(en)Disclosed herein are enzymes useful for the degradation of cellobiosan in materials such a pyrolysis oils. Methods of degrading cellobiosan using enzymes or organisms expressing the same are also disclosed.

1.ApplicationNumber: US-201615063197-A
1.PublishNumber: US-2016257980-A1
2.Date Publish: 20160908
3.Inventor: LINGER JEFFREY
BECKHAM GREGG T.

4.Inventor Harmonized: LINGER JEFFREY(US)
BECKHAM GREGG T(US)

5.Country: US
6.Claims:
(en)Disclosed herein are enzymes useful for the degradation of cellobiosan in materials such a pyrolysis oils. Methods of degrading cellobiosan using enzymes or organisms expressing the same are also disclosed.

7.Description:
(en)CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Provisional Application No. 62/129,384, filed Mar. 6, 2015, the contents of which are incorporated by reference in their entirety.


CONTRACTUAL ORIGIN
The United States Government has rights in this invention under Contract No. DE-AC36-08G028308 between the United States Department of Energy and Alliance for Sustainable Energy, LLC, the Manager and Operator of the National Renewable Energy Laboratory.


REFERENCE TO SEQUENCE LISTING
This application contains a Sequence Listing submitted as an electronic text file entitled “14-71_ST25.txt,” having a size in bytes of 36 kb and created on Mar. 7, 2016. Pursuant to 37 CFR §1.52(e)(5), the information contained in the above electronic file is hereby incorporated by reference in its entirety.
BACKGROUND
Thermal processes for biomass deconstruction, such as pyrolysis and liquefaction, offer rapid, effective methods for the depolymerization of plant cell wall components. These processes typically produce heterogeneous slates of compounds derived from polysaccharides and lignin that can potentially be upgraded simultaneously over chemical catalysts, integrated into petroleum refinery streams, or fractionated through a wide variety of approaches and subsequently upgraded in a more selective manner to a broader slate of fuels and chemicals. Cellobiosan, or 1,6-anhydro-β-D-glucose, is a major component of pyrolysis oil derived from cellulose and hemicellulose.
The foregoing examples of the related art and limitations related therewith are intended to be illustrative and not exclusive. Other limitations of the related art will become apparent to those of skill in the art upon a reading of the specification and a study of the drawings.
SUMMARY
The following embodiments and aspects thereof are described and illustrated in conjunction with systems, tools and methods that are meant to be exemplary and illustrative, not limiting in scope. In various embodiments, one or more of the above-described problems have been reduced or eliminated, while other embodiments are directed to other improvements.
Exemplary embodiments provide methods for degrading cellobiosan by contacting a cellobiosan-containing material with an enzyme capable of hydrolyzing β-1,4-glycosidic bonds to generate levoglucosan. In some embodiments, the enzyme comprises a Glycoside Hydrolase (GH) domain selected from the Glycoside Hydrolase Families GH1, GH3, GH5, GH9, GH30 or GH116.
In certain embodiments, the enzyme is a β-glucosidase or endoglucanase, such as a β-glucosidase or endoglucanase from a bacterium or fungus. In some embodiments, the bacterium is from the genus Agrobacterium (e.g., Agrobacterium sp.) or Thermotoga (e.g., Thermotoga maritima ). In others, the fungus is from the genus Aspergillus (e.g., A. niger ) or Phanerochaete (e.g., P. chrysosporium ).
In various embodiments, the β-glucosidase is Agrobacterium sp. abg, Thermotoga maritima bglA, Aspergillus niger bgl1, or Phanerochaete chrysosporium bgl1A.
In some embodiments, the cellobiosan-containing material is a pyrolysis oil, such as an oil derived from the pyrolysis of biomass, or a fraction obtained by fractionating a pyrolysis oil (e.g., an aqueous fraction).
In certain embodiments, the enzyme is expressed by a microorganism and the cellobiosan containing material is contacted with the microorganism to degrade the cellobiosan. In various embodiments, the microorganism expresses an exogenous gene encoding a levoglucosan kinase, such as the levoglucosan kinase LGK from Lipomyces starkeyi.
In some embodiments, the microorganism is a bacterium, such as a bacterium from the genus Pseudomonas (e.g., P. putida ). In further embodiments, an exogenous β-glucosidase may also be added to degrade the cellobiosan.
Additional exemplary embodiments provide methods for producing levoglucosan and glucose from cellobiosan by contacting the cellobiosan with at least one β-glucosidase and isolating at least one of the levoglucosan or glucose products.
In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by reference to the drawings and by study of the following descriptions.



