidirectional terminator 2 (SEQ ID NO:53); 2) a selectable marker expression cassette, which contained the blast gene from Aspergillus terreus codon optimized for N. gaditana (SEQ ID NO:54), driven by the N. gaditana TCTP promoter (SEQ ID NO:55) and followed by the EIF3 terminator (SEQ ID NO:56); and 3) a GFP reporter expression cassette, which contained the TurboGFP gene (Evrogen, Moscow, Russia) codon optimized for Nannochloropsis gaditana (SEQ ID NO:57), driven by the N. gaditana 4A-III promoter (SEQ ID NO:58) and followed by the N. gaditana bidirectional terminator 5 (SEQ ID NO:59).
The transformation mixture was plated onto PM074 agar medium containing 100 mg/L of blasticidin. Resulting colonies were patched onto selection media for analysis and archiving. A small bit of biomass was taken from the patches and completely resuspended in 300 μl of 1× Instant Ocean solution. Care was taken to not add too much biomass so that a light green resuspension was obtained. This liquid was directly analyzed by flow cytometry using a BD Accuri C6 flow cytometer, using a 488 nm laser and 530/10 nm filter to measure GFP fluorescence per cell. 10,000-30,000 events were recorded for each sample using the slow fluidics setting. The resulting histograms were overlaid with histograms of wild type cells (i.e., cells not expressing a fluorescent protein) run separately. Only strains with full penetrance expression in culture were carried forward; this meant that the flow cytometry histogram showed a single peak or bell-shaped curve in which the peak was fully shifted higher than the wild type auto fluorescence peak when plotted on a log scale. These strains were designated as “fully penetrant” Cas9 expressing strains, in that the expression of the GFP gene was found throughout the cells of a culture of the strain. That is, while at any given point in time the amount (and therefore fluorescence) of GFP might vary somewhat cell-to-cell, resulting in a bell-shaped curve, there was no subpopulation of cells exhibiting a distinct distribution of GFP expression. Thus, a fully penetrant strain was one in which there was a single peak (or bell-shaped curve having a peak) where the peak was separate from and at a higher fluorescence value than the background peak of non-expressing cells (e.g., cells not transformed with a GFP expression construct). Because the GFP gene was physically associated with the Cas9 gene, it was postulated that the Cas9 gene was also likely expressed throughout the cells of a culture of the strain in fully penetrant GFP strains.
Fully penetrant Cas9 strains demonstrating a single clearly shifted fluorescence peak with respect to nontransformed cells were subsequently tested by western blotting with an anti-FLAG antibody for evidence of Cas9 expression. A strain having a single fluorescence peak that was shifted to a fluorescence level higher than that demonstrated by wild-type cells and also demonstrating Cas9 protein expression by Western, designated strain GE-6791, was selected as a fully penetrant Cas9 strain resulting from transformation with pSGE-6202 for use in mutant generation by genome editing as described herein.
For targeting of the Bromo-1091 gene for disruption, a DNA molecule that included the sequence of a chimeric guide (SEQ ID NO:60) that included an 18 bp sequence (SEQ ID NO:61) corresponding to a sequence within the Bromo-1091 gene was synthesized (SGI-DNA, La Jolla, Calif.) downstream of a T7 promoter sequence. The construct was used in in vitro transcription reactions using the MEGAshortscript™ T7 Kit (Life Technologies # AM1354M) according to the manufacturer's instructions. The resulting RNA was purified using Zymo-Spin™ V-E columns (Zymo Research #C1024-25) according to manufacturer's protocol.
The donor fragment for insertion into the targeted Bromo-1091 locus included a selectable marker cassette that included the hygromycin resistance gene (HygR, SEQ ID NO:62) downstream of the N. gaditana EIF3 promoter (SEQ ID NO:63) and followed by N. gaditana bidirectional terminator 2 (SEQ ID NO:53), with the entire promoter-Hygromycin resistance gene terminator sequence flanked by 27 base pair identification sequences on the 5′ (SEQ ID NO:64) and 3′ (SEQ ID NO:65) ends to yield the DNA fragment referred to as the “Hyg Resistance Cassette” (SEQ ID NO:66).
For targeted knockout of the Bromo-1091 locus, SGIE-6791 was transformed by electroporation using 5 μg of purified chimeric guide RNA targeting the Bromo-1091 gene (SEQ ID NO:60) and 1 μg of the selectable donor DNA (Hyg Resistance Cassette; SEQ ID NO:66). Following electroporation, cells were plated on agar media containing hygromycin to select for transformants that incorporated the hygromycin resistance cassette. Transformants were screened by colony PCR for insertion of the donor fragment into the Bromo-1091 gene.
For colony PCR screening, a small amount of cells from a colony to be screened was suspended into 100 μl of 5% Chelex 100 Resin (BioRad)/TE solution and the suspension was boiled for boiled 10 minutes at 99° C., after which the tubes were briefly spun. One microliter of the lysate supernatant was added to a PCR reaction mix, in which the PCR mixture and reactions were set up and performed according to the QIAGEN Fast Cycling PCR Master Mix Protocol from the manufacturer (Handbook available at qiagen.com). Based on the PCR-based colony screening, one knockout strain, GE-8563, was selected for further analysis.
Example 4. Bromo-1091 Knockout Mutant in Batch Productivity Assay
To determine the effect of knocking out the Bromo-1091 gene on growth and lipid production, Bromo-1091 knockout strain GE-8563 and the wild type N. gaditana progenitor strain WT-3730 were grown in a batch productivity assay, in which cells were cultured for one week in 75 cm 2 rectangular tissue culture flasks containing 175 ml of culture medium PM123 that included 15 mM nitrate as the sole nitrogen source, i.e., the culture medium the starter culture was diluted into for productivity assays had no source of reduced nitrogen. Three flasks each of WT-3730 and GE-8563 were inoculated to an initial OD730 of 0.5 from starter cultures. Starter cultures of Bromo knockout strain GE-8563 used PM124 medium, which included 5 mM ammonium in addition to approximately 8.8 mM nitrate. Wild type WT-3730 starter cultures used PM074 nitrate-only medium. Typical dilutions of starter culture into the assay culture medium were from 15-35 mls of starter culture brought up to 175 mls total using PM123 (nitrate only) medium. Thus the initial concentration of ammonium in the batch assays ranged from approximately 0.4 mM to approximately 1 mM. Air that included 1% CO 2 was bubbled into the cultures by tubing connected to the cap of each flask and the lights, which were directed at the flasks from one narrow side (i.e., the light was introduced from the side along the narrowest width dimension of the culture flask), were programmed to simulate the intensities experienced on a spring day in Southern California, peaking at an intensity of approximately 1200 μE at “solar noon”. The diel cycle was 16 h light/8 h dark. The temperature was held at approximately 25° C. by positioning the culture flasks in a water bath. Cultures were inoculated on day 0 and samples (5 mls) were removed on days 3, 5, and 7 for assessing cell density, fatty acid methyl esters (FAME), and total organic carbon (TOC).
FAME analysis was performed on 2 mL samples that were dried using a GeneVac HT-4X. To the dried pellets the following was added: 500 μL of 500 mM KOH in methanol, 200 μL of tetrahydrofuran containing 0.05% butylated hydroxyl toluene, 40 μL of a 2 mg/ml C11:0 free fatty acid/C13:0 triglyceride/C23:0 fatty acid methyl ester internal standard mix and 500 μL of glass beads (425-600 μm diameter). The vials were capped with open top PTFE septa-lined caps and placed in an SPEX GenoGrinder at 1.65 krpm for 7.5 minutes. The samples were then heated at 80° C. for five minutes and allowed to cool. For derivatization, 500 μL of 10% boron trifluoride in methanol was added to the samples prior to heating at 80° C. for 30 minutes. The tubes were allowed to cool prior to adding 2 mL of heptane and 500 μL of 5 M NaCl. The samples were then vortexed for five minutes at 2 krpm and finally centrifuged for three minutes at 1 krpm. The heptane layer was sampled using a Gerstel MPS Autosampler. Quantitation used the 80 μg of C23:0 FAME internal standard.
Total organic carbon (TOC) was determined by diluting 2 mL of cell culture to a total volume of 20 mL with DI water. Three injections per measurement were injected into a Shimadzu TOC-Vcsj Analyzer for determination of Total Carbon (TC) and Total Inorganic Carbon (TIC). The combustion furnace was set to 720° C., and TOC was determined by subtracting TIC from TC. The 4 point calibration range was from 2 ppm to 200 ppm corresponding to 20-2000 ppm for non-diluted cultures with a correlation coefficient of r 2 >0.999.
The results of the FAME and TOC analyses of the strains cultured in batch mode in medium that included nitrate as the sole nitrogen source are provided in Tables 2-4.





