(en)A method for producing an L-amino acid comprising: a) cultivating in a medium containing glycerol a recombinant coryneform bacteria which produces the desired L-amino acid and which expresses at least one heterologous polynucleotide of glycerol metabolism, such as glpA, glpB, glpC, glpD, glpE, glpF, glpG, glpK, glpQ, gipT, glpX, gldA, dhaK, dhaL, dhaM, dhaR, fsa, and/or talC, and b) isolating the desired L-amino acid. Preferably, the pathways producing the desired L-amino acid in the coryneform bacteria are amplified.

1.ApplicationNumber: US-81653906-A
1.PublishNumber: US-9150827-B2
2.Date Publish: 20151006
3.Inventor: WENDISCH VOLKER F.
RITTMANN DORIS
SAHM HERMANN
KREUTZER CAROLINE

4.Inventor Harmonized: WENDISCH VOLKER F(DE)
RITTMANN DORIS(DE)
SAHM HERMANN(DE)
KREUTZER CAROLINE(DE)

5.Country: US
6.Claims:
(en)A method for producing an L-amino acid comprising: a) cultivating in a medium containing glycerol a recombinant coryneform bacteria which produces the desired L-amino acid and which expresses at least one heterologous polynucleotide of glycerol metabolism, such as glpA, glpB, glpC, glpD, glpE, glpF, glpG, glpK, glpQ, gipT, glpX, gldA, dhaK, dhaL, dhaM, dhaR, fsa, and/or talC, and b) isolating the desired L-amino acid. Preferably, the pathways producing the desired L-amino acid in the coryneform bacteria are amplified.

7.Description:
(en)The invention relates to recombinant coryneform bacteria in which is (are) expressed at least one or more of the heterologous genes of glycerol metabolism selected from the group glpA, glpB, glpC, glpD, glpE, glpF, glpG, glpK, glpQ, glpT, glpX, gldA, dhaK, dhaL, dhaM, dhaR, fsa and talC, and also to a method for the fermentative production of L-amino acids, in particular L-lysine and L-tryptophan, with the medium containing glycerol as carbon source, employing these bacteria. These bacteria exhibit the ability for utilization of glycerol and thereby for effective formation and accumulation of L-amino acids.
PRIOR ART
Chemical compounds, which are taken to mean, in particular, L-amino acids, vitamins, nucleosides and nucleotides and D-amino acids, are employed in human medicine, in the pharmaceutical industry, in cosmetics, in the food industry and in animal nutrition.
Many of these compounds are produced by fermentation of strains of coryneform bacteria, in particular Corynebacterium glutamicum . Because of the great importance, efforts are continuously being made to improve the production methods. Method improvements can relate to fermentation-related measures such as, for example, stirring and supply with oxygen, or the composition of the nutrient media such as, for example, the sugar concentration during fermentation, or workup to give the product form by, for example, ion-exchange chromatography, or the intrinsic performance characteristics of the microorganism itself.
To improve the performance characteristics of these bacteria, methods of mutagenesis, selection and mutant isolation are employed. In this manner strains are obtained which are resistant to antimetabolites such as the lysine analog 5-(2-aminoethyl)cysteine or the tryptophan analog 5-fluorotryptophan, for example, or are auxotrophic for metabolites of regulatory importance and produce L-amino acids.
For some years, methods of recombinant DNA technology have likewise been used for strain improvement of L-amino acid-producing strains of Corynebacterium glutamicum , by amplifying individual amino acid biosynthesis genes and studying the effect on L-amino acid production. A summarizing survey of very varied aspects of the genetics, metabolism and biotechnology of Corynebacterium glutamicum may be found in Pühler ((chief ed.) Journal of Biotechnology 104 (1-3), 1-338 (2003)) and Eggeling and Bott ((editors) Handbook of Corynebacterium glutamicum , CRC Press, Taylor & Francis Group, Boca Raton (2005)).
The entire fermentation industry for producing L-amino acids currently employs chiefly glucose or sucrose as carbon source, which sugars are obtained in the methoding of agricultural products. However, since the price of these carbon sources is increasing, an alternative which can be carried out by technological means for producing the L-amino acids, preferably L-lysine and L-tryptophan, the utilization of an inexpensive material as an alternative raw material for fermentation, is desirable.
Glycerol (propanetriol) is a natural component of oils and fats and, as a “bridge”, binds the fatty acid molecules in the triglycerides. The glycerol molecule is highly polar and therefore readily water soluble. As a coupling product, this valuable raw material is formed in biodiesel production (for example for rapeseed oil methyl ester, RME) and is used in cosmetics, pharmaceutical products, foods and for technical applications. A critical factor for the use of glycerol as a raw material for producing feedstuff components is its cheapness. It may be assumed that with increasing biodiesel production, glycerol will become of greater interest for production of feedstuff additives.
Corynebacterium glutamicum wild type uses a multiplicity of monomeric and oligomeric sugars such as glucose, sucrose or maltose as carbon source (Vahjen et al., FEMS Microbiology Ecology 18: 317-328 (1995)), but does not grow on glycerol as sole carbon source.
Corynebacterium glutamicum wild type possesses some genes having homology to known genes of glycerol metabolism, but it has not yet been possible to date to explain why growth on glycerol is nevertheless not possible.
OBJECT OF THE INVENTION
The inventors have set themselves the object of providing novel coryneform bacteria which are able to use glycerol as far as possible as sole carbon source. A further object in immediate conjunction therewith was to provide an improved method for fermentative production of L-amino acids, in particular L-lysine and L-tryptophan with the aid of such coryneform bacteria. In particular, glycerol should be made usable thereby for fermentative production of L-amino acids in the most economical manner possible.


DESCRIPTION OF THE INVENTION
This object and also other objects which are not explicitly mentioned but which can be derived or concluded without problems from the facts discussed herein are achieved by provision of coryneform bacteria according to embodiment 1 and also a method according to embodiment 19.
Embodiment 1
A recombinant coryneform bacterium in which is (are) expressed at least one or more of the heterologous polynucleotides of glycerol metabolism selected from the group glpA, glpB, glpC, glpD, glpE, glpF, glpG, glpK, glpQ, glpT, glpX, gldA, dhaK, dhal, dhaM, dhaR, fsa and talC.
Embodiment 19
A method for the fermentative production of L-amino acids, characterized in that the following steps are carried out:
a) culturing the recombinant coryneform bacteria producing the desired L-amino acid as described in embodiments 1 to 18 in a medium containing glycerol or, if appropriate, in addition one or more further carbon sources under conditions in which the desired L-amino acid accumulates in the medium or in the cells, and, if appropriate, b) isolating the desired L-amino acid, in which if appropriate components of the fermentation broth and/or biomass remain in the end product in their totality or in fractions (>0 to 100%).

Expedient modifications and developments of the invention are protected in the subclaims which refer back to embodiment 1 or embodiment 19, respectively.
The invention relates to recombinant coryneform bacteria which, in particular, already excrete L-amino acids and in which is (are) expressed at least one or more of the nucleotide sequence(s) encoding the heterologous gene products of glycerol metabolism selected from the group glpA, glpB, glpC, glpD, glpE, glpF, glpG, glpK, glpQ, glpT, glpX, gldA, dhaK, dhaL, dhaM, dhaR, fsa and talC. These bacteria exhibit the ability to utilize glycerol.
The bacteria employed include, in particular, coryneform bacteria in which at least one heterologous polynucleotide is expressed which encodes a polypeptide the amino acid sequence of which is at least 80% identical, or at least 90%, in particular at least 95%, preferably at least 98%, particularly preferably at least 99%, and very particularly preferably 100% identical, with 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. 10, 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 and SEQ ID No. 36.
Said bacteria preferably contain at least one heterologous polynucleotide selected from the group consisting of:
a) polynucleotide having the nucleotide sequence SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, 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 or SEQ ID No. 35 and nucleotide sequences complementary thereto b) polynucleotide having a nucleotide sequence which, in the context of degeneracy of the genetic code, corresponds to SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, 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 or SEQ ID No. 35; c) polynucleotide sequence having a sequence which hybridizes under stringent conditions to the sequence complementary to sequence SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, 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 or SEQ ID No. 35, in which the stringent conditions are preferably achieved by a wash step, in which the temperature varies over a range from 64° C. to 68° C. and the salt concentration of the buffer varies over a range from 2×SSC to 0.1×SSC; d) polynucleotide having a sequence SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, 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 or SEQ ID No. 35, which contains functionally neutral sense mutations,

the polynucleotides encoding enzymes of glycerol metabolism.


