# Patent

## Title
Method for producing an L-amino acid by fermentation using a bacterium having an enhanced ability to utilize glycerol

## Abstract

The present invention provides a method for producing an L-amino acid using a bacterium of the Enterobacteriaceae family, particularly a bacterium belonging to genus Escherichia or Pantoea, which has been modified to have glycerol kinase in which feedback inhibition by fructose-1,6-bisphosphate is desensitized, thereby having enhanced ability to utilize glycerol.

## Background
The present application is a continuation of PCT Patent Application No. PCT/JP2007/075348, filed on Dec. 21, 2007, which claims priority under 35 U.S.C. §119 to Russian Patent Application No. 2006145712, filed on Dec. 22, 2006, the entireties of which are incorporated by reference. Also, the Sequence Listing filed electronically herewith is hereby incorporated by reference (File name: US-310_Seq_List; File size: 95 KB; Date recorded: Jun. 19, 2009).


BACKGROUND OF THE INVENTION
1. Technical Field
The present invention relates to biotechnology, specifically to a method for producing L-amino acids by fermentation using glycerol, and more specifically to a method for producing L-amino acids using bacteria having enhanced ability to utilize glycerol. An inexpensive carbon source including glycerol could be utilized for commercial production of L-amino acids.
2. Background Art
Conventionally, L-amino acids have been industrially produced by a process of fermentation using strains of different microorganisms. The fermentation media for the process should contain sufficient amounts of different sources of carbon and nitrogen.
Traditionally various carbohydrates such as hexoses, pentoses, trioses; various organic acids and alcohols are used as a carbon source. Hexoses include glucose, fructose, mannose, sorbose, galactose and the like. Pentoses include arabinose, xylose, ribose and the like. But abovementioned carbohydrates and other traditional carbon sources, such as molasses, corn, sugarcane, starch, its hydrolysate, etc., used in the industry are still relatively expensive and a reduction in price of L-amino acid produced is desired.
Glycerol, especially glycerol obtained as by-product of biodiesel production, is a favorable feedstock for L-amino acid production because it is both readily available and less expensive than carbohydrates, corn, sugarcane or other sources of carbon. It is known also that bacteria can use glycerol as a carbon source for growth. (Ito T. et al, J Biosci Bioeng., 100, 3, 260-5 (2005)).
Two proteins, the glycerol facilitator and glycerol kinase, are involved in the entry of external glycerol into cellular metabolism. Glycerol kinase (EC2.7.1.30) encoded by the glpK gene is a component of regulatory network in E. coli by which glucose and other carbon sources control the utilization of glycerol and the gene expression that is needed for glycerol metabolism. (Escherichia coli and Salmonella. 2nd edition ASM Press Washington, D.C.). The proteins involved in glycerol metabolism are encoded by the glp regulon, which contains five operons located at three different chromosomal loci. Glucose modulation of glycerol utilization involves both regulation of transcription and posttranslational control of glycerol kinase catalytic activity. Transcription of the regulon is negatively controlled by a specific repressor encoded by the glpR gene.
It is known that cytoplasmic glycerol is immediately phosphorylated by the ATP-dependent glycerol kinase, which is present in its enzymatically active form associated with the glycerol facilitator GlpF (Voegele, R. T. et al, J. Bacteriol 175, 4, 1087-1094 (1993))). Furthermore, the glycerol kinase is subject to feedback inhibition by fructose-1,6-bisphosphate (fructose-1,6 diphosphate, FBP). Accordingly, the activity of glycerol kinase is rate-limiting in the metabolism of glycerol by cells of Escherichia coli. 
The glycerol facilitator is thought to act as a carrier or to form a selective pore in the cytoplasmic membrane, whereas the kinase traps the glycerol inside the cell as sn-glycerol-3-phosphate. It was found that the kinetics of glycerol uptake in a facilitator-minus strain are significantly different from the kinetics of glycerol uptake in the wild type. Free glycerol was not observed inside wild-type cells transporting glycerol, and diffusion of glycerol across the cytoplasmic membrane was not the rate-limiting step for phosphorylation in facilitator-minus mutants. Therefore, the kinetics of glycerol phosphorylation is different, depending on the presence or absence of the facilitator protein. It was concluded that there is an interaction between the glycerol facilitator protein and glycerol kinase that stimulates kinase activity, analogous to the hexokinase- and glycerol kinase-porin interactions in mitochondria (Voegele, R. T. et al, J. Bacteriol., 175, 4, 1087-1094 (1993)).