BRIEF DESCRIPTION OF THE DRAWINGS
Exemplary embodiments are illustrated in referenced figures of the drawings. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than limiting.
FIG. 1 shows a mechanism for cellobiosan cleavage via an exemplary enzyme disclosed herein to generate glucose and levoglucosan.
FIG. 2 shows the growth (A and B) and mcl-PHA production (C) of a P. putida strain expressing levoglucosan kinase. Glu indicates glucose and LG indicates levoglucosan.
FIG. 3 shows the cleavage of cellobiosan into glucose and levoglucosan increases with the concentration of β-glucosidase enzyme present in the reaction.
FIG. 4 shows the cleavage of cellobiosan into glucose and levoglucosan by several β-glucosidases. HPLC analysis shows the disappearance of cellobiosan and the appearance of glucose and levoglucosan with the addition of beta-glucosidase enzymes to the reaction. Cellobiosan molarity is reported as double its actual value to represent the molarity of the individual monosaccharide components (glucose plus levoglucosan) for clarity in comparisons.
FIG. 5 shows the cleavage of cellobiosan into glucose and levoglucosan by several β-glucosidases at different enzyme loadings.
FIG. 6 shows growth of P. putida on cellobiose and cellobiosan with or without the addition of exogenous β-glucosidase.
FIG. 7 shows the growth of P. putida on cellobiosan with or without the addition of exogenous β-glucosidase.
FIG. 8 shows an exemplary pyrolysis scheme.