TABLE 2



FAME (μg/ml) produced by wild

type and Bromo-1091 knockout cells








WT
Bromo-KO

% Increase









DAY

s.d.

s.d.
Difference
(Bromo v. wt)











3
105.03
9.71
139.66
5.56
34.63
32.97

5
140.01
13.48
167.71
4.58
27.70
19.78

7
198.49
2.035
199.08
20.60
0.58
0.29




































TABLE 3



Biomass (TOC) (μg/ml) produced by

wild type and Bromo-1091 knockout cells.








WT


% Difference








DAY

s.d.
Bromo -KO
Difference
(Bromo v. wt)











3
375.6
10.18
307.4
21.64
−68.2
−18.16

4
474.6
8.34
372.45
1.06
−102.15
−21.52

5
534.45
43.20
410.4
9.62
−124.05
−23.21

6
644.8
48.65
487.45
5.73
−157.35
−24.4

7
804.35
36.13
582.7
14.71
−221.65
−27.56






































TABLE 4



FAME/TOC ratios of Bromo-1091 knockout

mutant and wild type stains.








WT
Bromo -KO
Difference
% Increase









DAY

s.d.

s.d.
(Bromo v. wt)
(Bromo v. wt)











3
0.28
0.018
0.45
0.014
0.17
60.71

5
0.26
0.004
0.41
0.002
0.15
57.69

7
0.25
0.009
0.34
0.027
0.09
36.0































Table 2 shows that by Day 3 of the assay, the Bromo-1091 knockout mutant had produced approximately 33% more FAME lipids than wild type cells. The increase in lipid production with respect to wild type cells declined over the course of the assay, however, such that by day 5 of the assay, the mutant strain had produced just 20% more FAME/ml than was produced by wild type. By day 7 the Bromo-1091 knockout cultures had essentially the same amount of FAME/ml as the wild type cultures.
Table 3 shows that at every stage of the batch assay, the Bromo-1091 knockout mutant cultures had less biomass than the wild type cultures, and the Bromo-1091 knockout mutant cultures were falling further behind the wild type cultures in biomass accumulation as the assay progressed.
Table 4 shows that the wild type had typical non-induced FAME/TOC ratios of approximately 0.25 during the assay (the slightly higher value of 0.28 on day 3 may have been due to a stress response resulting from dilution into the assay flask, which results in a period of light stress the cells may have still been recovering from by day 3). The Brom-1091 knockout mutant however was induced for lipid on day 3 as evidenced by an elevated FAME/TOC ratio of 0.45, even though the Bromo-1091 knockout mutant continued to accumulate biomass throughout the assay, for example, the Bromo-1091 knockout mutant accumulated biomass to a level that throughout the assay was only about 20-25% less, e.g., 18-27.5% less, than the amount of biomass accumulated by wild type cells.
The Bromo-1091 mutant and wild type strain WT-3730 were cultured each in the same batch assay, except that the culture medium for the batch assay was PM124, which included both nitrate (8.8 mM) and ammonium (5 mM) as nitrogen sources. Samples were removed as described and analyzed for FAME and TOC as provided above. The results of the FAME and TOC analyses are provided in Tables 5-7.