The invention likewise relates to a method for the fermentative production of L-amino acids, in particular L-lysine and L-tryptophan with the medium containing glycerol as carbon source, with employment of recombinant coryneform bacteria which in particular already produce L-amino acids and in which at least one or more of the heterologous genes of glycerol metabolism selected from the group glpA, glpB, glpC, glpD, glpE, glpF, glpG, glpK, glpQ, glpT, glpX, gldA, dhaK, dhaL, dhaM, dhaR, fsa and talC or nucleotide sequences encoding their gene products is or are expressed.
Preferably, use is made of the bacteria according to the invention.
When hereinafter L-amino acids or amino acids are mentioned, they are to be taken to mean one or more of the proteinogenic amino acids including salts thereof selected from the group L-aspartic acid, L-asparagine, L-threonin, L-serine, L-glutamic acid, L-glutamine, L-glycine, L-alanine, L-cysteine, L-valine, L-methionin, L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-histidine, L-lysine, L-tryptophan, L-arginine and L-proline. Particular preference is given to L-lysine and L-tryptophan. The L-amino acids also include L-homoserine.
Proteinogenic amino acids are taken to mean the amino acids which occur in natural proteins, that is to say in proteins from microorganisms, plants, animals and humans.
Where amino acids are mentioned hereinafter, the term also comprises salts thereof such as, for example, lysine monohydrochloride or lysine sulfate, in the case of the amino acids L-lysine.
“Heterologous genes” or “heterologous nucleotide sequences” according to the invention can originate from any prokaryotic donor organism apart from representatives of the genus Corynebacterium . Preferably, use is made of the genes from Escherichia coli.
The phrase “expression of heterologous genes” in this context describes the cloning of corresponding genes and their expression in the heterologous system which leads to establishment of intracellular activity or concentration of one or more enzymes or proteins in a microorganism which are encoded by the corresponding DNA, by, for example, generating a vector which contains the desired gene or an allele of this gene and a promoter making possible the expression of the gene and transfers it into the microorganism by transformation, transduction or conjugation and, if appropriate, combines these measures.
Alleles are taken to mean alternative forms of a given gene. The forms are distinguished by differences in the nucleotide sequence.
The protein or the ribonucleic acid encoded by a nucleotide sequence, that is to say a gene or an allele, is termed gene product.
This invention also relates to a method for the fermentative production of L-amino acids, characterized in that the following steps are carried out:
a) culturing the recombinant coryneform bacteria producing the desired L-amino acid in which at least one or more of the heterologous genes of glycerol metabolism selected from the group glpA, glpB, glpC, glpD, glpE, glpF, glpG, glpK, glpQ, glpT, glpX, gldA, dhaK, dhaL, dhaM, dhaR, fsa and talC or nucleotide sequences or alleles encoding their gene products is or are expressed in a medium containing glycerol or, if appropriate, in addition one or more further carbon sources under conditions in which the desired L-amino acid accumulates in the medium or in the cells, and if appropriate, b) isolating the desired L-amino acid, in which if appropriate components of the fermentation broth and/or biomass remain in the end product in their totality or in fractions (>0 to 100%).

The coryneform bacteria used preferably already produce L-amino acids, in particular L-lysine and L-tryptophan, on conventional carbon sources such as, for example, glucose or sucrose, before the expression of one or more of the genes of glycerol metabolism. The glycerol used can be used individually or as a mixture, wherein the fraction of the glycerol should preferably be >10 to 100%.
It has been found that coryneform bacteria after heterologous expression of one or more of the genes of glycerol metabolism produce L-amino acids, in particular L-lysine and L-tryptophan, from glycerol as sole carbon source.
Recombinant bacteria of the invention are generated, for example, by transformation, transduction or conjugation, or a combination of these methods using a vector which contains the desired gene, an allele of this gene or parts thereof and a promoter making possible the expression of the gene. The heterologous expression is achieved, in particular, by integration of the gene or of the alleles into the chromosome of the microorganisms or an extrachromosomally replicating vector.
The promoter can be the inherent regulatory sequence situated upstream of the gene, or a promoter from coryneform bacteria is fused with the gene. An overview of known promoters from Corynebacterium glutamicum is described by Pátek et al. (Journal of Biotechnology 104, 311-323 (2003)).
The bacteria on which the measures of the invention are carried out and which are thereby starting point of the present invention can produce amino acids from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose, or from ethanol. These are representative of coryneform bacteria.
Among the coryneform bacteria, preference is given to the genus Corynebacterium . Particular preference is given to amino acid-excreting strains which are based on the following species:
Corynebacterium efficiens, such as, for example, strain DSM44549, Corynebacterium glutamicum , such as, for example, strain ATCC13032, Corynebacterium thermoaminogenes , such as, for example, strain FERM BP-1539, and Corynebacterium ammoniagenes, such as, for example, strain ATCC6871,

very particular preference being given to the species Corynebacterium glutamicum.


Some representatives of the species Corynebacterium glutamicum are also known under other species names in the prior art. These include, for example:
Corynebacterium acetoacidophilum ATCC13870 Corynebacterium lilium DSM20137 Corynebacterium melassecola ATCC17965 Brevibacterium flavum ATCC14067 Brevibacterium lactofermentum ATCC13869 and Brevibacterium divaricatum ATCC14020

Known representatives of amino acid-excreting strains of coryneform bacteria are, for example
the L-lysine-producing strains




Corynebacterium glutamicum DM58-1/pDM6 (=DSM4697) described in EP 0 358 940
Corynebacterium glutamicum MH20 (=DSM5714) described in EP 0 435 132
Corynebacterium glutamicum AHP-3 (=FermBP-7382) described in EP 1 108 790
Corynebacterium thermoaminogenes AJ12521 (═FERM BP-3304) described in U.S. Pat. No. 5,250,423

or the L-tryptophan-producing strains

Corynebacterium glutamicum K76 (=FermBP-1847) described in U.S. Pat. No. 5,563,052
Corynebacterium glutamicum BPS13 (=FermBP-1777) described in U.S. Pat. No. 5,605,818
Corynebacterium glutamicum FermBP-3055 described in U.S. Pat. No. 5,235,940




Information on taxonomic classification of strains of this group of bacteria may be found, inter alia, in Seiler (Journal of General Microbiology 129, 1433-1477 (1983)), Kämpfer and Kroppenstedt (Canadian Journal of Microbiology 42, 989-1005 (1996)), Liebl et al (International Journal of Systematic Bacteriology 41, 255-260 (1991)) and in U.S. Pat. No. 5,250,434.
Strains having the designation “ATCC” can be obtained from the American Type Culture Collection (Manassas, Va., USA). Strains having the designation “DSM” can be obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Brunswick, Germany). Strains having the designation “FERM” can be obtained from the National Institute of Advanced Industrial Science and Technology (AIST Tsukuba Central 6, 1-1-1 Higashi, Tsukuba Ibaraki, Japan). Said strain of Corynebacterium thermoaminogenes (FERM BP-1539) is described in U.S. Pat. No. 5,250,434.
The nucleotide sequences of the genes or open reading frames (ORF) of Escherichia coli are part of the prior art and can be taken from the genome sequence of Escherichia coli published by Blattner et al. (Science 277: 1453-1462 (1997)).
The nucleotide sequence for the genes glpA, glpB, glpC, glpD, glpE, glpF, glpG, glpK, glpQ, glpT and glpX, for example, are likewise known from Shigella flexneri (Accession No.: NC 004337 (sequence of the entire genome)) and Salmonella typhimurium (Accession No.: NC 003197 (sequence of the entire genome)) likewise belonging to the family Enterobacteriaceae. In addition, from Salmonella typhimurium (Accession No.: NC 003197 (sequence of the entire genome)) the nucleotide sequence for the genes gldA and talC, and from Shigella flexneri (Accession No.: NC 004337 (sequence of the entire genome)) the nucleotide sequence for the genes dhaK, dhaL, dham, dhaR and fsa is known.
The genes and activities of glycerol metabolism are also described as a summary in Lin (in: Neidhardt (ed), Escherichia coli and Salmonella , American Society for Microbiology, Washington, D.C., USA: 307-342 (1996)).
The glycerophosphate regulon (glp) containing the genes of glycerol transport and metabolism consists of five operons which lie on three different gene sites on the E. coli chromosome (Cozzarelli et al., Journal of Molecular Biology 31: 371-387 (1968)).
A regulon is a unit of genes which, although they are localized at various sites of a genome, their expression is controlled by the same regulator proteins. An operon is a unit of jointly regulated genes at one gene site.
The operon glpFKX (Cozzarelli and Lin, Journal of Bacteriology 91: 1763-1766 (1966); Weissenborn et al., Journal of Biological Chemistry 267: 6122-6131 (1992)), which encodes the glycerol facilitator GlpF and the glycerol kinase GlpK and a fructose-1,6-bisphosphatase II GlpX (Donahue et al., Journal of Bacteriology 182 (19): 5624-5627 (2000)) is situated at 88 min. In the operon mapped at 49 min, the genes glpT (glycerol-3-phosphate permease) and glpQ (periplasmic glycerol phosphodiesterase) are associated which are transcribed in the counterclockwise direction. The glpABC operon is arranged in the opposite direction and its genes encode the three subunits of the heterotrimer sn-glycerol-3-phosphate-dehydrogenase, active in the absence of atmospheric oxygen (anaerobic) (Cole et al., Journal of Bacteriology 170: 2448-2456 (1988); Ehrmann et al., Journal of Bacteriology 169: 526-532 (1987)). The glpDEG operon is situated at 75 min on the chromosome and encodes the glycerol-3-phosphate-dehydrogenase GlpD active in the presence of atmospheric oxygen (aerobic) (Cozzarelli et al., Journal of Molecular Biology 31: 371-387 (1968)), the sulfur transferase GlpE (Cozzarelli et al., Journal of Molecular Biology 31: 371-387 (1968)) and the glpG gene having an unknown function (Zeng et al., Journal of Bacteriology 178: 7080-7089 (1996)).
A short summary of the genes and activities of glycerol metabolism is given by the following listing:
glpA Gene:


Description: large subunit of the sn-glycerol-3-phosphate dehydrogenase (anaerobic)
Function: In an anaerobic environment, glycerol-3-phosphate, for energy production, is oxidized by an FAD-dependent glycerol-3-phosphate dehydrogenase (GlpABC) to dihydroxyacetone phosphate which can take part in glycolysis as an intermediate. The reduction equivalents released in this oxidation reaction are transferred by the flavoenzyme to a membrane-associated cytochrome complex, wherein fumarate or nitrate acts as terminal electron acceptor (Lin, in: Neidhardt (ed), Escherichia coli and Salmonella , American Society for Microbiology, Washington, D.C., USA: 307-342 (1996)). While the electrons are passed through the cytochrome complex, the energy being released is utilized for pumping protons through the membrane from the cytoplasmic side to the periplasmic side. With the proton gradient generated at the membrane, both the electrical and chemical potential change, which drives the membrane-bound ATPase and as a result ATP can be generated.
EC No.: 1.1.99.5
Reference: Blattner et al.; Science 277(5331): 1453-1474 (1997)
SEQ ID No.: 1
Accession No.: U00096 (region: 2350669-2352297)
Alternative gene name: b2241

glpB Gene:

Description: subunit for the membrane anchor of sn-glycerol-3-phosphate dehydrogenase (anaerobic)
Function: see glpA
EC No.: 1.1.99.5
Reference: Blattner et al.; Science 277(5331): 1453-1474 (1997)
SEQ ID No.: 3
Accession No.: U00096 (region: 2352287-2353546)
Alternative gene name: b2242

glpC Gene:

Description: small subunit of sn-glycerol-3-phosphate dehydrogenase (anaerobic)
Function: see glpA
EC No.: 1.1.99.5
Reference: Blattner et al.; Science 277(5331): 1453-1474 (1997)
SEQ ID No.: 5
Accession No.: U00096 (region: 2353543-2354733)
Alternative gene name: b2243

glpD Gene:

Description: glycerol-3-phosphate dehydrogenase (aerobic)
Function: GlpD was identified as an aerobic glycerol-3-phosphate dehydrogenase (Cozzarelli et al., Journal of Molecular Biology 31: 371-387 (1968)). In an aerobic environment, glycerol-3-phosphate, for energy production, is oxidized by this FAD-dependent glycerol-3-phosphate dehydrogenase (GlpD) to dihydroxyacetone phosphate which can participate in glycolysis as an intermediate. The reduction equivalents released in this oxidation reaction are transferred by the flavoenzyme to a membrane-associated cytochrome complex, wherein molecular oxygen or nitrate acts as terminal electron acceptor (Lin, in: Neidhardt (ed), Escherichia coli and Salmonella , American Society for Microbiology, Washington, D.C., USA: 307-342 (1996)) While the electrons are passed through the cytochrome complex, the energy being released is used for pumping protons through the membrane from the cytoplasmic side to the periplasmic side. With the proton gradient generated at the membrane both the electrical and also the chemical potential change, which drives the membrane-bound ATPase and as a result ATP can be generated. The open reading frame of the glpD gene consists of 501 codons and the translated sequence encodes a protein having a molecular weight of 57 kDa (Austin and Larson, Journal of Bacteriology 173: 101-107 (1991)).
EC No.: 1.1.99.5
Reference: Blattner et al.; Science 277(5331): 1453-1474 (1997)
SEQ ID No.: 7
Accession No.: U00096 (region: 3560036-3561541)
Alternative gene name: b3426, glyD

glpE Gene:

Description: sulfur transferase; acidic, cytoplasmic rhodanese
Function: GlpE was identified as a sulfur transferase (Cozzarelli et al., Journal of Molecular Biology 31: 371-387 (1968)). The acidic cytoplasmic rhodanese encoded by the glpE gene having a molecular weight of 12 kDa catalyzes as dimer the transfer of sulfur to the sulfur acceptor thioredoxin 1 (Ray et al., Journal of Bacteriology 182: 2277-2284 (2000)).
EC No.: 2.8.1.1
Reference: Blattner et al.; Science 277(5331): 1453-1474 (1997)
SEQ ID No.: 9
Accession No.: U00096 (region: 3559520-3559846)
Alternative gene name: b3425

glpF Gene:

Description: glycerol facilitator GlpF
Function: The facilitated diffusion of glycerol from the nutrient medium is catalyzed by the glycerol facilitator GlpF (Borgnia and Agre, Proc. Natl. Acad. Sci. U.S.A. 98: 2888-2893 (2001)) which forms a substrate-specific channel having a pore size of 0.4 nm (Heller et al., Journal of Bacteriology 144: 274-278 (1980)).
EC No.: —
Reference: Blattner et al.; Science 277(5331): 1453-1474 (1997)
SEQ ID No.: 11
Accession No.: U00096 (region: 4115268-4116113)
Alternative gene name: b3927

glpG Gene:

Description: gene of the glp regulon
Function: The physiological function of glpG is still unknown. The glpG gene product is a basic cytoplasmic or membrane-associated protein having a molecular weight of 28 kDa (Zeng et al., Journal of Bacteriology 178: 7080-7089 (1996)).
EC No.: —
Reference: Blattner et al.; Science 277(5331): 1453-1474 (1997)
SEQ ID No.: 13
Accession No.: U00096 (region: 3558645-3559475)
Alternative gene name: b3424

glpK Gene:

Description: glycerol kinase GlpK (ATP-dependent)
Function: Cytoplasmic glycerol is immediately phosphorylated by the ATP-dependent glycerol kinase K which in its enzymatically active form is present associated with the glycerol facilitator GlpF (Voegele et al., Journal of Bacteriology 175: 1087-1094 (1993)).
EC No.: 2.7.1.30
Reference: Blattner et al.; Science 277(5331): 1453-1474 (1997)
SEQ ID No.: 15
Accession No.: U00096 (region: 4113737-4115245)
Alternative gene name: b3926

glpQ Gene:

Description: Glycerol phosphodiesterase
Function: Glycerophosphate diesters, the deacetylated breakdown product of phospholipids (Lin, in: Neidhardt (ed), Escherichia coli and Salmonella , American Society for Microbiology, Washington, D.C., USA: 307-342 (1996)), are hydrolyzed in the periplasma by the phosphodiesterase GlpQ localized there to form alcohol and glycerol-3-phosphate (Larson et al., Journal of Biological Chemistry 258: 5428-5432 (1983)). Since the glycerol phosphodiesterase is thus ascribed an extracytoplasmic action the gene product derived from the heterologous glpQ gene in the claimed bacteria must comprise a leader peptide, as is typical of excreted proteins of Gram-positive bacteria (Nielsen et al., Protein Engineering Design and Selection 10:1-6 (1997)). This characteristic signal sequence enables export of the GlpQ protein mediated by the general secretion system (Sec system) via the cytoplasmic membrane (Kell and Young, Current opinion in microbiology 3:238-243 (2000); Mukamolova et al., Molecular Microbiology 46:611-621 (2002a); Molecular Microbiology 46:613-635 (2002b)).
EC No.: 3.1.4.46
Reference: Blattner et al.; Science 277(5331): 1453-1474 (1997)
SEQ ID No.: 17
Accession No.: U00096 (region: 2347957-2349033)
Alternative gene name: b2239

glpT Gene:

Description: Glycerol-3-phosphate permease
Function: Glycerol-3-phosphate is transported by the permease GlpT (Eiglmeier et al., Molecular Microbiology 1: 251-258 (1987); Larson et al., Journal of Bacteriology 152: 1008-1021 (1982)) into the cell interior in an exchange for inorganic phosphate (Auer et al., Biochemistry 40: 6628-6635 (2001)) The energy required for the transport is provided by this antiport and at the same time accumulation of the toxic phosphate is prevented (Xavier et al., Journal of Bacteriology 177: 699-704 (1995)).
EC No.: —
Reference: Blattner et al.; Science 277(5331): 1453-1474 (1997)
SEQ ID No.: 19
Accession No.: U00096 (region: 2349038-2350396)
Alternative gene name: b2240

glpX Gene:

Description: Fructose-1,6-bisphosphatase II
Function: To date, no physiological role of the enzyme GlpX has been found. A mutation in fbp, the gene for fructose-1,6-bisphosphatase I, was not complemented by GlpX. However, a functional importance nevertheless appears certain, since mutants which have a fault in glycolysis owing to a pfka (phosphofructokinase A) mutation grow much more slowly without GlpX (Donahue et al.; Journal of Bacteriology 182(19): 5624-5627 (2000)).
EC No.: 3.1.3.11
Reference: Blattner et al.; Science 277(5331): 1453-1474 (1997)
SEQ ID No.: 21
Accession No.: U00096 (region: 4112592-4113602)
Alternative gene name: 3925


In addition to the genes of glycerol transport and metabolism, the following genes of the breakdown of various intermediates and end products of said metabolism can be expressed heterologously:
gldA Gene:


Description: Glycerol dehydrogenase (NAD)
Function: Glycerol dehydrogenase catalyzes the reversible NAD-dependent reaction of glycerol to form dihydroxyacetone (Truniger and Boos, Journal of Bacteriology 176(6): 1796-1800 (1994)).
EC No.: 1.1.1.6
Reference: Blattner et al.; Science 277(5331): 1453-1474 (1997)
SEQ ID No.: 23
Accession No.: U00096 (region: 4135955-4137097)
Alternative gene name: b3945

dhaK Gene:

Description: Subunit of dihydroxyacetone kinase, N-terminal domain
Function: Dihydroxyacetone kinase, in its function as phosphoenolpyruvate (PEP)-dependent dihydroxyacetone phosphotransferase, catalyzes the reaction of dihydroxy-acetone to form dihydroxyacetone phosphate (Gutknecht et al., The EMBO Journal 20(10): 2480-2486 (2001)). DhaK carries the dihydroxyacetone binding site.
EC No.: 2.7.1.29
Reference: Blattner et al.; Science 277(5331): 1453-1474 (1997)
SEQ ID No.: 25
Accession No.: U00096 (region: 1248991-1250091)
Alternative gene name: dhaK1, ycgT, b1200

dhaL Gene:

Description: Subunit of dihydroxyacetone kinase, C-terminal domain
Function: Dihydroxyacetone kinase, in its function as a PEP-dependent dihydroxyacetone phosphotransferase, catalyzes the reaction of dihydroxyacetone to form dihydroxyacetone phosphate (Gutknecht et al., The EMBO Journal 20(10): 2480-2486 (2001)). DhaL carries ADP as cofactor for the transfer of phosphate from DhaM to dihydroxyacetone.
EC No.: 2.7.1.29
Reference: Blattner et al.; Science 277(5331): 1453-1474 (1997)
SEQ ID No.: 27
Accession No.: U00096 (region: 1248348-1248980)
Alternative gene name: dhaK2, ycgS, b1199

dhaM Gene:

Description: PTS protein subunit of dihydroxyacetone kinase, multiphosphoryl transfer protein
Function: Dihydroxyacetone kinase, in its function as a PEP-dependent dihydroxyacetone phosphotransferase, catalyzes the reaction of dihydroxyacetone to form dihydroxyacetone phosphate (Gutknecht et al., The EMBO Journal 20(10): 2480-2486 (2001)). DhaM consists of three domains having a similarity to the three domains of the phosphoenolpyruvate-dependent phosphotransferase system. Phosphorylated DhaM transfers the phosphate to the DhaL-bound ADP.
EC No.: 2.7.1.29
Reference: Blattner et al.; Science 277(5331): 1453-1474 (1997)
SEQ ID No.: 29
Accession No.: U00096 (region: 1246919-1248340)
Alternative gene name: dhaH, ycgC, b1198

dhaR Gene:

Description: Activator of the dha operon (dhaKLM)
Function: Transcriptional activator, stimulates the transcription of the dha operon from a sigma70 promoter (Bachler et al., The EMBO Journal 24(2): 283-293 (2005))
EC No.: —
Reference: Blattner et al.; Science 277(5331): 1453-1474 (1997)
SEQ ID No.: 31
Accession No.: U00096 (region: 1250280-1252208)
Alternative gene name: ycgU, b1201

fsa Gene:

Description: Fructose-6-phosphate aldolase I
Function: Fructose-6-phosphate aldolase I catalyzes an aldol cleavage of fructose-6-phosphate, substrates of the enzyme are dihydroxyacetone and also fructose-6-phosphate and glyceraldehyde-3-phosphate, and does not utilize fructose, fructose-1-phosphate, fructose-1,6-bisphosphate or dihydroxy-acetone phosphate (Schurmann and Sprenger, Journal of Biological Chemistry 276(14): 11055-11061 (2001))
EC No.: 4.1.2.—
Reference: Blattner et al.; Science 277(5331): 1453-1474 (1997)
SEQ ID No.: 33
Accession No.: U00096 (region: 862793-863527)
Alternative gene name: mipB, ybiZ, B0825

talC Gene:

Description: Fructose-6-phosphate aldolase II
Function: Fructose-6-phosphate aldolase II catalyzes an aldol cleavage of fructose-6-phosphate (Schurmann and Sprenger, Journal of Biological Chemistry 276(14): 11055-11061 (2001)). The talC gene is situated directly next to the gldA gene.
EC No.: 4.1.2.—
Reference: Blattner et al.; Science 277(5331): 1453-1474 (1997)
SEQ ID No.: 35
Accession No.: U00096 (region: 4137069-4137731)
Alternative gene name: fsaB, yijG, b3946