DETAILED DESCRIPTION
Disclosed herein are enzymes (and organisms expressing these enzymes) useful for the more efficient utilization of saccharides such as those found in pyrolysis oil. Methods of biologically cleaving cellobiosan found in feedstocks such as pyrolysis oil or fractions thereof into monosaccharides are also disclosed.
Pyrolysis offers a straightforward approach for the deconstruction of plant cell wall polymers into pyrolysis oil or bio-oil, which may be fractionated and subsequently used in biological approaches to selectively upgrade some of the resulting fractions. An exemplary pyrolysis set-up is demonstrated in FIG. 8 . One fraction of interest for biological upgrading contains polysaccharide-derived substrates including sugars and sugar dehydration products such as levoglucosan and cellobiosan, which are two of the most abundant pyrolysis products of cellulose. To date, however, the biological utilization of cellobiosan has not been demonstrated. Rather, cellobiosan is typically hydrolyzed to glucose by treatment with a strong acid such as sulfuric acid (H 2 SO 4 ).
In typical fast pyrolysis schemes, levoglucosan and cellobiosan are the most abundantly produced dehydration products of cellulose. Cellobiosan has been identified as a major component of anhydrosugars from the pyrolysis of Avicel (between 6-15% of the liquid product). The only utilization of cellobiosan and larger anhydrosugar oligomers derived from cellulose has been observed when acid hydrolysis is combined with subsequent conversion of the glucose (after hydrolysis of the glycosidic bonds for depolymerization and hydrolysis of levoglucosan to produce glucose).
Multiple tandem catalytic-biological schemes have been developed to fractionate levoglucosan-rich streams from bio-oil, hydrolyze it to glucose, and upgrade it to, for example, ethanol. For example, Lian et al., Bioresource Technology 101:9688-9699 (2010) demonstrate a fractionation process that used solvent fractionation to separate phenolics from pyrolytic sugars, hydrolyze the levoglucosan to glucose, and then use a biological step to either produce ethanol or fatty acids.
The selective fractionation of pyrolysis-derived substrates and use of biological approaches to selectively upgrade at least some of the resulting fractions are attractive as a means for efficient utilization of bio-oils. Given the high concentration of cellobiosan in pyrolysis oil, identifying a mechanism enabling the metabolic utilization of this compound is paramount to biological upgrading of pyrolysis oils. The present work represents a move towards a consolidated biological step for production of fuels or chemicals from pyrolytic sugar streams.
Cellobiosan-containing materials may be contacted with enzymes disclosed herein to degrade the cellobiosan to levoglucosan and glucose. A major component of the aqueous fraction of pyrolysis oil is cellobiosan, which consists of a disaccharide wherein the reducing end has undergone a dehydration reaction to form a levoglucosan connected to glucose. In order to derive value from the aqueous phase of pyrolysis oil, one route would be to biologically metabolize the primary components—including cellobiosan—to generate higher-value products. An exemplary reaction scheme utilizing a β-glucosidase to cleave cellobiosan into levoglucosan and glucose is provided in FIG. 1 .
Disclosed herein are methods of converting cellobiosan to levoglucosan and glucose with enzymes capable of cleaving the β-1,4-glycosidic bond in cellobiosan to liberate glucose and levoglucosan. Exemplary enzymes with this catalytic activity include β-glucosidases and endoglucanases. Being able to convert cellobiosan into its monomeric units enables the biological utilization of cellobiosan and enables the production of endless bio-products. Previously there were no known routes for the biological utilization of cellobiosan.
Suitable enzymes include those from Glycoside Hydrolase (GH) families 1, 3, 5, 9, 30, or 116. Exemplary enzymes also include those comprising a GH1 domain, a GH3 domain, a GH5 domain, a GH9 domain, a GH30 domain or a GH116 domain. Specific enzymes containing these GH domains are listed in the CAZY (Carbohydrate-Active enZYmes) database, grouped by GH family and available on the internet. Further examples include Bacillus thuringiensis GH5, Thermoanaerobacterium xylanolyticum GH116, Homo sapiens GH30, Mus musculus GH30 and Phytophthora infestans GH30.
Enzymes possessing a Glycoside Hydrolase Family 1 domain or a Glycoside Hydrolase Family 3 domain are typically referred to as β-glucosidases, which have previously not been shown to be active of cellobiosan substrates.
As used herein, “cellobiosan-containing materials” means any natural or processed materials comprising detectable amounts of cellobiosan. These may be derived from many sources, including oils derived from the pyrolysis of biomass of other lignocellulose or cellulose sources. Exemplary materials with high cellobiosan content include pyrolysis oil, bio-oil, fast pyrolysis oil and fractions thereof. Various fractions of pyrolysis oils may be obtained by, for example, extractions of the oils using various aqueous or organic solvents, or other processing steps. Also suitable are other sources of anhydrosugars, including cellobiosan in purified or semi-purified form. While use of pyrolysis oils is exemplified here, any source of cellobiosan may be suitable for use with the methods herein, including cellobiosan derived from liquefaction or other high temperature methods to break down cellulose.
The Examples below demonstrate that β-glucosidases are capable of hydrolyzing the β-1,4-glycosidic bond in cellobiosan to liberate glucose and levoglucosan as products. While β-glucosidases are exemplified, additional enzymes disclosed herein (including endoglucanases) are suitable for use in the methods. β-glucosidases from many families may be used to degrade cellobiosan. Generally, enzymes with the ability to cleave β-1,4-glycosidic bonds (such as those containing a Glycoside Hydrolase (GH) Family 1 (GH1) domain or GH Family 3 (GH3) domain) are suitable for use in the methods disclosed herein. In various embodiments, the enzyme is a GH1 family enzyme, a GH3 family enzyme, a β-glucosidase, an endoglucanase, or combinations thereof.