A mutant strain which produces a glycerol kinase resistant to inhibition by fructose-1,6-bisphosphate grows faster than its wild-type parent on glycerol as the sole source of carbon. Pittigrew et al. identified the Escherichia coli glycerol kinase mutation G304S which lost sensitivity to inhibition by FBP (Pettigrew, D. W., Liu, W. Z., Holmes, C., Meadow, N. D., and Roseman, S., J. Bacteriol. 178, 10, 2846-52 (1996)). Honisch et. al. identified the mutation G231D in glycerol kinase of an adaptively evolved strain, and kinetically characterized wild type glycerol kinase and G231D mutant. Kinetic studies for G231D variant show a 12-fold increase in glycerol kinase activity and simultaneous increase in tolerance toward the allosteric inhibitor fructose-1,6-bisphosphate (Honisch, C. et. al., Genome Research, 14: 2495-2502 (2004)).
Furthermore, adaptation of E. coli to glycerol media was tested, and some mutations which allowed for growth in the glycerol medium were determined (Herring C. D. et al, Nat. Genet., 38 (12): 1406-1412 (2006). Epub 2006 Nov. 5).
It is known also that attenuation of glpR is effective for producing L-amino acid production by fermentation in a glycerol containing medium. (EP1715056A1)
However, such known mutants are not sufficient for L-amino acid production from glycerol.
Furthermore, at present, there are no reports describing bacteria having a synergetic effect by combination of a mutation in glycerol kinase and attenuation of glpR on L-amino acid production from glycerol.
SUMMARY OF THE INVENTION
The present invention provides an L-amino acid producing bacterium which has an enhanced ability to utilize glycerol, and a method for producing L-amino acids by fermentation of glycerol using the bacterium. A fermentation feedstock containing glycerol obtained, for example, as by-product of biodiesel production, can be used as a carbon source for the culture medium.
This aspect was achieved by finding that a bacterium which comprises glycerol kinase in which feedback inhibition by fructose-1,6 bisphosphate is desensitized shows increased production of L-amino acid in the medium containing glycerol.
It is an aspect of the present invention to provide a method for producing an L-amino acid including;
a) culturing in a culture medium containing glycerol an L-amino acid producing bacterium of the Enterobacteriaceae family, and
b) collecting L-amino acid from the culture,
wherein said bacterium comprises glycerol kinase in which feedback inhibition by fructose-1,6-bisphosphate is desensitized, and said glycerol kinase comprises mutation to replace at least one amino acid with another amino acid in the region corresponding to the positions from 233 to 235 of the amino acid sequence of a wild type glycerol kinase shown in SEQ ID NO: 4.
It is a further aspect of the present invention to provide the method as described above, where the glycerol kinase further comprises mutation to replace at least one amino acid with another amino acid in the region corresponding to the positions from 231 to 232.
It is a further aspect of the present invention to provide the method as described above, wherein the glycerol kinase comprises mutation selected from the group consisting of replacing Gly residue at position 234 with Asp residue at position 234 with Asp residue, replacing Lys-Gly-Gly at positions from 233 to 235 with Arg-Ile-Pro, and replacing Gly-Gly-Lys-Gly-Gly at positions from 231 to 235 with Arg-Ala-His-Leu-Ala.
It is a further aspect of the present invention to provide the method as described above, wherein the bacterium is further modified so that activity of glycerol facilitator and/or glycerol 3-phosphate dehydrogenase is enhanced.
It is a further aspect of the present invention to provide the method as described above, wherein the expression of the protein is increased by increasing the copy number of the gene or modifying an expression control sequence of the gene so that the expression of the gene is enhanced.
It is a further aspect of the present invention to provide the method as described above, wherein the bacterium is further modified so that activity of triose phosphate isomerase is enhanced.
It is a further aspect of the present invention to provide the method as described above, wherein the expression of the protein is increased by increasing the copy number of the gene or modifying an expression control sequence of the gene so that the expression of the gene is enhanced.
It is a further object of the present invention to provide the method as described above, wherein the L-amino acid is selected from the group consisting of an aromatic L-amino acid and a non-aromatic L-amino acid.
It is a further object of the present invention to provide the method as described above, wherein the aromatic L-amino acid is selected from the group consisting of L-phenylalanine, L-tyrosine, and L-tryptophan.
It is a further object of the present invention to provide the method as described above, wherein the non-aromatic L-amino acid is selected from the group consisting of L-threonine, L-lysine, L-cysteine, L-methionine, L-leucine, L-isoleucine, L-valine, L-histidine, glycine, L-serine, L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, L-proline, and L-arginine.
It is a further object of the present invention to provide the method as described above, wherein the bacterium belongs to the genus Escheriahia or Pantoea. 