TABLE 5



FAME (μg/ml) produced by wild type and Bromo-

1091 knockout cells pre-cultured in and batch

cultured in nitrate plus ammonium medium.








WT
Bromo-KO
Difference
% Difference









DAY

s.d.

s.d.
(Bromo v. wt)
(Bromo v. wt)











3
93.03
6.94
69.74
3.65
−23.29
−25.03

4
120.14
8.43
101.93
1.44
−18.21
−15.16

5
121.31
0.79
117.93
3.37
−3.37
−2.8

6
169.70
6.07
165.57
4.24
−4.13
−2.43

7
198.11
7.95
210.79
4.74
12.68
+6.4







































TABLE 6



Biomass (TOC) (μg/ml) produced by wild type and Bromo-1091

knockout cells batch cultured in nitrate plus ammonium medium.








WT
Bromo -KO

% Difference









DAY

s.d.

s.d.
Difference
(Bromo v. wt)











3
321.5
35.07
186.1
13.86
135.4
−42.12

4
392.3
16.69
298.3
10.47
94
−23.96

5
464
4.38
399.75
12.80
64.25
−13.85

6
556.45
20.15
504.1
7.50
52.35
−9.41

7
679.95
6.01
635.35
38.82
44.6
−6.56






































TABLE 7



FAME/TOC ratios of Bromo-1091 knockout mutant and wild type

stains in Batch Assay with nitrate plus ammonium medium.








WT
Bromo -KO
Difference
% Difference









DAY

s.d.

s.d.
(Bromo v. wt)
(Bromo v. wt)











3
0.29
0.0100
0.38
0.0476
0.11
37.93

4
0.31
0.0085
0.34
0.0072
0.03
9.68

5
0.26
0.0008
0.30
0.0010
0.04
15.38

6
0.31
0.02196
0.33
0.0035
0.02
6.45

7
0.29
0.0091
0.33
0.0128
0.04
13.79

































The results of FAME analysis provided in Table 2 and Table 5 are depicted in FIG. 4A , in which it can be seen that the Bromo-1091 knockout cultures, when grown on nitrate as the sole nitrogen source (but not when the culture medium included ammonium), had increased FAME with respect to wild type at the outset of the culture (on day 3) but actually produced less FAME per day with respect to wild type on days 3-7, such that the amount of FAME in the cultures was highly similar by day 7. The lower FAME productivity could be attributed to a slower overall growth rate, as evidenced in FIG. 4B , which provides a graph of biomass accumulation during the assay. Table 6 shows that biomass accumulation by the Bromo-1091 knockout mutant grown in the presence of ammonium and nitrate tracked FAME accumulation of the same culture (Table 5), with both being similar to wild type by day 5 of the assay. The FAME/TOC ratio of the Bromo-1091 knockout mutant was elevated approximately 60% with respect to wild type (non-induced) cells when both the mutant and wild type were cultured in nitrate only medium (Table 4). A much smaller increase (38%) in the FAME/TOC ratio was observed on day 3 of the Bromo-1091 knockout mutant cultures that included ammonium in the culture medium; however this increase with respect to wild type cells lessened considerably over the next 4 days to only 10-15% greater than the wild type FAME/TOC ratios. Thus, as evidenced by the FAME/TOC ratios over the course of the batch assay ( FIG. 4C ) the Bromo-1091 knockout mutant was induced for lipid biosynthesis on nitrate-only medium, but not when ammonium was present in the culture medium.
Example 5. Growth and Lipid Biosynthesis of the Bromo-1091 Knockout Mutant in Semi-Continuous Culture
Bromo-1091 Cas9 knockout strain GE-8563 was also assayed in the semi-continuous productivity assay. In the continuous productivity assay PM074 (nitrate only) medium in a 225 cm 2 flask was inoculated with Nannochloropsis seed culture so that the initial 550 ml (inoculated final volume) culture had an initial OD 730 of 0.15. A typical dilution used approximately 150 mls of starter culture in PM124 medium (containing 5 mM ammonium) which was brought up to 550 mls using PM074 medium, such that the starting concentration of ammonium in the semi-continuous assay was less than 1.5 mM. Daily dilutions with PM074 medium further reduced the ammonium concentration as the assay progressed. Three cultures were initiated per strain. The flasks included stir bars and had stoppers having tubing connected with syringe filters for delivering CO 2 enriched air (1% CO 2 , flow rate, 100 ml per min) that was bubbled through the cultures. The flasks were set on stir plates set to 450 rpm. The flasks were aligned with the width (narrowest dimension) against an LED light bank that was programmed with a light/dark cycle and light profile that increased until “solar noon” and then declined to the end of the light period. The “depth” dimension of the flasks, extending back from the light source, was 13.7 cm. Taking into account the positioning of the flasks the farthest distance of the cells in the flasks from the surface of the light source was approximately 15.5 cm. The light profile was designed to mimic a spring day in Southern California: 16 h light: 8 h dark, with the light peaking at approximately 2000 μE. The culture were diluted daily at the middle (peak) of the light period by removing 30% (150 ml) of the culture volume and replacing it with fresh PM074 media diluted (66 ml di H 2 O to 1 L PM074 medium) to adjust for the increase in salinity due to evaporation occurring in the cultures. Samples for FAME and TOC analysis were taken from the culture removed for the dilution. Continuous assays were typically run for 7-14 days. Tables 8-10 show the results of FAME and TOC analysis of knockout and wild type cultures run in the semi-continuous assay. Averages of three cultures are provided with the standard deviation of each value in parentheses.





TABLE 8



Daily production of FAME (μg/ml) by wild type and Bromo-1091 knockout

cells in semi-continuous culture with daily dilution in nitrate-only medium.