The nucleic acid sequences can be taken from the databases of the National Center for Biotechnology Information (NCBI) of the National Library of Medicine, (Bethesda, Md., USA), the nucleotide sequence database of the European Molecular Biologies Laboratories (EMBL, Heidelberg, Germany and Cambridge, UK) or the DNA database of Japan (DDBJ, Mishima, Japan).
For the sake of better clarity, the known sequences for the genes in question of Escherichia coli are described SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, 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 and SEQ ID No. 35.
The open reading frames described in the text positions cited can be used according to the invention. In addition, use can be made of alleles of the genes or open reading frames which result from the degeneracy of the genetic code or owing to functionally neutral sense mutations.
Alleles of the treated genes which contain functionally neutral sense mutations include, inter alia, those which lead to at most 13, or at most 10, preferably at most 7, or at most 5, very particularly preferably at most 3 or at most 2, or to at least one conservative amino acid replacement in the protein encoded by them.
Among the aromatic amino acids, conservative replacements are considered to be when phenylalanine, tryptophan and tyrosine are exchanged for one another. Among the hydrophobic amino acids, conservative replacements are considered to be when leucine, isoleucine and valine are exchanged for one another. Among the polar amino acids, conservative replacements are considered to be when glutamine and asparagine are exchanged for one another. Among the basic amino acids, conservative replacements are considered to be when arginine, lysine and histidine are exchanged for one another. Among the acidic amino acids, conservative replacements are considered to be when aspartic acid and glutamic acid are exchanged for one another. Among the hydroxyl-containing amino acids, conservative replacements are considered to be when serine and threonine are exchanged for one another.
In the same manner, use can also be made of those nucleotide sequences which encode variants of said proteins which, in addition at the N or C terminus have an extension or shortening by at least one (1) amino acid. This extension or shortening is no more than 13, 10, 7, 5, 3 or 2 amino acids or amino acid radicals.
It is known that host-specific enzymes, what are termed aminopeptidases, remove the terminal methionine in protein synthesis.
The suitable alleles also include those which encode proteins in which at least one (1) amino acid is inserted or deleted. The maximum number of such changes termed indels can be 2, 3, 4, 5, but in no case more than 6 amino acids.
The suitable alleles include, in addition, those which are obtainable by hybridization, in particular under stringent conditions or use of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, 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 or SEQ ID No. 35 or parts thereof, in particular the coding regions, or of the sequences complementary thereto.
Instructions for identifying DNA sequences by means of hybridization may be found by those skilled in the art, inter alia, in the handbook “The DIG System Users Guide for Filter Hybridization” from Boehringer Mannheim GmbH (Mannheim, Germany, 1993) and in Liebl et al. (International Journal of Systematic Bacteriology 41: 255-260 (1991)). Hybridization takes place under stringent conditions, that is to say hybrids are formed only for which probe and target sequence, that is to say the polynucleotides treated with the probe, are at least 70% identical. It is known that the stringency of hybridization including the wash steps is affected or determined by varying the buffer composition, the temperature and the salt concentration. The hybridization reaction is generally carried out at relatively low stringency compared with the wash steps (Hybaid Hybridisation Guide, Hybaid Limited, Teddington, UK, 1996).
For the hybridization reaction, for example use can be made of a buffer equivalent to 5×SSC buffer at a temperature of approximately 50° C.-68° C. In this case probes can also hybridize with polynucleotides which have less than 70% identity to the sequence of the probe. Such hybrids are less stable and are removed by washing under stringent conditions. This can be achieved, for example, by lowering the salt concentration to 2×SSC and if appropriate subsequently 0.5×SSC (The DIG System User's Guide for Filter Hybridisation, Boehringer Mannheim, Mannheim, Germany, 1995), a temperature of approximately 50° C.-68° C., approximately 52° C.-68° C., approximately 54° C.-68° C., approximately 56° C.-68° C., approximately 58° C.-68° C., approximately 60° C.-68° C., approximately 62° C.-68° C., approximately 64° C.-68° C., approximately 66° C.-68° C. being set. Temperature ranges of approximately 64° C.-68° C. or approximately 66° C.-68° C. are preferred. It is if appropriate possible to lower the salt concentration to a concentration equivalent to 0.2×SSC or 0.1×SSC. By stepwise elevation of the hybridization temperature in steps of approximately 1-2° C. from 50° C. to 68° C., polynucleotide fragments can be obtained which have, for example, at least 70%, or at least 80% or 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%, or at least 99% identity to the sequence of the probe used or to the nucleotide sequences represented in SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, 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 or SEQ ID No. 35. Further guidance on hybridization is obtainable on the market in the form called kits (for example DIG Easy Hyb from Roche Diagnostics GmbH, Mannheim, Germany, Catalog No. 1603558).
Use can also be made of polynucleotides from Bacillus subtilis (Accession No.: NC 000964 (sequence of the entire genome)) and Streptomyces coelicolor (Accession No.: NC 003888 (sequence of the entire genome)) which have, for example, at least 70%, or at least 80%, or 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%, or at least 99% identity to the nucleotide sequences represented in SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, 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 or SEQ ID No. 35 and have the functions described. The nucleotide sequences of the genes and ORFs known from the genome projects are part of the prior art and can be taken from various publications, patent applications and also the database of the National Center for Biotechnology Information (NCBI) of the National Library of Medicine (Bethesda, Md., USA).
Expression of the genes of glycerol metabolism can be detected with the aid of 1- and 2-dimensional protein gel separation and subsequent optical identification of the protein concentration in the gel using corresponding evaluation software. A customary method for preparation of the protein gels in the case of coryneform bacteria and for identification of the proteins is the procedure described by Hermann et al. (Electrophoresis, 22:1712-23 (2001)). The protein concentration can likewise be analyzed by Western blot hybridization using an antibody specific for the protein to be detected (Sambrook et al., Molecular cloning: a laboratory manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) and subsequent optical evaluation using corresponding software for determination of concentration (Lohaus and Meyer (1998) Biospectrum 5:32-39; Lottspeich (1999) Angewandte Chemie 111:2630-2647). The activity of DNA-binding proteins can be measured by means of DNA band shift assays (also termed gel retardation) (Wilson et al. (2001) Journal of Bacteriology 183:2151-2155). The effect of DNA-binding proteins on the expression of other genes can be detected by various well described methods of the reporter gene assay (Sambrook et al., Molecular cloning: a laboratory manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). The intracellular enzymatic activities can be determined by various described methods (Donahue et al. (2000) Journal of Bacteriology 182(19):5624-5627; Ray et al. (2000) Journal of Bacteriology 182(8):2277-2284; Freedberg et al. (1973) Journal of Bacteriology 115(3): 816-823).
The heterologous genes are expressed, for example, with the aid of episomal plasmids. Suitable plasmids are those which are replicated in coryneform bacteria. Numerous known plasmid vectors such as, for example, pZ1 (Menkel et al., Applied and Environmental Microbiology (1989) 64: 549-554), pEKEx1 (Eikmanns et al., Gene 102:93-98 (1991)) or pHS2-1 (Sonnen et al., Gene 107:69-74 (1991)) are based on the cryptic plasmids pHM1519, pBL1 or pGA1. Other plasmid vectors such as, for example, those based on pCG4 (U.S. Pat. No. 4,489,160), or pNG2 (Serwold-Davis et al., FEMS Microbiology Letters 66, 119-124 (1990)), or pAG1 (U.S. Pat. No. 5,158,891) can be used in the same manner. A survey of native plasmids from amino acid-producing coryneform bacteria is available in the Handbook of Corynebacterium glutamicum (Tauch, Chapter 4, 57-80, editors: Eggeling and Bott, CRC Press, Taylor & Francis Group, Boca Raton (2005)).
In addition, suitable plasmid vectors are those with the aid of which the method of gene amplification by integration into the chromosome can be employed, for instance as has been described, for example, by Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)) for duplication or amplification of the hom-thrb operon in WO03/040373. In this method the gene is cloned into a plasmid vector which can replicate in a host (typically E. coli ), but not in C. glutamicum . Vectors which come into consideration are, for example, pSUP301 (Simon et al., Bio/Technology 1, 784-791 (1983)), pK18mob or pK19mob (Schäfer et al., Gene 145, 69-73 (1994)), PGEM-T (Promega corporation, Madison, Wis., USA), pCR2.1-TOPO (Shuman (1994), Journal of Biological Chemistry 269: 32678-84; U.S. Pat. No. 5,487,993), pCR®Blunt (from Invitrogen, Groningen, Netherlands; Bernard et al., Journal of Molecular Biology, 234: 534-541 (1993)), pEM1 (Schrumpf et al, 1991, Journal of Bacteriology 173:4510-4516) or pBGS8 (Spratt et al., 1986, Gene 41: 337-342). The plasmid vector which contains the heterologous gene to be amplified, if appropriate including the expression and/or regulation signals, and the edge regions of a non-essential homologous gene, is subsequently transferred into the desired strain of C. glutamicum by conjugation or transformation. The conjugation method is described, for example, in Schäfer et al. (Applied and Environmental Microbiology 60, 756-759 (1994)). Methods for transformation are described, for example, in Thierbach et al. (Applied Microbiology and Biotechnology 29, 356-362 (1988)), Dunican and Shivnan (Bio/Technology 7, 1067-1070 (1989)) and Tauch et al. (FEMS Microbiology Letters 123, 343-347 (1994)). After homologous recombination by means of a crossover event, the resultant strain contains a copy of the heterologous gene including the plasmid vector at the desired gene site of the C. glutamicum chromosome which was predetermined via the homologous nucleotide sequences on the plasmid. By means of a suitable second, excision-causing cross-over event in the target gene or in the target sequence, incorporation of only the heterologous gene is achieved. In this case, at the respective natural gene site, no nucleotide sequence capable of episomal replication in the microorganisms remains, no nucleotide sequence capable of transposition and no nucleotide sequence conferring resistance to antibiotics.
In addition, it can be advantageous for the production of L-amino acids, in addition to the functional expression of one or more of the heterologous genes of glycerol metabolism selected from the group glpA, glpB, glpC, glpD, glpE, glpF, glpG, glpK, glpQ, glpT, glpX, gldA, dhaK, dhaL, dhaM, dhaR, fsa and talC, to amplify, in particular overexpress, or attenuate, in particular reduce the expression, of one or more enzymes of the metabolic pathways which increase or decrease the formation of the desired amino acid, such as, for example, the biosynthetic pathway, glycolysis, anaplerosis, the citric acid cycle, the pentosphosphate cycle, amino acid export and if appropriate regulatory proteins.
The expression “amplification” or “amplify” describes in this context the increase in intracellular activity or concentration of one or more enzymes or proteins in a microorganism which are encoded by the corresponding DNA by, for example, increasing the number of copies of the gene or genes, using a strong promoter or a gene or allele which encodes a corresponding enzyme or protein having a high activity and if appropriate combining these measures.
By the measures of amplification, in particular overexpression, the activity or concentration of the corresponding protein is generally increased by at least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%, at most to 1000% or 2000%, based on that of the wild type protein or the activity or concentration of the protein in the starting microorganism.
To achieve an overexpression, the copy number of the corresponding genes can be increased, or the promoter and regulation region or the ribosome binding site which is situated upstream of the structural gene can be mutated. Expression cassettes which are incorporated upstream of the structural gene act in the same manner. By inducible promoters it is, in addition, possible to increase the expression in the course of the fermentative amino acid production. By measures for extending the life of m-RNA, likewise expression is improved. In addition, by hindering the breakdown of the enzyme protein, likewise the enzyme activity is amplified. The genes or gene constructs can be present either in plasmids with different copy numbers or be integrated in the chromosome and amplified. Alternatively, in addition, overexpression of the genes in question can be achieved by altering the media composition and culture procedure.
Instructions in this respect can be found by those skilled in the art, inter alia, in Martin et al. (Bio/Technology 5, 137-146 (1987)), in Guerrero et al. (Gene 138, 35-41 (1994)), Tsuchiya and Morinaga (Bio/Technology 6, 428-430 (1988)), in Eikmanns et al. (Gene 102, 93-98 (1991)), in European patent 0 472 869, in U.S. Pat. No. 4,601,893, in Schwarzer and Pühler (Bio/Technology 9, 84-87 (1991), in Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)), in LaBarre et al. (Journal of Bacteriology 175, 1001-1007 (1993)), in the patent application WO 96/15246, in Malumbres et al. (Gene 134, 15-24 (1993)), in the Japanese laid-open publication JP-A-10-229891, in Jensen and Hammer (Biotechnology and Bioengineering 58, 191-195 (1998)), in Makrides (Microbiological Reviews 60:512-538 (1996)) and in known textbooks of genetics and molecular biology.
For amplification, genes are overexpressed, for example, with the aid of episomal plasmids. Suitable plasmids are those which are replicated in coryneform bacteria. Numerous known plasmid vectors such as, for example, pZ1 (Menkel et al., Applied and Environmental Microbiology (1989) 64: 549-554), pEKEx1 (Eikmanns et al., Gene 102:93-98 (1991)) or pHS2-1 (Sonnen et al., Gene 107:69-74 (1991)) are based on the cryptic plasmids pHM1519, pBL1 or pGA1. Other plasmid vectors such as, for example, those based on pCG4 (U.S. Pat. No. 4,489,160), or pNG2 (Serwold-Davis et al., FEMS Microbiology Letters 66, 119-124 (1990)), or pAG1 (U.S. Pat. No. 5,158,891), can be used in the same manner.
In addition, suitable plasmid vectors are those with the aid of which the method of gene amplification by integration into the chromosome can be employed, for instance as has been described, for example, by Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994) for the duplication or amplification of the hom-thrB operon. In this method the complete gene is cloned into a plasmid vector which can replicate in a host (typically E. coli ), but not in C. glutamicum . Vectors which come into consideration are, for example, pSUP301 (Simon et al., Bio/Technology 1, 784-791 (1983)), pK18mob or pK19mob (Schäfer et al., Gene 145, 69-73 (1994)), pGEM-T (Promega corporation, Madison, Wis., USA), pCR2.1-TOPO (Shuman (1994), Journal of Biological Chemistry 269: 32678-84; U.S. Pat. No. 5,487,993), pCR®Blunt (from Invitrogen, Groningen, Netherlands; Bernard et al., Journal of Molecular Biology, 234: 534-541 (1993)), pEM1 (Schrumpf et al, 1991, Journal of Bacteriology 173:4510-4516) or pBGS8 (Spratt et al., 1986, Gene 41: 337-342). The plasmid vector which contains the gene to be amplified is subsequently transferred into the desired strain of C. Glutamicum by conjugation or transformation. The method of conjugation is described, for example, in Schäfer et al. (Applied and Environmental Microbiology 60, 756-759 (1994)). Methods for transformation are described, for example, in Thierbach et al. (Applied Microbiology and Biotechnology 29, 356-362 (1988)), Dunican and Shivnan (Bio/Technology 7, 1067-1070 (1989)) and Tauch et al. (FEMS Microbiology Letters 123, 343-347 (1994). After homologous recombination by means of a crossover event, the resultant strain contains at least two copies of the gene in question.
A customary method of incorporating one or more additional copies of a gene of C. glutamicum into the chromosome of the desired coryneform bacterium is the method of gene doubling described in WO03/014330 and WO03/040373. For this, in WO03/040373 the nucleotide sequence of the desired ORF, gene or allele, if appropriate including the expression and/or regulation signals is isolated and two copies, preferably in tandem arrangement, are cloned into a vector which is not replicative for C. glutamicum such as, for example, pK18mobsacB or pK19mobsacB (Jäger et al., Journal of Bacteriology 174: 5462-65 (1992)). The vector is subsequently transferred to the desired coryneform bacterium by transformation or conjugation. After homologous recombination by means of a first crossover event causing integration and a suitable second excision-causing cross-over event in the target gene or in the target sequence, incorporation of the additional gene copy is achieved. Thereafter, those bacteria are isolated in which there are two copies of the ORF, gene or allele at the respective natural site instead of the original singular copy present. In this case, at the respective natural gene site, there remains no nucleotide sequence which is capable of/enables episomal replication in microorganisms, no nucleotide sequence which is capable of, enables transposition and no nucleotide sequence which imparts resistance to antibiotics.
The expression “attenuation” in this context describes the reduction or switching off of the intracellular activity of one or more enzymes (proteins) in a microorganism which are encoded by the corresponding DNA by, for example, using a weak promoter or a gene or allele which encodes a corresponding enzyme or protein having a low activity or which inactivates the corresponding gene or enzyme (protein) and if appropriate combining these measures.
By the attenuation measures, the activity or concentration of the corresponding protein is generally lowered to 0 to 75%, 0 to 50%, 0 to 25%, 0 to 10%, or 0 to 5% of the activity or concentration of the wild type protein, or of the activity or concentration of the protein in the starting microorganism.
The elevation or lowering of the protein concentration is detectable via the methods already mentioned hereinbefore (Hermann et al., Electrophoresis, 22:1712-23 (2001); Lohaus and Meyer, Biospektrum 5:32-39 (1998); Lottspeich, Angewandte Chemie 111:2630-2647 (1999); Wilson et al., Journal of Bacteriology 183:2151-2155 (2001)).
The use of endogenous genes is generally preferred. “Endogenous genes” or “endogenous nucleotide sequences” is taken to mean genes or nucleotide sequences present in the population of a species.
Thus, for example for the production of L-lysine, in addition to the expression of one or more heterologous genes of glycerol metabolism selected from the group glpA, glpB, glpC, glpD, glpE, glpF, glpG, glpK, glpQ, glpT, glpX, gldA, dhaK, dhaL, dhaM, dhaR, fsa and talC, one or more of the genes selected from the group of the genes or alleles of lysine production are amplified, in particular overexpressed. “Genes or alleles of lysine production” are taken to mean all, preferably endogenous, open reading frames, genes or alleles, the amplification/overexpression of which can cause an improvement in lysine production.
The following genes or alleles, inter alia, can be used for this:
accBC, accDA, cstA, cysD, cysE, cysH, cysK, cysN, cysQ, dapA, dapB, dapC, dapD, dapE, dapF, ddh, dps, eno, gap, gap2, gdh, gnd, lysC, lySCFBR, lysE, msiK, opcA, oxyR, ppc, ppcFBR, pgk, pknA, pknB, pknD, pknG, ppsA, ptsH, ptsI, ptsM, pyc, pyc P458S, sigC, sigD, sigE, sigH, sigM, tal, thyA, tkt, tpi, zwa1, zwf and zwf A243T. These are summarized and explained in Table 1.