It is a further object of the present invention to provide the method as described above, wherein the glycerol is obtained as a by-product of biodiesel production.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the alignment of the primary sequences of glycerol kinase from Escherichia coli (P0A6F3, SEQ ID NO: 4), Shigella sonnei (Q3YV52, SEQ ID NO: 43), Yersinia pestis (Q8D1T6, SEQ ID NO: 44), Pseudomonas aeruginosa (Q51390, SEQ ID NO: 45), and Bacillus subtilis (P18157, SEQ ID NO: 46). The alignment was done by using the PIR Multiple Alignment program (http://pir.georgetown.edu). The identical amino acids are indicated by an asterisk (*), similar amino acids are indicated by a colon (:).
FIG. 2 shows the relative positions of primers P3 and P4, and P11 and P12 on plasmid pMW118-attL-Cm-attR which is used as a template for PCR amplification of the cat gene.
FIG. 3 shows the construction of the chromosomal DNA fragment containing the inactivated glpK/glpR gene.



DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
1. Bacterium
Exemplary bacteria of the present invention can include an L-amino acid-producing bacterium of the Enterobacteriaceae family which has been modified to contain glycerol kinase in which feedback inhibition by fructose-1,6-bisphosphate is desensitized, and in which at least one amino acid is replaced with another amino acid in the region corresponding to the positions from 233 to 235 of the amino acid sequence of a wild type glycerol kinase shown in SEQ ID NO: 4, thereby having an enhanced ability to utilize glycerol. The glycerol kinase which has the mutation as described above is referred to as “mutant glycerol kinase”.
The phrase “to utilize glycerol” means that bacterium grows on a medium containing glycerol, and wherein glycerol is a carbon source. The phrase “enhanced ability to utilize glycerol” means that the modified bacterium can utilize glycerol more effectively as compared with an unmodified bacterium, or can grow faster than an unmodified bacterium on the culture medium containing glycerol.
A culture medium can contain glycerol as the sole carbon source, or can be in a mixture with other carbon sources By way of example, the other carbon sources can be different carbohydrates, such as glucose, sucrose, xylose, arabinose, etc., alcohols, such as ethanol, methanol, etc.
“L-amino acid-producing bacterium” means a bacterium which can have an ability to produce and excrete an L-amino acid into a medium, when the bacterium is cultured in the medium.
The term “L-amino acid-producing bacterium” also means a bacterium which can produce and can cause accumulation of an L-amino acid in a culture medium in an amount larger than a wild-type or parental strain of the bacterium, for example, E. coli, such as E. coli K-12. “L-amino acid producing bacterium” also can mean that the bacterium can cause accumulation in a medium of an amount not less than 0.5 g/L, or, alternatively, not less than 1.0 g/L, of the target L-amino acid.
The term “L-amino acid” includes L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine.
The term “aromatic L-amino acid” includes L-phenylalanine, L-tyrosine, and L-tryptophan.
The term “non-aromatic L-amino acid” includes L-threonine, L-lysine, L-cysteine, L-methionine, L-leucine, L-isoleucine, L-valine, L-histidine, glycine, L-serine, L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, L-proline, L-arginine, L-phenylalanine, and L-tryptophan.
The Enterobacteriaceae family can include bacteria belonging to the genera Escherichia, Enterobacter, Raoultella, Erwinia, Klebsiella, Pantoea, Photorhabdus, Providencia, Salmonella, Serratia, Shigella, Morganella, Yersinia, etc. Specifically, those classified into the Enterobacteriaceae according to the taxonomy used by the NCBI (National Center for Biotechnology Information) database (http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=91347) can be used. In an exemplary embodiment in accordance with the presently disclosed subject matter, the bacterium can belong to the genus Escherichia or Pantoea. 
The phrase “a bacterium belonging to the genus Escherichia” means that the bacterium can be classified into the genus Escherichia according to the classification known to a person skilled in the art of microbiology. Examples of a bacterium belonging to the genus Escherichia as used in the present invention can include, but are not limited to, Escherichia coli (E. coli).
The bacterium belonging to the genus Escherichia that can be used in accordance with the presently disclosed subject matter is not particularly limited. For example, bacteria in accordance with the presently disclosed subject matter can encompass bacteria described by Neidhardt, F. C. et al. (Escherichia coli and Salmonella typhimurium, American Society for Microbiology, Washington D.C., 1208, Table 1).