Suitable enzymes may be derived from both prokaryotic and eukaryotic organisms, or from mesophilic, thermophilic or hyperthermophilic organisms. Enzymes may be derived from microorganisms such as bacteria, fungi, yeast or the like via cell lysis and isolation techniques, or produced recombinantly. Additional enzymes include any enzyme able to cleave β-1,4-glycosidic bonds, regardless of species. For example, β-glucosidases are highly conserved across diverse biological organisms, from bacteria to higher eukaryotes such as mice and humans. Suitable enzymes may be from the kingdoms/domains Bacteria, Archaea or Eucarya.
In some embodiments, a microorganism expressing the enzyme may be used to directly as a biocatalyst to degrade the cellobiosan sample. Exemplary bacteria include those from the genera Agrobacterium (e.g., Agrobacterium sp.) and Thermotoga (e.g., T. maritima ), while exemplary fungi include those from the genera Aspergillus (e.g., A. niger, A. nidulans, A. awamori, or A. aculeatus ) and Phanerochaete (e.g., P. chrysosporium ). Other species of the genus Thermotoga include T. elfii, T. hypogeal, T. lettingae, T. naphthophila, T. neapolitana, T. petrophila, T. subterranean, and T. thermarum.
Enzymes described herein may be used as purified recombinant enzyme or as culture broths from cells that naturally produce the enzyme or that have been engineered to produce the enzyme. β-Glucosidases or endoglucanases can be added exogenously, or may be expressed and secreted directly from a microbial biocatalyst. In some embodiments, the enzymes may be expressed in a bacterium from the genus Pseudomonas, such as P. putida or a strain thereof such as P. putida KT2440. Additional host organisms include those disclosed below.
Certain embodiments disclosed herein involve microorganisms that have been genetically modified to express a gene encoding levoglucosan kinase (LGK). The amino acid sequence of LGK from Lipomyces starkeyi can be found at GenBank Accession No. EU751287 and SEQ ID NO:2. Additional examples of levoglucosan kinases are known in the art, including the microbial examples found in Dai et al., World J Microbiol Biotechnol 25:1589-1595 (2009).
Levoglucosan can be converted to glucose-6-phosphate (G6P) through the activity of a LGK. Expression of exogenous LGK by a microorganism allows the microbe to more fully utilize levoglucosan present in a feedstock, including levoglucosan derived from cellobiosan as described herein. An exemplary LGK enzyme is the lgk gene from Lipomyces starkeyi, which is active for conversion of levoglucosan to G6P. The lgk gene may be expressed in microbial biocatalysts such as P. putida KT2440 (see Example 1).
The addition of β-glucosidase or endoglucanase to an LGK strain of P. putida KT2440, for example, enables complete utilization of cellobiosan via hydrolytic cleavage of the glycosidic linkage, liberating glucose and levoglucosan. β-glucosidases or endoglucanases can be added exogenously or may be expressed and secreted directly from the microbial biocatalyst.
Hybrid processing requires biocatalysts that span the breadth of molecules present in thermochemical-derived substrates as well as detoxification strategies that enable growth of a given biocatalyst. P. putida KT2440 represents a promising strain for hybrid processing applications given its inherent ability to tolerate toxic environments, the ability to engineer the microbe, and the large suite of endogenous substrate specificity beyond sugars especially for aromatic catabolism. Strains of P. putida may be engineered to catabolize a broad range of substrates including xylose and phenol, among others. P. putida strains may also be engineered to express exogenous levoglucosan kinase, thereby enabling the utilization of levoglucosan from biomass-derived feedstocks, including thermochemical-derived substrates and lignin-derived streams. In addition, microbes such as Rhodococcus jostii RHA1, Acinetobacter sp. ADP1, and Amycolatopsis sp. may be similarly engineered and used as biocatalysts.
Bio-oils and other cellobiosan-containing materials may be contacted with enzymes at a concentration and a temperature for a time sufficient to achieve the desired amount of cellobiosan degradation. The enzymes disclosed herein may be used at any temperature, but may also well suited for higher temperature digestions. For example, the enzymes or cocktails may be used at temperatures ranging from about 30° C. to about 100° C., or from about 40° C. to about 80° C., or from about 50° C. to about 65° C., depending on the thermophilic nature of the enzyme
Suitable times for cellobiosan degradation range from a few hours to several days, and may be selected to achieve a desired amount of degradation. Exemplary digestion times include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours; and 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5 or 15 days. In some embodiments, digestion times may be one or more weeks.
The resulting products after cellobiosan degradation may also be converted to products such as ethanol via fermentation or upgraded to products other than ethanol. Examples include conversion to higher alcohols, hydrocarbons, or other advanced fuels via biological or chemical pathways, or combination thereof. Products (such as levoglucosan or glucose) may be recovered or isolated from the cellobiosan degradation reactions by standard separation techniques for further upgrading. Products may also be further processed by a biocatalyst into additional fuels or chemicals.
Cellulose containing materials may be subjected to pyrolysis processes to generate oils rich in cellobiosan. Exemplary cellulose-containing materials include bioenergy crops, agricultural residues, municipal solid waste, industrial solid waste, sludge from paper manufacture, yard waste, wood and forestry waste. Examples of biomass include, but are not limited to, corn grain, corn cobs, crop residues such as corn husks, corn stover, corn fiber, grasses, wheat, wheat straw, barley, barley straw, hay, rice straw, switchgrass, waste paper, sugar cane bagasse, sorghum, soy, components obtained from milling of grains, trees, branches, roots, leaves, wood (e.g., poplar) chips, sawdust, shrubs and bushes, vegetables, fruits, flowers and animal manure.