TABLE 1



Genes and alleles of lysine production







Description of the coded enzyme or

Accession

Name
protein
Reference
number



accBC
Acyl-CoA Carboxylase
Jäger et al.
U35023


EC 6.3.4.14
Archives of



(acyl-CoA carboxylase)
Microbiology




(1996) 166: 76-82




EP1108790;
AX123524



WO0100805
AX066441

accDA
Acetyl-CoA Carboxylase
EP1055725



EC 6.4.1.2
EP1108790
AX121013


(acetyl-CoA carboxylase)
WO0100805
AX066443

cstA
Carbon Starvation Protein A
EP1108790
AX120811


(carbon starvation protein A)
WO0100804
AX066109

cysD
Sulfate adenylyltransferase subunit II
EP1108790
AX123177


EC 2.7.7.4




(sulfate adenylyltransferase small




chain)



cysE
Serine acetyltransferase
EP1108790
AX122902


EC 2.3.1.30
WO0100843
AX063961


(serine acetyltransferase)



cysH
3′-Phosphoadenylsulfate reductase
EP1108790
AX123178


EC 1.8.99.4
WO0100842
AX066001


(3′-phosphoadenosine 5′-phosphosulfate




reductase)



cysK
Cysteine synthase
EP1108790
AX122901


EC 4.2.99.8
WO0100843
AX063963


(cysteine synthase)



cysN
Sulfate adenylyltransferase subunit I
EP1108790
AX123176


EC 2.7.7.4

AX127152


(sulfate adenylyltransferase)



cysQ
Transport protein CysQ
EP1108790
AX127145


(transporter cysQ)
WO0100805
AX066423

dapA
Dihydrodipicolinate synthase
Bonnassie et al.
X53993


EC 4.2.1.52
Nucleic Acids



(dihydrodipicolinate synthase)
Research 18: 6421




(1990)