The phrase “a bacterium belonging to the genus Pantoea” means that the bacterium can be classified as the genus Pantoea according to the classification known to a person skilled in the art of microbiology. Some species of Enterobacter agglomerans have been recently re-classified into Pantoea agglomerans, Pantoea ananatis, Pantoea stewartii or the like, based on the nucleotide sequence analysis of 16S rRNA, etc. (Int. J. Syst. Bacteriol., 43, 162-173 (1993)).
The phrase “glycerol kinanse in which feedback inhibition by fructose-1,6-bisphosphate (FBP) is desensitized” means that glycerol kinase in the presence of high concentrations of fructose-1,6-bisphosphate can be increased as compared with a wild-type strain or non-mutated strain. Whether feedback inhibition of glycerol kinase by FBP is desensitized can be confirmed by measurement of glycerol kinase activity in the presence of FBP (Pettigrew, D. W. et al. J. Bacteriol. 178, 10, 2846-52 (1996)). Effect of desentization of feedback inhibition of glycerol kinase by FBP on glycerol utilization can be confirmed by comparing the growth of modified bacterium and unmodified bacterium in glycerol medium.
The glpK gene of E. coli (synonyms: ECK3918, b3926) encodes glycerol kinase (synonym B3926). The glpK gene (nucleotides complementary to nucleotides in positions 4,113,737 to 4,115,245; GenBank accession no. NC—000913.2; gi: 49175990) is located between the glpX gene and the glpF gene on the chromosome of E. coli K-12. The nucleotide sequence of the glpK gene and the amino acid sequence of GlpK encoded by the glpK gene are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.
The mutation of glycerol kinase to desensitize feedback inhibition by FBP can be the replacement of one or more amino acids in the region corresponding to the positions from 233 to 235 in the amino acid sequence of a wild type glycerol kinase presented by SEQ ID NO: 4 with another amino acid. The mutant glycerol kinase can further contain mutations such as the replacement of at least one amino acid with another amino acid in the region corresponding to the positions from 231 to 232. The “another amino acid” is not particularly limited so long as it is different from the original amino acid and feedback inhibition of glycerol kinase by FBP is desensitized by the replacement. However, “another amino acid” in the positions 231, 232, 234, 235 means other than Gly, and the substitution of Lys, Glu, Thr, Val, Leu, Ile, Ser, Asp, Asn, Gln, Arg, Cys, Met, Phe, Trp, Tyr, Ala, Pro or His are exemplary. “Another amino acid” in the position 233 means other than Lys, and the substitution of Glu, Thr, Val, Leu, Ile, Ser, Asp, Asn, Gln, Arg, Cys, Met, Phe, Trp, Tyr, Ala, Pro, Gly or His are exemplary. In an exemplary embodiment in accordance with the presently disclosed subject matter, Gly at position 231 can be replaced with an basic amino acid such as Arg, Lys or an acidic amino acid such as Asp, Glu.
In another exemplary embodiment in accordance with the presently disclosed subject matter, Gly at position 232 can be replaced with an aliphatic amino acid such as Ala, Val, Leu or an acidic amino acid such as Asp or Glu.
In another exemplary embodiment in accordance with the presently disclosed subject matter, Lys at position 233 can be replaced with an amino acid such as Arg, Thr, Ala, or His.
In another exemplary embodiment in accordance with the presently disclosed subject matter, Gly at position 234 can be replaced with an amino acid such as Asp, Ile, Leu, Thr, or Arg.
In another exemplary embodiment in accordance with the presently disclosed subject matter, Gly at position 235 can be replaced with an amino acid such as Leu, Glu, Phe, Pro, or Ala.
Exemplary mutations in accordance with the disclosed subject matter can include replacing the Gly residue at position 234 with an Asp residue, replacing Lys-Gly-Gly at positions 233 to 235 with Arg-Ile-Pro, or replacing Gly-Gly-Lys-Gly-Gly at positions 231 to 235 with Arg-Ala-His-Leu-Ala. The last two described mutations can be more advantageous than the first described mutation. Mutations other than those described above can also occur, as long as the properties of the mutant are maintained, for example, amino acids that do not influence the enzymatic activity, can be replaced by one or several other amino acids. “Several” as used herein can include a range of 1 to 20, a range of 1 to 10, or a range of 1 to 5.
Furthermore, mutation of the known mutant of glycerol kinase in which feedback inhibition by FBP is desensitized can be applied to the mutant glycerol kinase in accordance with the presently disclosed subject matter, in addition to the mutations as described above.
For example, the mutation can be thereplacement of the Gly at position 231 with another amino acid. (Honisch, C. et. al., Genome Research, 14:2495-502 (2004)). In another exemplary embodiment in accordance with the presently disclosed subject matter, Gly at position 231 can be replaced with an acidic amino acid such as Asp, Glu.