Table 1 below provides GenBank accession numbers for the nucleic acid and amino acid sequences for exemplary β-glucosidases for use in the disclosed methods. “Nucleic acid” or “polynucleotide” as used herein refers to purine- and pyrimidine-containing polymers of any length, either polyribonucleotides or polydeoxyribonucleotide or mixed polyribo-polydeoxyribonucleotides. This includes single-and double-stranded molecules (i.e., DNA-DNA, DNA-RNA and RNA-RNA hybrids) as well as “protein nucleic acids” (PNA) formed by conjugating bases to an amino acid backbone. This also includes nucleic acids containing modified bases. Also included are cDNA molecules.
Nucleic acids referred to herein as “isolated” are nucleic acids that have been removed from their natural milieu or separated away from the nucleic acids of the genomic DNA or cellular RNA of their source of origin (e.g., as it exists in cells or in a mixture of nucleic acids such as a library), and may have undergone further processing. Isolated nucleic acids include nucleic acids obtained by methods described herein, similar methods or other suitable methods, including essentially pure nucleic acids, nucleic acids produced by chemical synthesis, by combinations of biological and chemical methods, and recombinant nucleic acids that are isolated.
Nucleic acids referred to herein as “recombinant” are nucleic acids which have been produced by recombinant DNA methodology, including those nucleic acids that are generated by procedures that rely upon a method of artificial replication, such as the polymerase chain reaction (PCR) and/or cloning into a vector using restriction enzymes. Recombinant nucleic acids also include those that result from recombination events that occur through the natural mechanisms of cells, but are selected for after the introduction to the cells of nucleic acids designed to allow or make probable a desired recombination event. Portions of isolated nucleic acids that code for polypeptides having a certain function can be identified and isolated by, for example, the method disclosed in U.S. Pat. No. 4,952,501.
An isolated nucleic acid molecule can be isolated from its natural source or produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning) or chemical synthesis. Isolated nucleic acid molecules can include, for example, genes, natural allelic variants of genes, coding regions or portions thereof, and coding and/or regulatory regions modified by nucleotide insertions, deletions, substitutions, and/or inversions in a manner such that the modifications do not substantially interfere with the nucleic acid molecule's ability to encode a polypeptide or to form stable hybrids under stringent conditions with natural gene isolates. An isolated nucleic acid molecule can include degeneracies. As used herein, nucleotide degeneracy refers to the phenomenon that one amino acid can be encoded by different nucleotide codons. Thus, the nucleic acid sequence of a nucleic acid molecule that encodes a protein or polypeptide can vary due to degeneracies.
Unless so specified, a nucleic acid molecule is not required to encode a protein having protein activity. A nucleic acid molecule can encode a truncated, mutated or inactive protein, for example. In addition, nucleic acid molecules may also be useful as probes and primers for the identification, isolation and/or purification of other nucleic acid molecules, independent of a protein-encoding function.
Suitable nucleic acids include fragments or variants that encode a functional enzyme. For example, a fragment can comprise the minimum nucleotides required to encode a functional β-glucosidase or endoglucanase. Nucleic acid variants include nucleic acids with one or more nucleotide additions, deletions, substitutions, including transitions and transversions, insertion, or modifications (e.g., via RNA or DNA analogs). Alterations may occur at the 5′ or 3′ terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
Embodiments of the nucleic acids include those that encode the polypeptides that functions as β-glucosidases, endoglucanases or functional equivalents thereof. A functional equivalent includes fragments or variants of these that exhibit the ability to function as a β-glucosidase or endoglucanase. As a result of the degeneracy of the genetic code, many nucleic acid sequences can encode a given polypeptide with a particular enzymatic activity. Such functionally equivalent variants are contemplated herein.
Nucleic acids may be derived from a variety of sources including DNA, cDNA, synthetic DNA, synthetic RNA, or combinations thereof. Such sequences may comprise genomic DNA, which may or may not include naturally occurring introns. Moreover, such genomic DNA may be obtained in association with promoter regions or poly (A) sequences. The sequences, genomic DNA, or cDNA may be obtained in any of several ways. Genomic DNA can be extracted and purified from suitable cells by means well known in the art. Alternatively, mRNA can be isolated from a cell and used to produce cDNA by reverse transcription or other means.
Also disclosed herein are recombinant vectors, including expression vectors, containing nucleic acids encoding enzymes. A “recombinant vector” is a nucleic acid molecule that is used as a tool for manipulating a nucleic acid sequence of choice or for introducing such a nucleic acid sequence into a host cell. A recombinant vector may be suitable for use in cloning, sequencing, or otherwise manipulating the nucleic acid sequence of choice, such as by expressing or delivering the nucleic acid sequence of choice into a host cell to form a recombinant cell. Such a vector typically contains heterologous nucleic acid sequences not naturally found adjacent to a nucleic acid sequence of choice, although the vector can also contain regulatory nucleic acid sequences (e.g., promoters, untranslated regions) that are naturally found adjacent to the nucleic acid sequences of choice or that are useful for expression of the nucleic acid molecules.
The nucleic acids described herein may be used in methods for production of enzymes and enzyme cocktails through incorporation into cells, tissues, or organisms. In some embodiments, a nucleic acid may be incorporated into a vector for expression in suitable host cells. The vector may then be introduced into one or more host cells by any method known in the art. One method to produce an encoded protein includes transforming a host cell with one or more recombinant nucleic acids (such as expression vectors) to form a recombinant cell. The term “transformation” is generally used herein to refer to any method by which an exogenous nucleic acid molecule (i.e., a recombinant nucleic acid molecule) can be inserted into a cell, but can be used interchangeably with the term “transfection.”