DAY
1
2
3
4
5
6
7












WT
46.89
44.91
45.40
43.89
46.15
47.92
50.40


(1.24)
(0.43)
(1.35)
(0.91)
(0.93)
(1.10)
(1.78)

Bromo-KO
68.20
63.56
60.64
59.34
60.86
63.53
62.18


(2.24)
(1.17)
(2.80)
(2.74)
(2.24)
(2.14)
(3.02)

% Increase
45.45
41.53
33.57
35.20
31.87
32.58
23.37

(Bromo v. WT)



































TABLE 9



Daily production of TOC (μg/ml) by wild type and Bromo-1091 knockout

cells in semi-continuous culture with daily dilution in nitrate-only medium.










DAY
1
2
3
4
5
6
7












WT
184.07
173.07
179.23
165.4
162.9
162.6
179.23


(3.99)
(2.18)
(11.47)
(6.82)
(4.59)
(7.37)
(14.35)

Bromo -KO
192.63
182.37
189.4
183.2
171.7
170.1
190.03


(5.25)
(3.95)
(13.78)
(10.11)
(8.73)
(5.98)
(11.06)

Difference
8.56
9.3
10.17
17.8
8.8
7.5
10.8

% Difference
4.65
5.37
5.67
10.76
5.40
4.61
6.03

(Bromo v. WT)




































TABLE 10



Daily FAME/TOC ratios of wild type and Bromo-1091 knockout cells in

semi-continuous culture with daily dilution in nitrate-only medium.










DAY
1
2
3
4
5
6
7












WT
0.21
0.21
0.21
0.21
0.23
0.23
0.23


(0.003)
(0.004)
(0.005)
(0.006)
(0.002)
(0.006)
(0.007)

Bromo -KO
0.29
0.28
0.26
0.26
0.29
0.30
0.27


(0.003)
(0.001)
(0.004)
(0.001)
(0.005)
(0.003)
(0.014)




























In the semi-continuous assay, performed with nitrate-only culture medium, the Bromo-1091 knockout mutant demonstrated a higher FAME productivity with respect to the wild type strain, with daily productivities ranging from about 20% to about 50% more than the FAME productivities of the wild type cells (Table 8 and FIG. 5A ). Biomass (TOC) accumulation by the Bromo-1091 knockout mutant was, however, surprisingly in line with wild type cells (Table 9 and FIG. 5B ). The increased partitioning of carbon to lipids was clear from the FAME/TOC ratio of the Bromo-1091 knockout mutant over the course of the assay (Table 10 and FIG. 5C ) which showed that the mutant had a FAME/TOC ratio of from about 0.26 to about 0.30 over the course of the assay, whereas the FAME/TOC ratio of the wild type assayed under identical culture conditions varied between about 0.21 to 0.23.
Example 6. Cas9 Bromo-1091 Knockdown Constructs
To test the productivity of additional mutant alleles that might have decreased but not eliminated expression of a Bromo-1091 gene, multiple insertions into the promoter region of the gene were generated using Cas9 ( FIG. 6A , see arrows labeled 1-5) using the methods provided in Example 3 and guide RNAs targeting the 5′ promoter of the gene as provided in Table 11.





TABLE 11



Guide RNA Sequences used to generate promoter

insertions in Bromo-1091 Gene







N18 portion of guide 


Guide RNA
(crRNA homologous to

Strain
Sequence
genomic sequence)



GE-13027
SEQ ID NO: 67
ACTGAAAGGGCAGAGTG

Bromo BASH-1

(SEQ ID NO: 68)



GE-13030
SEQ ID NO: 69
TGTGGACGCTAGTACAGG

Bromo BASH-4

(SEQ ID NO: 70)



GE-13032
SEQ ID NO: 71
AAAAGCGCCGTCTCGGAA

Bromo BASH-5

(SEQ ID NO: 72)































Chimeric guide DNA constructs were synthesized and purified as described in Example 3. Each chimeric guide RNA was individually transformed into Nannochloropsis Editor strain GE-6791 along with the donor fragment that included a Hyg resistance (“HygR”) cassette (SEQ ID NO:67) as described in Example 3. Hygromycin resistant colonies were selected and screened by colony PCR as described using primers adjacent to the targeted region of the Bromo-1091 gene (Primers Br-promoter-FP (ATTGCTAGCCGTGCTTTCAAC; SEQ ID NO:73) and Br-promoter-RP (GTCGGTTTGGAGACCCTAGA; SEQ ID NO:74) to confirm donor fragment insertion into the 5′ region of the Bromo-1091 gene.
Quantitative reverse transcription-PCR (qRT-PCR) was performed on RNA isolated from these “basher” lines to determine whether expression of the Bromo-1091 gene was in fact reduced in these lines. The Bromo-1091 Bash knockdown strains were grown under standard nitrogen replete conditions (PM074 (nitrate-only) medium) and harvested during early stationary phase, and total RNA was isolated from the cells\ using methods provided in Example 1, above. RNA was converted to cDNA BioRad's iScript™ Reverse Transcription Supermix reaction mixture kit for reverse transcription according to the manufacturer's protocol. For PCR, Ssofast™ EvaGreen® Supermix PCR reaction mixture (Bio-Rad, Hercules, Calif.) was used along with gene-specific primers. The PCR reaction was carried out on C1000 Thermal Cycler coupled with a CFX Real-time System (BioRad). Primer and cDNA concentrations were according to the manufacturer's recommendation. Primers for amplifying a sequence of the Bromo-1091 transcript were SEQ ID NO:75 and SEQ ID NO:76 (Table 12).
Transcript levels for each sample were normalized against a housekeeping gene with consistent expression levels under different culture conditions (gene 1T5001704) using primers 1704-F (SEQ ID NO:77) and 1704-R (SEQ ID NO:78) and relative expression levels were calculated using the ddCT method using CFX Manager software from BioRad.