Additionally, the mutation can be the replacement of the Asp at position 73, the Val at position 62, the Met at position 272, the Gln at position 38, the Gly at position 231 with another amino acid. In another exemplary embodiment in accordance with the presently disclosed subject matter, the Asp residue at position 73 can be replaced with Val, the Val residue at position 62 can be replaced with Leu, the Met residue at position 272 can be replaced with Ile, the Gln residue at position 38 can be replaced with Pro, and/or the Gly at position 231 can be replaced with Asp (Herring C. D. et al, Nat. Genet., 38 (12): 1406-1412 (2006). Epub 2006 Nov. 5). Additionally, the 9 bp duplication of nucleotide can be inserted at position 705 in SEQ ID NO. 3, which causes duplication of Lys-Gly-Gly (Herring C. D. et al, Nat. Genet., 38 (12): 1406-1412 (2006). Epub 2006 Nov. 5).
A method for obtaining a mutant gene which encodes such a mutant glycerol kinase can be as follows. The mutant gene can be obtained by randomly introducing a mutation into the glpK gene in vitro, introducing the mutated gene into a bacterium of the Enterobacteriaceae family, and screening for strains which can grow on a medium containing glycerol and which produce an L-amino acid during cultivation in the medium containing glycerol. To screen for a mutant-type glpK gene, a glpK-deficient strain can be used. In vitro mutagenesis of the glpK gene can be performed as follows. First, glpK can be cloned into a plasmid that can replicate in a bacterium of Enterobacteriaceae family and a marker gene such as an antibiotic resistance gene. The obtained glpK gene-carrying plasmid can be dissolved in a buffer containing mutagens, for example, 400 mM hydroxylamine and eacted to introduce a mutation into the glpK gene. After mutagenesis, the plasmid can be desalted with SUPREC-02 (Takara Bio INC.) or the like, and then introduced into a ΔglpK strain with an ability to produce an L-amino acid, and transformants can be screened in a medium containing an antibiotic corresponding to the marker gene. As a control, a glpK gene-carrying plasmid which has not undergone mutagenesis can be introduced into the ΔglpK strain. The transformants which emerge can be inoculated into a medium containing glycerol and cultured with shaking, and then the concentration of the accumulated L-amino acid can be determined. The strain with the mutant glpK gene carried on a plasmid will produce a larger amount of the L-amino acid in the medium than the wild-type strain which contains the plasmid with the wild-type glpK gene. Whether the strain caries a mutant-type glpK gene or not can be confirmed by extracting the plasmid from the strain and determining the nucleotide sequence of the glpK gene.
Alternatively, a mutant-type glpK gene can be obtained by artificially introducing mutations into the glpK gene by methods such as error prone PCR, DNA shuffling, and StEP-PCR (Firth A E, A. E. and Patrick W. M., Bioinformatics, 21, 3314-3315 (2005)).
The methods of introducing a mutation into the glpK gene on the chromosome can include, in addition to the above-mentioned method, mutagenizing spontaneously or by treatment of a bacterium of Enterobacteriaceae family with irradiation of X-rays or ultraviolet rays or with a mutagen such as N-methyl-N′-nitro-N-nitrosoguanidine, and selecting a strain which grows on glycerol faster than a parent strain. Whether the mutant-type glpK gene has been introduced or not can be confirmed by determining the nucleotide sequence of the glpK gene on the chromosome.
Introduction of a mutant glpK gene can include introducing a mutation into a chromosomal glpK gene, introducing a plasmid containing a mutant glpK gene, and replacement of a chromosomal glpK gene with a mutant glpK gene. The method for introduction of mutant glpK can be done by referring to the following method for enhancement of the expression of enzyme.
Furthermore, a bacterium in accordance with the presently disclosed subject matter can be further modified so that the expression of the repressor of glp regulon is attenuated, in addition to desensitization of glycerol kinase. The phrase “repressor of the glp regulon” means the protein encoded by glpR. The glpR gene of E. coli (synonyms: ECK3409, b3423) encodes the repressor of the glycerol-3-phosphate regulon; specifically binding to operator sites of the glpD, glpFK, glpTQ, and glpACB operons. The attenuation of glpR gene can lead to overexpression of the glpACB, glpFK, glpD genes.
The glpR gene (nucleotides complementary to nucleotides in positions 3557870.3558628, GenBank accession no. NC—000913.2.[gi:16131297]) is located between the rtcR gene and the glpT gene on the chromosome of E. coli K-12. The nucleotide sequence of the glpR gene and the amino acid sequence of GlpR encoded by the glpR gene are shown in SEQ ID NO:31 and SEQ ID NO: 32, respectively.