Non-limiting examples of suitable host cells include cells from microorganisms such as bacteria, yeast, fungi, and filamentous fungi. Exemplary microorganisms include, but are not limited to, bacteria such as E. coli, filamentous fungi from the genera Trichoderma (e.g., T. reesei, T. viride, T. koningii, or T. harzianum ), Penicillium (e.g., P. funiculosum ), Humicola (e.g., H. insolens ), Chrysosporium (e.g., C. lucknowense ), Gliocladium, Aspergillus (e.g., A. niger, A. nidulans, A. awamori, or A. aculeatus ), Fusarium, Neurospora, Hypocrea (e.g., H. jecorina ), and Emericella; yeasts from the genera Saccharomyces (e.g., S. cerevisiae ), Pichia (e.g., P. pastoris ), or Kluyveromyces (e.g., K. lactis ). Cells from plants such as Arabidopsis, barley, citrus, cotton, maize, poplar, rice, soybean, sugarcane, wheat, switch grass, alfalfa, miscanthus, and trees such as hardwoods and softwoods are also contemplated herein as host cells.
Host cells can be transformed, transfected, or infected as appropriate by any suitable method including electroporation, calcium chloride-, lithium chloride-, lithium acetate/polyene glycol-, calcium phosphate-, DEAE-dextran-, liposome-mediated DNA uptake, spheroplasting, injection, microinjection, microprojectile bombardment, phage infection, viral infection, or other established methods. Alternatively, vectors containing the nucleic acids of interest can be transcribed in vitro, and the resulting RNA introduced into the host cell by well-known methods, for example, by injection. Exemplary embodiments include a host cell or population of cells expressing one or more nucleic acid molecules or expression vectors described herein (for example, a genetically modified microorganism). The cells into which nucleic acids have been introduced as described above also include the progeny of such cells.
Vectors may be introduced into host cells such as those from filamentous fungi by direct transformation, in which DNA is mixed with the cells and taken up without any additional manipulation, by conjugation, electroporation, or other means known in the art. Expression vectors may be expressed by filamentous fungi or other host cells episomally or the gene of interest may be inserted into the chromosome of the host cell to produce cells that stably express the gene with or without the need for selective pressure. For example, expression cassettes may be targeted to neutral chromosomal sites by recombination.
Host cells carrying an expression vector (i.e., transformants or clones) may be selected using markers depending on the mode of the vector construction. The marker may be on the same or a different DNA molecule. In prokaryotic hosts, the transformant may be selected, for example, by resistance to ampicillin, tetracycline or other antibiotics. Production of a particular product based on temperature sensitivity may also serve as an appropriate marker.
Host cells may be cultured in an appropriate fermentation medium. An appropriate, or effective, fermentation medium refers to any medium in which a host cell, including a genetically modified microorganism, when cultured, is capable of growing or expressing the polypeptides described herein. Such a medium is typically an aqueous medium comprising assimilable carbon, nitrogen and phosphate sources, but can also include appropriate salts, minerals, metals and other nutrients. Microorganisms and other cells can be cultured in conventional fermentation bioreactors and by any fermentation process, including batch, fed-batch, cell recycle, and continuous fermentation. The pH of the fermentation medium is regulated to a pH suitable for growth of the particular organism. Culture media and conditions for various host cells are known in the art. A wide range of media for culturing filamentous fungi, for example, are available from ATCC. Exemplary culture/fermentation conditions and reagents are provided in the Examples that follow.
The nucleic acid molecules described herein encode the enzymes with amino acid sequences such as those represented by Table 1. As used herein, the terms “protein” and “polypeptide” are synonymous. “Peptides” are defined as fragments or portions of polypeptides, preferably fragments or portions having at least one functional activity as the complete polypeptide sequence. “Isolated” proteins or polypeptides are proteins or polypeptides purified to a state beyond that in which they exist in cells. In certain embodiments, they may be at least 10% pure; in others, they may be substantially purified to 80% or 90% purity or greater. Isolated proteins or polypeptides include essentially pure proteins or polypeptides, proteins or polypeptides produced by chemical synthesis or by combinations of biological and chemical methods, and recombinant proteins or polypeptides that are isolated. Proteins or polypeptides referred to herein as “recombinant” are proteins or polypeptides produced by the expression of recombinant nucleic acids.
Proteins or polypeptides encoded by nucleic acids as well as functional portions or variants thereof are also described herein. Polypeptide sequences may be identical to the amino acid sequences presented in Table 1, or may include up to a certain integer number of amino acid alterations. Such protein or polypeptide variants retain functionality as β-glucosidases or endoglucanases, and include mutants differing by the addition, deletion or substitution of one or more amino acid residues, or modified polypeptides and mutants comprising one or more modified residues. The variant may have one or more conservative changes, wherein a substituted amino acid has similar structural or chemical properties (e.g., replacement of leucine with isoleucine). Alterations may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence.