TABLE 12



Primers used for qRT/PCR





Gene
Primer
Sequence



Bromo-1091
JLC-1091-RT-F
GAATAGGCGGTTCAGAATGTAGG



(SEQ ID NO: 75)



Bromo-1091
JLC-1091-RT-R
ATATTTTGTGGGCGTTGCTG



(SEQ ID NO: 76)



Housekeeping
JLC-RT-1704-F
GAGGAAGCGGAAGAGGATG

gene

(SEQ ID NO: 77)

T5001704





Housekeeping
JLC-RT-1704-R
TCAAGTACCAGTTCCACACG

gene

(SEQ ID NO: 78)

T5001704

































FIG. 6B shows that several of the strains had reduced levels of Bromo-1091 transcript. Of these, strains GE-13027 (Bromo-1091 BASH-1), GE-13030 (Bromo BASH-4), and GE- GE-13032 (Bromo-1091 BASH-5), targeting the 5′ end of the Bromo-1091 gene, expressed the Bromo-1091 gene at about 35%, 20%, and 50% of the wild type level of the gene, respectively.
Example 7. Knockdown Constructs in Batch Assay
The Bromo-1091 knockdown “basher” strains GE-13027, GE-13030, and GE-13032 that demonstrated attenuated expression of the Bromo-1091 gene (see FIGS. 6A and 6B ) were tested in the batch productivity assay described in Example 4 by scaling up the cultures in culture medium PM124 (which includes both NH 4 and NO 3 as nitrogen sources) and by carrying out the assay in PM123 culture medium that includes nitrate as the sole nitrogen source. In addition, the wild type N. gaditana strain WT-3730 and the Bromo-1091 knockout mutant GE-8563 were tested in the same assay.
FAME and TOC were analyzed as provided in Example 4. The results of these analyses are provided in Tables 13-16, with the values provided being the average of three cultures, and the standard deviations provided in parentheses.





TABLE 13



FAME productivity of Bromo-1091 Knockdown Strains Compared to Wild

Type in Batch Assay with NO 3 -only Culture Medium (mg/L culture)








BASH-1
BASH-4
BASH-5
Bromo-1091 KO


(GE-13027)
(GE-13030)
(GE-13032)
(GE-8563)












DAY
WT

% incr

% incr

% incr

% diff














3
106.56
153.39
43.95
127.13
19.30
162.19
52.21
139.66
31.06


(3.89)
(4.23)

(0.09)

(1.12)

(5.56)

5
154.22
208.36
35.11
185.68
20.40
225.23
46.04
167.71
8.75


(4.72)
(12.21)

(2.83)

(6.14)

(4.58)

7
210.97
264.03
25.15
248.60
17.84
290.4
37.65
199.08
−5.64


(15.43)
(5.65)

(0.53)

(16.58)

(20.60)








































TABLE 14



TOC productivity of Bromo-1091 Knockdown Strains Compared to Wild

Type in Batch Assay with NO 3 -only Culture Medium (mg/L culture)








BASH-1
BASH-4
BASH-5
Bromo-1091 KO


(GE-13027)
(GE-13030)
(GE-13032)
(GE-8563)












DAY
WT

% diff

% diff

% diff

% diff














3
455.45
502.4
10.31
441.75
−3.0
461.7
1.37
292.1
−35.87


(13.93)
(5.09)

(12.23)

(1.70)

5
678.75
663.55
−2.24
651.3
−4.0
646.8
−4.7
417.2
−38.53


(22.98)
(22.98)

(6.93)

(10.32)

7
811.75
772.5
−4.84
773.6
−4.7
756.6
−6.8
593.1
−26.94


(52.54)
(18.67)

(0.99)

(8.06)








































TABLE 15



FAME/TOC ratios of Bromo-1091 Knockdown Strains Compared

to Wild Type in Batch Assay with NO 3 -only Culture Medium








BASH-1
BASH-4
BASH-5
Bromo-1091 KO


(GE-13027)
(GE-13030)
(GE-13032)
(GE-8563)












DAY


% incr

% incr

% incr

% incr














3
0.23
0.31
34.78
0.29
26.09
0.35
52.17
0.46
100


(0.0014)
(0.0115)

(0.0082)

(0.0011)

(0.0139)

5
0.23
0.31
34.78
0.29
26.09
0.35
52.17
0.41
78.26


(0.0007)
(0.0075)

(0.0013)

(0.0039)

(0.0016)

7
0.26
0.34
30.77
0.32
23.08
0.38
46.15
0.34
30.77


(0.0022)
(0.001)

(0.0003)

(0.0178)

(0.0267)



