The phrase “attenuation of expression of repressor of the glp regulon” means that the bacterium has been modified in such a way that the modified bacterium contains a reduced amount of the repressor of the glp regulon, as compared with an unmodified bacterium, or is unable to synthesize the repressor of the glp regulon.
The phrase “inactivation of the glpR gene” means that the modified DNA region is unable to naturally express the gene due to a deletion of a part of the gene or deletion of the gene entirely, or the modification of an adjacent region of the gene, including sequences controlling gene expression, such as promoters, enhancers, attenuators, etc.
Expression of the gene can be attenuated by introducing a mutation into the gene on the chromosome so that the intracellular amount of the protein encoded by the gene is decreased as compared to an unmodified strain. Such a mutation can be introduced by insertion of a drug-resistance gene into the target gene, or deletion of a part of the gene or the entire gene (Qiu, Z. and Goodman, M. F., J. Biol. Chem., 272, 8611-8617 (1997); Kwon, D. H. et al, J. Antimicrob. Chemother., 46, 793-796 (2000)). Expression of the glpR gene also can be attenuated by modifying an expression regulating sequence such as the promoter, the Shine-Dalgarno (SD) sequence, etc. (WO95/34672, Carrier, T. A. and Keasling, J. D., Biotechnol Prog 15, 58-64 (1999)).
For example, the following methods can be employed to introduce a mutation by gene recombination. A mutant gene can be prepared, and a bacterium can be transformed with a DNA fragment containing the mutant gene. Then, the native gene on the chromosome can be replaced with the mutant gene by homologous recombination, and the resulting strain can be selected. Such gene replacement by homologous recombination can be conducted by employing a linear DNA, which is known as “Red-driven integration” (Datsenko, K. A. and Wanner, B. L., Proc. Natl. Acad. Sci. USA, 97, 12, p 6640-6645 (2000)), or by methods employing a plasmid containing a temperature-sensitive replication (U.S. Pat. No. 6,303,383 or JP 05-007491A). Furthermore, the incorporation of a site-specific mutation by gene substitution using homologous recombination such as set forth above also can be conducted with a plasmid lacking the ability to replicate in the host.
Expression of the gene also can be attenuated by insertion of a transposon or an IS factor into the coding region of the gene (U.S. Pat. No. 5,175,107), or by conventional methods, such as a mutagenesis treatment using UV irradiation or nitrosoguanidine (N-methyl-N′-nitro-N-nitrosoguanidine) treatment.
An exemplary bacterium of the present invention can be further modified so that the activity of a glycerol facilitator is enhanced, in addition to desensitization of glycerol kinase and attenuation of repressor of glp regulon. The glpF gene encoding glycerol facilitator can be a glp regulon, therefore, the activity of glycerol facilitator can be enhanced by the attenuation of glpR gene.
The phrase “the activity of glycerol facilitator is enhanced.” means that the bacterium has been modified in such a way that the modified bacterium can have increased activity of said protein(s) per cell, as compared with an unmodified bacterium. Examples of such modifications can include increasing the number of glycerol facilitator molecules per cell, increasing the specific activity per molecule of protein, etc. Furthermore, a wild-type strain that can be used for comparison includes, for example, Escherichia coli K-12. The amount of accumulated L-amino acid, for example, L-threonine, L-lysine, L-histidine, L-phenylalanine, L-arginine, L-tryptophan, or L-glutamic acid, can be increased in a culture medium as the result of enhancing the intracellular activity of one or several proteins of glycerol facilitator.
A glycerol facilitator in accordance with the presently disclosed subject matter can have the function of facilitating diffusion of glycerol into the cell. It can be a member of the major intrinsic protein (MIP) family of transmembrane channel proteins. The activity of glycerol facilitator can be measured by transport assay (Voegele, R. T., Sweet, G. D., and Boos, W. J. Bacteriol. 175:1087-1094 (1993)) The glycerol facilitator activity can be enhanced within a range of not less than 1.5-fold, preferably a range of not less than 2-fold, or within a range of not less than 3-fold as compared to an unmodified strain or a wild-type strain.
The glpF gene of E. coli (synonyms: ECK3919, b3927) can encode the GlpF protein-glycerol facilitator (synonym B3927). The glpF gene (nucleotides complementary to nucleotides in positions 4,115,268 to 4,116,113; GenBank accession no. NC—000913.2; gi: 49175990) is located between the glpK gene and the yliU ORF on the chromosome of E. coli K-12. The nucleotide sequence of the glpF gene and the amino acid sequence of GlpF encoded by the glpF gene are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
A bacterium in accordance with the presently disclosed subject matter can be further modified so that the activity of glycerol-3-phosphate dehydrogenase is enhanced, in addition to the desensitization of a glycerol kinase, attenuation of glpR, and enhancement of a glycerol facilitator.