Pisabarro
Z21502



et al., Journal




of Bacteriology




175: 2743-2749




(1993)




EP1108790




WO0100805




EP0435132




EP1067192
AX123560



EP1067193
AX063773

dapB
Dihydrodipicolinate reductase
EP1108790
AX127149


EC 1.3.1.26
WO0100843
AX063753


(dihydrodipicolinate reductase)
EP1067192
AX137723



EP1067193
AX137602



Pisabarro et
X67737



al., Journal of
Z21502



Bacteriology




175: 2743-2749




(1993)




JP1998215883
E16749



JP1997322774
E14520



JP1997070291
E12773



JP1995075578
E08900

dapC
N-Succinylaminoketopimelate
EP1108790
AX127146


transaminase
WO0100843
AX064219


EC 2.6.1.17
EP1136559



(N-succinyl-diaminopimelate




transaminase)



dapD
Tetrahydrodipicolinate succinylase
EP1108790
AX127146


EC 2.3.1.117
WO0100843
AX063757


(tetrahydrodipicolinate succinylase)
Wehrmann et al.
AJ004934



Journal of




Bacteriology




180: 3159-3165




(1998)


dapE
N-Succinyldiaminopimelate desuccinylase
EP1108790
AX127146


EC 3.5.1.18
WO0100843
AX063749


(N-succinyl-diaminopimelate
Wehrmann et al.
X81379


desuccinylase)
Microbiology




140: 3349-3356




(1994)


dapF
Diaminopimelate epimerase
EP1108790
AX127149


EC 5.1.1.7
WO0100843
AX063719


(diaminopimelate epimerase)
EP1085094
AX137620

ddh
Diaminopimelate dehydrogenase
EP1108790
AX127152


EC 1.4.1.16
WO0100843
AX063759


(diaminopimelate dehydrogenase)
Ishino et al.,
Y00151



Nucleic Acids




Research




15: 3917-3917




(1987)




JP1997322774




JP1993284970
E14511



Kim et al.,
E05776



Journal of
D87976



Microbiology and




Biotechnology




5: 250-256 (1995)


dps
DNA protection protein
EP1108790
AX127153


(protection during starvation protein)



eno
Enolase
EP1108790
AX127146


EC 4.2.1.11
WO0100844
AX064945


(enolase)
EP1090998
AX136862



Hermann et al.,




Electrophoresis




19: 3217-3221




(1998)


gap
Glyceraldehyde-3-phosphate
EP1108790
AX127148


dehydrogenase
WO0100844
AX064941


EC 1.2.1.12
Eikmanns et al.,
X59403


(glyceraldehyde-3-phosphate
Journal of



dehydrogenase)
Bacteriology




174: 6076-6086




(1992)


gap2
Glyceraldehyde-3-phosphate
EP1108790
AX127146


dehydrogenase
WO0100844
AX064939


EC 1.2.1.12




(glyceraldehyde-3-phosphate




dehydrogenase 2)



gdh
Glutamate dehydrogenase
EP1108790
AX127150


EC 1.4.1.4
WO0100844
AX063811


(glutamate dehydrogenase)
Boermann et al.,
X59404



Molecular
X72855



Microbiology




6: 317-326 (1992)


gnd
6-Phosphogluconate dehydrogenase
EP1108790
AX127147


EC 1.1.1.44

AX121689


(6-phosphogluconate dehydrogenase)
WO0100844
AX065125

lysC
Aspartate kinase
EP1108790
AX120365


EC 2.7.2.4
WO0100844
AX063743


(aspartate kinase)
Kalinowski et
X57226



al., Molecular




Microbiology




5: 1197-204




(1991)


lysE
Lysine exporter
EP1108790
AX123539


(lysine exporter protein)
WO0100843
AX123539



Vrljić et al.,
X96471



Molecular




Microbiology




22: 815-826




(1996)


msiK
Sugar importer
EP1108790
AX120892


(multiple sugar import protein)



opcA
Glucose-6-phosphate dehydrogenase
WO0104325
AX076272


(subunit of glucose-6-phosphate




dehydrogenase)



oxyR
Transcription regulator
EP1108790
AX122198


(transcriptional regulator)

AX127149

ppc FBR
Phosphoenolpyruvate carboxylase
EP0723011



feedback resistant
WO0100852



EC 4.1.1.31




(phosphoenolpyruvate carboxylase




feedback resistant)



ppc
Phosphoenolpyruvate carboxylase
EP1108790
AX127148


EC 4.1.1.31

AX123554


(phosphoenolpyruvate carboxylase)
O'Reagan et al.,
M25819



Gene 77(2): 237-251




(1989)


pgk
Phosphoglycerate kinase
EP1108790
AX121838


EC 2.7.2.3

AX127148


(phosphoglycerate kinase)
WO0100844
AX064943



Eikmanns,
X59403



Journal of




Bacteriology




174: 6076-6086




(1992)


pknA
Protein kinase A
EP1108790
AX120131


(protein kinase A)

AX120085

pknB
Protein kinase B
EP1108790
AX120130


(protein kinase B)

AX120085

pknD
Protein kinase D
EP1108790
AX127150


(protein kinase D)

AX122469




AX122468

pknG
Protein kinase G
EP1108790
AX127152


(protein kinase G)

AX123109

ppsA
Phosphoenolpyruvate synthase
EP1108790
AX127144


EC 2.7.9.2

AX120700


(phosphoenolpyruvate synthase)

AX122469

ptsH
Phosphotransferase system protein H
EP1108790
AX122210


EC 2.7.1.69

AX127149


(phosphotransferase system component H)
WO0100844
AX069154

ptsI
Phosphotransferase system enzyme I
EP1108790
AX122206


EC 2.7.3.9

AX127149


(phosphotransferase system enzyme I)



ptsM
Glucose-specific phosphotransferase
Lee et al.,
L18874


system enzyme II
FEMS



EC 2.7.1.69
Microbiology



(glucose-phosphotransferase-system
Letters 119(1-2):



enzyme II)
137-145




(1994)


pyc
Pyruvate carboxylase
WO9918228
A97276


EC 6.4.1.1
Peters-Wendisch
Y09548


(pyruvate carboxylase)
et al.,




Microbiology




144: 915-927




(1998)


pyc
Pyruvate carboxylase
EP1108790


P458S
EC 6.4.1.1




(pyruvate carboxylase)




Amino acid exchange P458S



sigC
Sigma factor C
EP1108790
AX120368


EC 2.7.7.6

AX120085


(extracytoplasmic function




alternative sigma factor C)



sigD
RNA polymerase sigma factor D
EP1108790
AX120753


EC 2.7.7.6

AX127144


(RNA polymerase sigma factor)



sigE
Sigma factor E
EP1108790
AX127146


EC 2.7.7.6

AX121325


(extracytoplasmic function




alternative sigma factor E)



sigH
Sigma factor H
EP1108790
AX127145


EC 2.7.7.6

AX120939


(sigma factor SigH)



SigM
Sigma factor M
EP1108790
AX123500


EC 2.7.7.6

AX127153


(sigma factor SigM)



tal
Transaldolase
WO0104325
AX076272


EC 2.2.1.2




(transaldolase)



thyA
Thymidylate synthase
EP1108790
AX121026


EC 2.1.1.45

AX127145


(thymidylate synthase)



tkt
Transketolase
Ikeda et al.,
AB023377


EC 2.2.1.1
NCBI



(transketolase)



tpi
Triose-phosphate isomerase
Eikmanns,
X59403


EC 5.3.1.1
Journal of



(triose-phosphate isomerase)
Bacteriology




174: 6076-6086




(1992)


zwa1
Cell growth factor 1
EP1111062
AX133781


(growth factor 1)



zwf
Glucose-6-phosphate-1-dehydrogenase
EP1108790
AX127148


EC 1.1.1.49

AX121827


(glucose-6-phosphate-1-dehydrogenase)
WO0104325
AX076272

zwf
Glucose-6-phosphate-1-dehydrogenase
EP1108790


A213T
EC 1.1.1.49




(glucose-6-phosphate-1-dehydrogenase)




Amino acid exchange A213T
































































































































































































































































In addition, it can be advantageous for production of L-lysine when, in addition to the functional expression of one or more of the heterologous genes of glycerol metabolism selected from the group glpA, glpB, glpC, glpD, glpE, glpF, glpG, glpK, glpQ, glpT, glpX, gldA, dhaK, dhaL, dhaM, dhaR, fsa and talC, at the same time one or more of the genes selected from the group of genes or alleles which are not essential for growth or lysine production is or are attenuated, in particular switched off, or their expression decreased.
The following open reading frames, genes or alleles, inter alia, can be used for this: aecD, ccpA1, ccpA2, citA, citB, cite, fda, glua, gluB, gluC, gluD, luxR, luxS, lysR1, lysR2, lysR3, menE, mqo, pck, pgi, poxB and zwa2, which are summarized and explained in Table 2.