In certain embodiments, the polypeptides may be at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequences set forth in Table 1 and possess enzymatic function. Percent sequence identity can be calculated using computer programs (such as the BLASTP and TBLASTN programs publicly available from NCBI and other sources) or direct sequence comparison. Polypeptide variants can be produced using techniques known in the art including direct modifications to isolated polypeptides, direct synthesis, or modifications to the nucleic acid sequence encoding the polypeptide using, for example, recombinant DNA techniques.
Polypeptides may be retrieved, obtained, or used in “substantially pure” form, a purity that allows for the effective use of the protein in any method described herein or known in the art. For a protein to be most useful in any of the methods described herein or in any method utilizing enzymes of the types described herein, it is most often substantially free of contaminants, other proteins and/or chemicals that might interfere or that would interfere with its use in the method (e.g., that might interfere with enzyme activity), or that at least would be undesirable for inclusion with a protein.
EXAMPLES
Example 1
Plasmid and Strain Construction and Levoglucosan Utilization
The levoglucosan kinase (lgk) gene from Lipomyces starkeyi was codon optimized using Gene Designer software from DNA 2.0 and synthesized as a gBlock (SEQ ID NO:1). This fragment was cloned into plasmid pMFL76, which is derived from the commercial PCR-Blunt II Topo vector (ThermoFisher), with the addition of two 1-kb genomic regions located in proximity of the rpoS region of the genome from Pseudomonas putida KT2440 to enable homologous recombination mediated genomic integration into strain KT2440. The Ptac-lgk was inserted in between these 1 kb homology regions and transformed into KT2440. A single kanamycin-resistant transformant was isolated and growth in M9 levoglucosan was confirmed, to generate strain FJPO3.
FIG. 2 shows growth, metabolism and polyhydroxyalkanoate production of strain FJPO3. Panel A shows growth curves of FJPO3 in M9 medium supplemented with either 7.5 g/L glucose or 7.5 g/L levoglucosan. Growth profiles in these carbon sources at this concentration are similar, suggesting efficient utilization of levoglucosan in strain FJPO3. Panel B shows that total levoglucosan utilization is similar to glucose utilization and FJPO3 has the capacity to grow at concentrations as high as 60 g/L.
P. putida produces medium chain-length polyhydroxy-alkanoates (mcl-PHAs), which are high-value polymers that can serve as plastics, adhesive precursors, and as precursors to chemical building blocks or hydrocarbons. As mcl-PHAs represent a potential target to generate from pyrolytic sugars, the generation of mcl-PHAs by P. putida using levoglucosan as its sole carbon source as investigated. As shown in Panel C of FIG. 2 , Nile Red fluorescence demonstrates that FJPO3 produces mcl-PHAs in N-limited medium [M9 1mM (NH 4 ) 2 SO 4 ] containing levoglucosan as a sole carbon source, but not in the N- and C-rich LB medium.
Example 2
Media and Growth Conditions
Most growths were in a modified formulation of M9 minimal salts. Briefly, 6.78 g Na 2 PO 4 (anhydrous), 3 g KH 2 PO 4 , and 0.5 g NaCl were dissolved in 750 mL deionized H 2 O. The pH was adjusted to 7.4 with 10 N NaOH and brought to 900 mL with H 2 O. The solution was autoclaved for 15 minutes at 121° C. and allowed to cool. One hundred μL of 1 M CaCl 2 (100 μM final) and 2 mL of 1 M MgSO 4 -7H 2 O (2 mM Final), 1 mL of 100 mM FeSO 4 , (100 μM final) and 10 mL 1 M (NH 4 ) 2 SO 4 (10 mM final) was added. In the case of FIG. 2 , 1 mM (NH 4 ) 2 SO 4 , was used to induce mcl-PHA production.
Example 3
Growth Curves and Growth Rate Analysis
Growth curves were generated using a Bioscreen C automated microbiology growth curve analysis system from Growth Curves USA. Overnight cultures were diluted to 0.02 OD in a total volume of 300 μL per well. Incubations were performed at 30° C. with continuous shaking and turbidity measurements (OD 420-580 ) were collected every 15 minutes for the duration of the experiments. Spectrophotometric blanks were subtracted from the measured values and each growth curve represents the average of three independent cultures. The maximum specific growth rate (μ max ) was calculated as the maximum slope of the log phase of the growth curve over any given 4 hour period during the course of the experiment. In the case of the growths containing β-glucosidases, relevant cultures were spiked with twenty micrograms of agl ( Agrobacterium sp. β-glucosidase, Megazyme) prior to inoculation in Bioscreen C Honeycomb plates. Plates were held at 40° C. with shaking for 1 hour immediately prior to inoculation.
Example 4
Microscopic Visualization of mcl-PHA Production
One mL of saturated cultures was centrifuged at 5,000×g for 5 minutes. The growth medium was removed via aspiration and the cell pellet was washed twice in phosphate-buffered saline (PBS). Cells were suspended in 100 μL of PBS with 10 μg/mL Nile Red (Molecular Probes, ThermoFisher) and incubated for 15 minutes at room temperature in the dark. Cells were centrifuged, washed once in PBS and immobilized on microscope slides by mixing with 1% low-melting-temperature agarose in a 1:1 ratio. Images were acquired using a Nikon Eclipse 80i microscope. Nile Red fluorescence was detected between 560 and 590 nm using band-pass filtering.
Example 5
High Performance Liquid Chromatography
Following the β-glucosidase reaction period, the tubes were placed on ice, filtered through a 0.2 μm filter and analyzed via High-Performance Liquid Chromatography (HPLC; Agilent 1100 series system (Agilent USA, Santa Clara, Calif.)) using a Shodex SP0810 carbohydrate column with de-ashing guard cartridges (BioRad Laboratories, Hercules, Calif.) run at 85° C. with ultra-pure water as the isocratic mobile phase at a flow rate of 0.6 mL/min. A refractive index detector was used for compound detection. By-products were identified by co-elution at the same retention time with pure compounds. Standard curves for substrate (cellobiosan) and products (glucose and levoglucosan) were also generated in order to quantify results. Additionally, enzymes were analyzed via HPLC in buffer alone (without cellobiosan) to ensure no carry-over products inherent to the enzyme preparations.
Example 6
β-Glucosidase Mediated Cleavage of Cellobiosan
The enzymes listed in Table 1 below were used in the cellobiosan cleavage reactions.