Glycerol-3-phosphate dehydrogenase can catalyze the oxidation from glycerol-3-phosphate (G3P) to dihydroxyacetone phosphate (EC 1.1.99.5). The activity of glycerol-3-phosphate dehydrogenase can be measured by the method of Spector and Pizer (Methods Enzymol. 41:249-254 (1975)). The glycerol-3-phosphate dehydrogenase activity can be enhanced within a range of not less than 1.5-fold, within a range of not less than 2-fold, or within a range of not less than 3-fold as compared to an unmodified strain or a wild-type strain.
In E. coli, glycerol-3-phosphate dehydrogenase can be encoded by glpABC and glpD genes.
The glpA gene of E. coli (synonyms: ECK2233, b2241) can encode the GlpA protein-subunit of glycerol 3-phosphate dehydrogenase (anaerobic)(synonym B2241). The glpA gene (nucleotides in positions 2,350,669 to 2,352,297; GenBank accession no. NC—000913.2; gi: 49175990) is located between the glpT gene and the glpB gene on the chromosome of E. coli K-12. The nucleotide sequence of the glpA gene and the amino acid sequence of GlpA encoded by the glpA gene are shown in SEQ ID NO: 5 and SEQ ID NO:6, respectively.
The glpB gene of E. coli (synonyms: ECK2234, psi-51, b2242) can encode the GlpB protein-subunit of glycerol 3-phosphate dehydrogenase (anaerobic)(synonym B2242). The glpB gene (nucleotides in positions 2,352,287 to 2,353,546; GenBank accession no. NC—000913.2; gi: 49175990) is located between the glpA gene and the glpC gene on the chromosome of E. coli K-12. The nucleotide sequence of the glpB gene and the amino acid sequence of GlpB encoded by the glpB gene are shown in SEQ ID NO: 7 and SEQ ID NO:8, respectively.
The glpC gene of E. coli (synonyms: ECK2235, b2243) can encode the GlpC protein-subunit of glycerol 3-phosphate dehydrogenase (anaerobic)(synonym B2243). The glpC gene (nucleotides in positions 2,353,543 to 2,354,733; GenBank accession no. NC—000913.2; gi: 49175990) is located between the glpB gene and the yfaD ORF on the chromosome of E. coli K-12. The nucleotide sequence of the glpC gene and the amino acid sequence of GlpC encoded by the glpC gene are shown in SEQ ID NO: 9 and SEQ ID NO:10, respectively.
The glpD gene of E. coli (synonyms: ECK3412, b3426, glvD, glyD) can encode the GlpD protein-subunit of glycerol 3-phosphate dehydrogenase (aerobic)(synonyms: B3426, GlvD, GlyD). The glpD gene (nucleotides in positions 3,560,036 to 3,561,541; GenBank accession no. NC—000913.2; gi: 49175990) is located between the glpE gene and the yzgL ORF on the chromosome of E. coli K-12. The nucleotide sequence of the glpD gene and the amino acid sequence of GlpD encoded by the glpD gene are shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively.
A bacterium in accordance with the presently disclosed subject matter can be further modified so that activity of triose phosphate isomerase is enhanced, in addition to the desensitization of a glycerol kinase, attenuation of glpR, enhancement of a glycerol facilitator, and enhancement of glycerol 3-phosphate dehydrogenase.
Triose phosphate isomerase can catalyze reversible conversion between dihydroxyacetone phosphate and glyceraldehydes-3-phosphate (EC:5.3.1.1). The activity of triose phosphate isomerase can be measured by the method of Anderson and Cooper (FEBS Lett. 4: 19-20 (1969)). The triose phosphate isomerase activity can be enhanced within a range of not less than 1.5-fold, within a range of not less than 2-fold, or within a range of not less than 3-fold as compared to an unmodified strain or a wild-type strain.
The tpiA gene of E. coli (synonyms: ECK3911, b3919, G102, tpi) can encode the TpiA protein—triose phosphate isomerase (synonyms: B3946, Tpi). The tpiA gene (nucleotides complementary to nucleotides in positions 4,108,763 to 4,109,530; GenBank accession no. NC—000913.2; gi: 16131757) is located between the cdh gene and the yijQ ORF on the chromosome of E. coli K-12. The nucleotide sequence of the tpiA gene and the amino acid sequence of TpiA encoded by the tpiA gene are shown in SEQ ID NO: 33 and SEQ ID NO: 34, respectively.