Table 13 shows that all of the gene attenuation knockdown strains that had reduced expression of the Bromo-1091 gene, GE-13027, GE-13030, and GE-13032, produced more lipid than the wild type strain as measured on days 3, 5, and 7 of the assay. These knockdown strains were more consistent lipid producers than knockout strain GE-8563, which produced at least 30% more lipid than wild type by day 3 of the assay, but by day 7 of the assay was slightly behind wild type in lipid production. Table 14 shows that GE-13027, GE-13030, and GE-13032 did not decrease their biomass production as much as did knockout strain GE-8563 over the course of the assay. In fact, by day 7 of the assay, GE-13027 and GE-13030 were producing within 5% of the amount of biomass produced by wild type cells in the same assay, and strain GE-13032 produced only about 7% less biomass than wild type strain WT-3730. The amount of biomass produced by the knockdown strains was significantly greater than the biomass produced by knockout strain GE-8563 on each day of the assay, in other words, the knockdown strains had only a slight deficit in biomass production with respect to the wild type strain, as compared with an about 25%-40% reduction in biomass production by the knockout strain GE-8563 in relation to the wild type biomass production level over the course of the assay. Table 15 provides the FAME/TOC ratios of the mutant and wild type cultures on days 3, 5, and 7 of the batch assay. With respect to the wild type strain, knockout strain GE-8563 on day 3 had a FAME/TOC ratio that was double that of the wild type (i.e., 100% higher) but this ratio decreased over the course of the assay to about 30% greater than the wild type FAME/TOC ratio. The knockdown strains began with more modest increases in the FAME/TOC ratio than was seen for GE-8563, but the increase over wild type was more consistent over the course of the assay, ranging from 30-35% for GE-13027, from 23-26% for GE13030, and from 45-53% for GE-13032.
The results are depicted graphically in FIGS. 7A-C . FIG. 7A shows that all Bromo-1091 gene knockdown attenuation mutants (GE-13027 (bisected X), GE-13030 (plus sign), and GE-13032 (X)), produced FAME in amounts greater than wild type (circles) when cultured with nitrate as the sole nitrogen source, with the increase in lipid production being greatest at the end of the assay. This is in contrast to the knockout mutant (GE-8563, triangles) cultured in nitrate-only medium, in which an increase in FAME with respect to wild type on day 3 disappeared by the end of the assay (day 7). Thus, in overall FAME production, the Bromo-1091 knockdown mutants were a significant improvement over the knockout mutant. FIG. 7B provides even more surprising results—unlike the knockdown strain (triangles), in batch culture with nitrate as the sole nitrogen source the knockdown mutants (GE-13027 (bisected X), GE-13030 (plus sign), and GE-13032 (X)) were close to wild type levels of biomass production throughout the culture assay. The knockdown mutant (triangles), however, as also seen in FIG. 4B , had consistently significantly reduced TOC accumulation when compared with the wild type strain (circles).
FIG. 7C shows the increased FAME/TOC ratio of the knockout strain with respect to the wild type strain when both strains are batch-cultured in nitrate-only medium (as also seen in FIG. 4C ). The Figure also shows that FAME/TOC ratios of the knockdown mutants are enhanced with respect to the wild type strain and for the most part intermediate between wild type and knockout strain ratios.
Example 8. Bromo-1091 Knockdown Mutants in the Semi-Continuous Productivity Assay
Bromo-1091 attenuation (BASH) strains GE-13127, GE-13130, and GE-13132 were then assayed in the semi-continuous productivity assay described in Example 5, in which the assay medium, PM074, included nitrate as the sole nitrogen source and the knockdown strains were pre-cultured in PM124 medium that included 5 mM ammonium in addition to 8.8 mM nitrate.
The starter cultures were used to inoculate 225 cm 2 rectangular tissue culture flasks, each of which contained a final total volume of 550 ml of culture after inoculation. The cultures were inoculated so that each 550 ml culture had an initial OD 730 of 0.9. A typical inoculum volume was approximately 200 ml of scale-up culture that was added to approximately 350 ml of assay culture medium, which was PM074 (nitrate-only medium). Cultures were diluted daily at mid-day, when the light intensity was at its peak, by removing 30% of the volume (165 mls) and replacing it with the same volume of the assay medium (PM074) plus an additional 10 ml of deionized water to make up for evaporation (included in the make-up medium). Thus, assay cultures inoculated from scale-up cultures that included 5 mM ammonium in the culture medium (PM124 medium) started out with a significant amount of ammonium (e.g., less than 2 mM ammonium) that was progressively diluted out further during the course of the assay. After the cultures reached equilibrium (the growth rate of the cultures equaled the dilution rate of 30% as determined by cell counts and optical density) the semi-continuous assays were run for 10 days. Daily lipid and biomass productivities were only calculated for cultures that had reached steady state (where the increase in growth was equal to the dilution factor for the assay). Three cultures were run for each strain.
The results of the assay are provided in FIGS. 8A-F . The table provided in FIG. 8A shows the daily FAME productivities of the semi-continuous cultures over a ten day period in which they were diluted by 30% each day, with each value being the average of three cultures (standard deviations are provided in parentheses). Each of the Bromo-1091 knockdown mutants has significantly higher FAME productivity than the wild type strain, with GE-13127 and GE-13132 each demonstrating at least 50% greater FAME productivity than wild type over the course of the ten day assay in which nitrate was the sole nitrogen source for the cells. The graph in FIG. 8B shows clearly the largely consistent production of FAME by the knockdown mutants at levels considerably higher than wild type.
FIG. 8C provides the daily biomass productivity, measured as TOC of the semi-continuous cultures of the same assay depicted in FIGS. 8A and 8B , with each value being the average of three cultures (standard deviations are provided in parentheses). Astonishingly, although the mutants produced more lipid than wild type cells every day of the assay ( FIGS. 8A and 8B ), they did not produce significantly less biomass (measured as TOC). Thus, unlike classical lipid induction that occurs when cells are starved for nitrogen, the cultures experience no loss of biomass as compared to wild type cells while producing much more lipid than wild type. FIG. 8D shows this clearly: while the cultures show minor variations in biomass production from day to day, on average the wild type (circles) does not significantly outperform the Bromo-1091 attenuation mutants.
FIG. 8E provides the daily FAME/TOC ratios of the cultures. Over the course of the entire culture period, the wild type cultures had an average FAME/TOC ratio of 0.29, whereas the cultures of strains having attenuated expression of the Bromo-1091 gene had average FAME/TOC ratios of 0.39 (strain GE-13030), 0.42 (strain GE-13032), and 0.50 (strain GE-13127) over the culture period. Strain GE-13030 had an average FAME/TOC value that was 33% higher than that of wild type cultures, strain GE-13132 had an average FAME/TOC value that was 43% higher than that of wild type cultures, and strain GE-13027 had an average FAME/TOC value that was 72% higher than that of wild type cultures, over the course of the ten day assay. The graph in FIG. 8F shows clearly the largely consistent FAME/TOC ratio of the knockdown mutants at ratios considerably higher than wild type.
Example 9. Proximate Analysis of Bromo-1091 Knockdown Mutants
To determine the overall biomass composition of the Bromo-1091 attenuation mutants, quantitative analysis of samples from cultures of Example 8 was performed to determine the lipid, protein, and carbohydrate content of the cells in semi-continuous culture.