TABLE 2



Genes and alleles which are not essential for lysine production






Gene
Description of the coded enzyme

Accession

name
or protein
Reference
number



aecD
beta C-S Lyase
Rossol et al., Journal
M89931


EC 2.6.1.1
of Bacteriology



(beta C-S lyase)
174(9): 2968-77 (1992)


ccpA1
Catabolite control protein
WO0100844
AX065267


(catabolite control protein A1)
EP1108790
AX127147



WO 02/18419


ccpA2
Catabolite control protein
WO0100844
AX065267


(catabolite control protein A2)
EP1108790
AX121594

citA
Sensor kinase CitA
EP1108790
AX120161


(sensor kinase CitA)



citB
Transcription regulator CitB
EP1108790
AX120163


(transcription regulator CitB)



citE
Citrate lyase
WO0100844
AX065421


EC 4.1.3.6
EP1108790
AX127146


(citrate lyase)



fda
Fructose bisphosphate aldolase
von der Osten et al.,
X17313


EC 4.1.2.13
Molecular Microbiology



(fructose-1,6-bisphosphate
3(11): 1625-37 (1989)



aldolase



gluA
Glutamate transport ATP-binding
Kronemeyer et al.,
X81191


protein
Journal of Bacteriology



(glutamate transport ATP-
177(5): 1152-8 (1995)



binding protein)



gluB
glutamate binding protein
Kronemeyer et al.,
X81191


(glutamate binding protein)
Journal of Bacteriology




177(5): 1152-8 (1995)


gluC
Glutamate transport permease
Kronemeyer et al.,
X81191


(glutamate transport system
Journal of Bacteriology



permease)
177(5): 1152-8 (1995)


gluD
Glutamate transport permease
Kronemeyer et al.,
X81191


(glutamate transport system
Journal of Bacteriology



permease)
177(5): 1152-8 (1995)


luxR
Transcription regulator LuxR
WO0100842
AX065953


(transcription regulator LuxR)
EP1108790
AX123320

luxS
Histidine kinase LuxS
EP1108790
AX123323


(histidine kinase LuxS)

AX127153

lysR1
Transcription regulator lysR1
EP1108790
AX064673


(transcription regulator LysR1)

AX127144

lysR2
Transcription activator LysR2
EP1108790
AX123312


(transcription regulator LysR2)



lysR3
Transcription regulator LysR3
WO0100842
AX065957


(transcription regulator LysR3)
EP1108790
AX127150

menE
O-Succinylbenzoate-CoA-ligase
WO0100843
AX064599


EC 6.2.1.26
EP1108790
AX064193


(O-succinylbenzoate-CoA-ligase

AX127144

mqo
Malate-quinone-oxidoreductase
Molenaar et al., Eur.
AJ224946


(malate-quinone-oxidoreductase)
Journal of Biochemistry




1; 254(2): 395-403 (1998)


pck
Phosphoenolpyruvate
WO0100844
AJ269506


carboxykinase
EP-A-1094111
AX065053


(phosphoenolpyruvate




carboxykinase)



pgi
Glucose-6-phosphate isomerase
EP1087015
AX136015


EC 5.3.1.9
EP1108790
AX127146


(glucose-6-phosphate isomerase)
WO01/07626


poxB
Pyruvate oxidase
WO0100844
AX064959


EC 1.2.3.3
EP1096013
AX137665


(pyruvate oxidase)



zwa2
Cell growth factor 2
EP1106693
AX113822


(growth factor 2)
EP1108790
AX127146

















































































It is also possible to amplify endogenous polynucleotides from Corynebacterium glutamicum (Accession No.: NC 006958 and NC 003450 (sequence of the entire genome)), which have, for example, at least 45%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or 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% or at least 99% identity with the nucleotide sequences described in SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, 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 or SEQ ID No. 35 and which have the functions described. The nucleotide sequences of the genes and ORFs known from the genome projects are part of the prior art and can be taken from various publications, patent applications and also the database of the National Center for Biotechnology Information (NCBI) of the National Library of Medicine (Bethesda, Md., USA).
It has been found that coryneform bacteria, also after shared overexpression of the endogenous polynucleotides glpK (58% identity with SEQ ID No. 15), encoding the ATP-dependent glycerol kinase K, and glpD (47% identity with SEQ ID No. 7), encoding the glycerol-3-phosphate dehydrogenase of glycerol metabolism produce L-amino acids, in particular L-lysine and L-tryptophan, from glycerol as sole carbon source.
For the sake of better clarity, the known sequences of the treated genes of Corynebacterium glutamicum are described under SEQ ID No. 37 and SEQ ID No. 39.
The bacteria of the invention can be cultured continuously, such as, for example, in the method described in PCT/EP2004/008882, or discontinuously in the batch method or in the fed batch method or repeated fed batch method for the purposes of production of L-amino acids. A summary of known culture methods is available in the textbook by Chmiel (Bioprozesstechnik 1. Einfuhrung in die Bioverfahrenstechnik [Biomethod technology 1. Introduction to biomethod engineering] (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktoren und periphere Einrichtungen [Bioreactors and peripheral equipment] (Vieweg Verlag, Brunswick/Wiesbaden, 1994)).
The culture medium to be used must meet the requirements of the respective strains in a suitable manner. Descriptions of culture media for various microorganisms are contained in the handbook “Manual of Methods for General Bacteriology” of the American Society for Bacteriology (Washington D.C., USA, 1981).
The carbon source used is glycerol. This can be used individually or as a mixture. Sugars and carbohydrates such as, for example, glucose, sucrose, lactose, fructose, maltose, molasses, starch and if appropriate cellulose, oils and fats such as, for example, soybean oil, sunflower seed oil, peanut oil and coconut fat, fatty acids such as, for example, palmitic acid, stearic acid and linoleic acid, alcohols such as, for example, ethanol and methanol, and organic acids such as, for example, acetic acid, can be added, in which case the fraction of glycerol is at least greater than or equal to (≧) 10%, or at least ≧25%, or at least ≧50%, or at least ≧75%, or at least ≧90%, or at least ≧95%, at least greater ≧99%, preferably 100%.
As nitrogen source, use can be made of organic nitrogenous compounds such as peptones, yeast extract, meat extract, malt extract, corn steep water, soybean meal and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. The nitrogen sources can be used individually or as a mixture.
As phosphorus source, use can be made of phosphoric acid, potassium dihydrogenphosphate or dipotassium hydrogenphosphate or the corresponding sodium-containing salts. The culture medium must in addition contain salts of metals such as, for example, magnesium sulfate or iron sulfate, which are necessary for growth. Finally, in addition to the above-mentioned substances, use can be made of essential growth substances such as amino acids and vitamins. In addition, suitable precursors can be added to the culture medium. Said feed materials can be added to the culture in the form of a single batch or can be fed in during the culture in a suitable manner.
For pH control of the culture, use is made, in a suitable manner, of basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water or acidic compounds such as phosphoric acid or sulfuric acid. For control of foam development, use can be made of antifoams such as, for example, fatty acid polyglycol esters. To maintain the stability of plasmids, suitable selectively acting substances such as, for example, antibiotics can be added to the medium. To maintain aerobic conditions, oxygen or oxygen-containing gas mixtures such as, for example, air, are introduced into the culture. The temperature of the culture is usually 20° C. to 45° C. and preferably 25° C. to 40° C. The culture is continued until a maximum of the desired product has formed. This target is usually achieved within 10 hours to 160 hours. In continuous methods, longer culture times are possible.
Methods of determining L-amino acids are known from the prior art. The analysis can proceed as described in Spackman et al. (Analytical Chemistry, 30, (1958), 1190) by anion-exchange chromatography with subsequent ninhydrin derivatization, or it can proceed by reversed-phase HPLC, as described in Lindroth et al. (Analytical Chemistry (1979) 51: 1167-1174).