TABLE 1



Tested β-glucosidases









GH
GenBank


Enzyme
Organism
Family
Accession No.
Seq ID No.



bgl1

A. niger

GH3
AJ132386
SEQ ID NO: 3

bgl1A

P. chrysosporium

GH3
AAC26489
SEQ ID NO: 4

bglA

T. maritima

GH1
CAA52276.1
SEQ ID NO: 5

abg
Agrobacterium sp.
GH1
AAA22085.1
SEQ ID NO: 6

























Each reaction was set up in 1.5 mL microcentrifuge tubes using cellobiosan at a concentration of 2 mg/mL in the following buffers: 50 mM sodium maleate, pH 6.5 ( T. maritima and Agrobacterium sp.), and 100 mM sodium acetate, pH 5.0 ( P. chrysosporium and A. niger ). For each reaction, 2.5 to 20 μg of enzyme was loaded and the total reaction volume was 400 μL. An additional enzyme-free reaction was run simultaneously to ensure cellobiosan cleavage was enzyme-dependent. Reactions were incubated at 40° C. using a dry-block for 90 minutes. In a separate reaction evaluating the substrate concentration, 0.125 μM of purified A. niger β-glucosidase was incubated at 40° C. in a 96-well microtiter plate with 0, 0.5, 1, 2, 4, 8, 16 or 32 mM cellobiose or cellobiosan. Reactions were initiated by the addition of enzyme and quenched by boiling in a thin walled PCR tube at 0.5, 1, 5 and 10 minutes.
Following the reaction periods, the tubes were placed on ice, filtered through a 0.2 filter and analyzed via High-Performance Liquid Chromatography (HPLC; Agilent1100 series system (Agilent USA, Santa Clara, Calif.)) using a Shodex SP0810 carbohydrate column with de-ashing guard cartridges (BioRad Laboratories, Hercules, Calif.) run at 85° C. with ultra-pure water as the isocratic mobile phase at a flow rate of 0.6 ml min −1 . A refractive index detector was used for compound detection. By-products were identified by co-elution at the same retention time with pure compounds. Standard curves for substrate (cellobiosan) and products (glucose and levoglucosan) were also generated in order to quantify results. Additionally, enzymes were analyzed via HPLC in buffer alone (without the addition of cellobiosan) to ensure no carry-over products inherent to the enzyme preparations.
In order to visualize the data more effectively, numbers were converted to molarity (as depicted in FIGS. 3 and 4 ). Additionally, cellobiosan molarity numbers were doubled in order to show the total molarity of monosaccharide units. The results of this assay show that β-glucosidases are capable of hydrolyzing the β-1,4-glycosidic bond in cellobiosan to liberate glucose and levoglucosan as products. Additionally, it demonstrates that this activity is conserved from β-glucosidases derived from multiple GH families (GH1 and GH3), derived from both prokaryotic ( Agrobacterium sp. and T. maritima ) and eukaryotic organisms ( P. chrysosporium and A. niger ), and from mesophilic ( A. niger and Agrobacterium sp.) thermophilic ( P. chrysosporium ) and hyperthermophilic organisms ( T. maritima ).
The exemplary enzymes comprise two representatives each from GH Families 1 and 3 as well as two fungal and two bacterial enzymes: Aspergillus niger bgl1 (GH Family 3), Phanerochaete chrysosporium bgl1A (GH Family 3, Thermotoga maritima bglA (GH Family 1), and Agrobacterium sp. abg (GH Family 1). As shown in FIG. 5 , all four β-glucosidases are able to cleave cellobiosan to generate glucose and levoglucosan at four different enzyme loadings. HPLC analysis shows the disappearance of cellobiosan and the appearance of glucose and levoglucosan with the addition of beta-glucosidase enzymes to the reaction. Percent utilization is shown wherein the cellobiosan molarity is reported as double its actual value to represent the molarity of the individual monosaccharide components (glucose plus levoglucosan) for clarity in comparisons.
Example 7
β-Glucosidase Enables Growth of P Putida-lgk on Cellobiosan
P. putida strains FJPO3 (which expresses the levoglucosan kinase (lgk) gene; see Example 1) and KT2440 were grown using various carbon sources with and without the addition of the Agrobacterium sp. abg β-glucosidase As shown in FIG. 6 , growth profiles of KT2440 and FJPO3 are virtually indistinguishable in M9 medium containing cellobiose with the addition of abg, demonstrating equivalent glucose usage between the strains (Panel A). Conversely, in the cellobiosan plus abg conditions, while both FJPO3 and KT2440 are capable of utilizing the glucose component of cellobiosan, FJPO3 continues growth at or near the time when KT2440′s growth ceases. No growth of either strain was observed in either cellobiose or cellobiosan without the addition of abg. For clarity, these data are not depicted on the growth curves of Panel A. The maximum specific growth rates (μ max ) for all conditions are shown in Panel B, along with the max growth rate in M9 media containing levoglucosan and glucose as the sole carbon source.
Example 8
Generation and Fractionation of Pyrolysis Oils
Pyrolysis oils may be generated by many means, including through the scheme set forth in FIG. 8 . Examples can be found in Rover et al., ChemSusChem 7:1662-1668 (2014) or Pollard et al., Journal of Analytical and Applied Pyrolysis 93:129-138 (2012), among others.
The Examples discussed above are provided for purposes of illustration and are not intended to be limiting. Still other embodiments and modifications are also contemplated.
While a number of exemplary aspects and embodiments have been discussed above, those of skill in the art will recognize certain modifications, permutations, additions and sub combinations thereof. It is therefore intended that the following appended claims and claims hereafter introduced are interpreted to include all such modifications, permutations, additions and sub-combinations as are within their true spirit and scope.