FAME lipids were analyzed as described in Example 4 for strains cultured in the semi-continuous assay of Example 8, with the results provided in FIG. 8A .
To determine protein content, isolated biomass samples were hydrolyzed and the amino acids were derivatized to propoxycarbonyl propyl esters (AAPE's), analyzed via GC/MS, and quantitated against an internal standard, as detailed below.
0.5 ml aliquots of wild type N. gaditana (WT-3730) and Bromo-1091 attenuation (BASH) strains GE-13127 (“Bromo Bash-1”) and GE-13132 (“Bromo Bash-5”) from the cultures of Example 8 (taken from the same dilution samples that were used to determine FAME content) were centrifuged and the pellets were washed twice with phosphate buffered saline (PBS). The cells were finally resuspended to a final volume of 0.5 ml (the starting volume) and transferred to a 4 ml glass vial. To the culture sample, 800 μl of 6M HCl with TGA was added (400 μl of thioglycolic acid (TGA) was added to 19.6 ml of 6M HCl just before use). Ten μl of beta mercaptoethanol was then added to the vial, followed by 200 μl of 20 mM norvaline, used as an internal standard. Each vial was blanketed with N2 for 10 seconds, after which the vials were vortexed for 1 min at 2500 rpm to homogenize the samples. The vials were then placed in a 110° C. oven for 22 hours.
At the end of the hydrolysis incubation, the vials were vortexed for 10 min at 2500 rpm, and then centrifuged up to 1000 rpm after which the centrifuge was stopped. A 50 aliquot was removed from each vial and dried by placing in an acid safe EZ-2 Genevac which was run on the HCl method for at least 3 hours prior to derivatization.
For derivativization, 250 μl of milli-Q H 2 O was added to the dried acid hydrolysates, followed by 10 μl of antioxidant mix and then 120 μl of 0.5M NaOH. The antioxidant mix was made by adding 0.25 ml of n-propanol, 50 μl of thiodiglycol, and a few granules of phenol to 2.20 ml of Milli-Q H 2 O, and vortexing. 80 μl of the catalyst, a 4:1 mix of pyridine and n-propanol was then added, and the vial was capped and vortexed at 2500 rpm for 1 min After a 1 min incubation, the vial was again vortexed at 2500 rpm for 1 min 500 μl of a 4:1 mixture of isooctane and chloroform was then added to the vial which was again capped and vortexed at 2500 rpm for 1 min. The rack of sample vials was then covered with another sample rack and shaken 20 times to ensure emulsion of the samples. The samples were then centrifuged until the centrifuge reached 1000 rpm and then the centrifuge was stopped. 200 μl of the organic layer was removed into a new GC vial with a glass insert and analyzed by GC/MS.
The samples were analyzed by GC/MS using a ZB-AAA 10×0.25 mm ID Amino Acid Analysis GC column and quantitated using the internal norvaline standard. The needle Wash 1 solvent was acetone and the needle Wash 2 solvent was isooctane/chloroform (80/20) with a program of 110° C., hold 0 min, 30° C./min to 320° C., hold 0.5 min, using a 4 μl injection at 15:1 split, 250° C. at 1.1 ml/min with a 300° C. transfer line.
The GC-MS data was multiplied by 0.0005 L to obtain μmol values, and multiplied by the molecular weight of the amino acid. The value was divided by 5 to correct for the volume to obtain μg/ml of each amino acid. Asparagine is converted to aspartic acid during acid hydrolysis, thus asparagine plus aspartic acid are determined as aspartic acid in these methods. Tryptophan is not measured by these methods but does not make up a significant fraction of the amino acids in Nannochloropsis proteins.
For total carbohydrate quantitation, biomass was hydrolyzed for one hour in 6N hydrochloric acid to convert polysaccharides to monosaccharides. The resulting monosaccharides were converted to trimethylsilyl ethers using MSTFA N-methyl-N-trinethylsilyltrifluoroacetamide with 1% trimethylchlorosilane, and the ethers were resolved and quantitated using GC-MS analysis. In this analysis, we are able to quantitate arabinose, rhamnose, xylose, mannose, galactose, mannitol, and glucose, which encompass the majority of the sugars present in Nannochloropsis sp.
For acid hydrolysis of culture samples, 500 μl of Milli-Q H 2 O was added to 500 μl culture samples in 4 ml vials, or, where the culture sample was more concentrated (higher TOC), 800 μl of Milli-Q H 2 O was added to 200 μl of culture sample. 20 μl of 2.5 mg/ml ribitol and U- 13 C-glucose as an internal standard was added to the 1 ml diluted culture samples in 4 ml vials. 1 ml of concentrated HCl was then added to each of the vials, the vials were capped and placed in a 105° C. dry bath for 1 hour. The samples were then allowed to cool to room temperature, and 100 μl was transferred to a glass insert inside a 1.5 ml microcentrifuge tube.
For derivatization, the microfuge tubes that included glass inserts containing the samples were place in an acid safe EZ-2 Genevac which was run on the HCl method for at least 3 hours. After drying, 100 μl of the derivatization reagent, which consisted of 800 μl of dry pyridine added to 1 ml of freshly opened MFSTA-1% TMCS, was added to each sample. The samples were incubated for 1 hour at 40° C. while mixing at 1000 rpm in an Eppendorf Thermomixer. Following incubation, the samples were directly analyzed by GC/MS.
The samples were analyzed by GC/MS using a DB5-MS 30 m×250 μm×25 μm GC column and quantitated using the internal U- 13 C-glucose standard. The needle wash solvent was pyridine with a program of 1 min equilibration, 170° C. for 8 min, 10° C./min to 210° C. for 0 min, then 50° C./min to 325° C. for 2 min (total run time 16.3 min).
FIG. 9 shows the results of this analysis on the wild type N. gaditana strain as well as the Bromo-1091 gene attenuation knockdown mutants GE-13127 and GE-13132. As expected from the elevated FAME/TOC ratios of the “basher” mutants with respect to wild type (see, for example, Table 15 and FIG. 9E ), the knockdown mutants had an increased percentage of their total organic carbon as lipid with respect to wild type. Interestingly, the mutants, which as demonstrated in Example 8 had essentially the same total organic carbon accumulation as wild type in the semi-continuous assay, were reduced by about the same extent in protein and carbohydrate content, demonstrating an approximately 20% reduction in both protein and carbohydrate with respect to wild type levels.
Example 10. Chlorophyll Content of Bromo-1091 Knockdown Mutants
Interestingly, Bromo-1091 gene attenuation mutant cultures were noticeably paler than corresponding wild type cultures. Chlorophyll a content (chlorophyll a is the only chlorophyll present in Nannochloropsis ) of the GE-13127 and GE-13132 mutants was determined along with that of wild type cells cultured under the same conditions (the semi-continuous productivity cultures of Example 8) by extracting cells with DMSO/acetone, and analyzing the extraction supernatants by spectrophotometry.
Briefly, 200-250 μl aliquots of culture were pipetted into 2.0 ml microfuge tubes and pelleted using a table top microcentrifuge at 12,000 rpm for 3 minutes. The supernatants were immediately aspirated off of the pellets, and each pellet was resuspended in 1 ml of 1:1 DMSO:acetone. Samples were then vortexed for 2-5 min, and the cell debris was pelleted using the table top microcentrifuge at 12,000 rpm for 3 min at room temperature. The resulting pellets were white.
Cuvettes containing 1 ml 1:1 DMSO:acetone were blanked at 663 nm and 720 nm and the supernatants of the DMSO:acetone extracted cells were read (using at least 750 of sample supernatant).
To calculated the chlorophyll a present in each sample, the absorption at 720 nm was subtracted from the absorption at 663 nm. The absorption value was multiplied by the dilution factor (e.g., 5 for a 200 μl aliquot of cell culture) and extinction coefficient of 20.15 to determine the ug/ml concentration of chlorophyll a or by 18.01 to determine the μmol/ml concentration.
The results of the analysis are provided in FIG. 10 , where it can be seen that the GE-13027 and GE-13032 Bromo-1091 gene attenuation mutants had an approximately 55% reduction in total chlorophyll per cell.





TABLE 16



Chlorophyll content of Bromo-1091 Knockdown Strains

Compared to Wild Type strain in Semi-continuous

Assay with NO 3 -only Culture Medium








avg pg



Sample
chl/cell
% difference










WE-3730
0.183
0%



(0.012)


HAT BASH-1
0.077
−58%


GE-13027
(0.003)


HAT BASH-4
0.105
−43%


GE-13030
(0.003)


HAT BASH-5
0.090
−51%


GE-13032
(0.009)



































Example 11. Transcriptomic Analysis of LION Mutants
To determine what genes were regulated by the Bromo-1091 polypeptide, as well as the ZnCys regulator disclosed in commonly-owned and co-pending U.S. patent application Ser. No. 15/210,845 filed Jul. 14, 2016, a transcriptomic analysis was performed of GE-13032, the Bromo-1091 5′ BASH-5 mutant, the ZnCys-2845 knockout mutant GE-8564 (US Provisional application filed Jul. 14, 2015), and a nitrate reductase (NR) loss-of-function (knockout) mutant. GE-13032 and GE-8564 are referred to herein as Lipid Induced on Nitrate or “LION” mutants. GE-8564 is given the designation LION1, referring to mutants relating to attenuation, disruption, or mutation in the ZnCys-2845 gene disclosed in U.S. patent application Ser. No. 15/210,845 and orthologs of other species, and GE-13032 is given the designation LION2, referring to mutants relating to attenuation, disruption, or mutantion in the Bromo gene disclosed herein and orthologs of other species. The GE-13032 and GE-8564 strains and appropriate parental controls were grown in batch assay using PM074 medium containing only nitrate as the nitrogen source. Samples were harvested during the 5 th day of the 7 day assay and RNA was extracted and sequenced using the NextSeq platform available in-house. Pairwise correlation, principal component and replicate clustering analyses of total reads obtained from the NextSeq (quantified as fragments per kilobase of exon per million reads mapped, FPKM) indicated that the biological replicates clustered together, thus validating the experimental setup. As expected, the parental controls (Cas9 Editor strains and wild type) displayed one transcriptional pattern and the mutants displayed a distinct transcriptional pattern. Interestingly, the ZnCys-2845 knockout mutant, the Bromo-1091 5′ bash mutant, and the nitrate reductase mutant each showed a distinct pattern.
FIGS. 11A-11B provides a “heat map” diagram of transcriptional profiling of the ZnCys-2845 knockout, the Bromo-1091 5′ bash promoter disruption mutant, and nitrate reductase loss-of-function mutants (ZnCys-KO, Bromo-5′ Bash, and NR-KO, respectively) grown in batch assay on nitrate-based medium. Genes were hierarchically clustered based on their abundance (measured as FPKM) using Pearson's correlation. The color intensity corresponds to the FPKM value, with darker bands representing higher values and lighter bands representing lower values, respectively. Biological replicates clustered together for each mutant line, thus validating the experimental setup. As expected, the parental controls (Cas9 enabled strains) grouped into a “wild-type” Glade and the mutants formed distinct separate clades. Interestingly, within the mutant Glade, both the ZnCys-2845 knockout and the Bromo-1091 mutants demonstrated a different gene expression pattern from the nitrate reductase mutant, and differed significantly from one another in their gene expression patterns. The Bromo-1091 knockout mutant was observed to upregulate 1895 genes at least 2-fold with respect to the Cas9 parental strain while 1025 genes were observed to be down-regulated at least 2-fold with respect to the Cas9 parental strain (FDR<0.01).