Since there can be some differences in DNA sequences between the genera or strains of the Enterobacteriaceae family, the glpF, glpK, glpA, glpB, glpC, glpD, glpR and tpi genes to be modified (or the expression amount thereof to be increased) on the chromosome are not limited to the genes shown 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: 31 and SEQ ID NO: 33., but may include genes homologous 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: 31 and SEQ ID NO: 33.
Therefore, the protein variant encoded by the glpF, glpK, glpA, glpB, glpC, glpD, glpR and tpi genes can have a homology range of not less than 80%, a homology range of not less than 90%, or a homology range of not less than 95%, with respect to the entire amino acid sequence shown in 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: 32 and SEQ ID NO: 34 respectively as long as the native activity of the protein is maintained. The phrase “variant protein” as used in in accordance with the presently disclosed subject matter means a protein which has changes in the sequence, whether they are deletions, insertions, additions, or substitutions of amino acids, but still maintains the desired activity at a useful level, for example, useful for the enhanced production of an L-amino acid. The number of changes in the variant protein depends on the position in the three dimensional structure of the protein or the type of amino acid residue. The number of changes can be in a range of 1 to 30, a range of 1 to 15, or a range of 1 to 5. These changes can occur in regions of the protein which are not critical for the function of the protein. This is because some amino acids have high homology to one another so the three dimensional structure or activity is not affected by such a change. These changes in the variant protein can occur in regions of the protein which are not critical for the function of the protein. Activity of glycerol kinase can be measured by the method described by Hayashi, S. I. and Lin, E. C. (J. Biol. Chem. 242:1030-1035 (1967)). Activity of glycerol 3-phosphate dehydrogenase can be measured by the method described by Kistler, W. S. and Lin, E. C. (J. Bacteriol. 108: 1224-1234 (1971) and J. Bacteriol. 112: 539-547 (1972)).
Moreover, the glpF, glpK, glpA, glpB, glpC, glpD, glpR and tpi genes can be a variant which hybridizes with a complementary strand of the nucleotide sequence shown 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: 31 and SEQ ID NO: 33, or a probe which can be prepared from the nucleotide sequence under stringent conditions. “Stringent conditions” can include those under which a specific hybrid, for example, a hybrid having homology range of not less than 80%, a homology range of not less than 90%, a homology range of not less than 95%, a homology range of not less than 97%, or a homology range of not less than 98%, is formed and a non-specific hybrid, for example, a hybrid having homology lower than the above, is not formed. For example, stringent conditions can be exemplified by washing one time or more, or two or three times at an exemplary salt concentration of 1×SSC, 0.1% SDS in accordance with the presently disclosed subject matter. This exemplary salt concentration of 0.1×SSC, 0.1% SDS can occur within a first temperature range of 60-68° C., or within a second temperature range of 64-68° C. Duration of washing can depend on the type of membrane used for blotting and, as a rule, should be what is recommended by the manufacturer. For example, the recommended duration of washing for the HybondTM N+ nylon membrane (Amersham) under stringent conditions is 15 minutes. Preferably, washing can be performed 2 to 3 times. The length of the probe can, depend on the hybridization conditions, and can be 100 bp to 1 kbp in an exemplary embodiment in accordance with the presently disclosed subject matter.
Homology between two amino acid sequences can be determined using any method known to one skilled in the art, for example, the computer program BLAST 2.0.
The substitution, deletion, insertion, or addition of one or several amino acid residues can be conservative mutation(s) so that the activity can be maintained. The representative conservative mutation can be a conservative substitution. Examples of conservative substitutions can include: substitution of Ala with Ser or Thr, substitution of Arg with Gln, His, or Lys, substitution of Asn with Glu, Gln, Lys, His, or Asp, substitution of Asp with Asn, Glu, or Gln, substitution of Cys with Ser or Ala, substitution of Gln with Asn, Glu, Lys, His, Asp, or Arg, substitution of Glu with Gly, Asn, Gln, Lys, or Asp, substitution of Gly with Pro, substitution of His with Asn, Lys, Gln, Arg, or Tyr, substitution of Ile with Leu, Met, Val, or Phe, substitution of Leu with Ile, Met, Val, or Phe, substitution of Lys with Asn, Glu, Gln, His, or Arg, substitution of Met with Ile, Leu, Val, or Phe, substitution of Phe with Trp, Tyr, Met, Ile, or Leu, substitution of Ser with Thr or Ala, substitution of Thr with Ser or Ala, substitution of Trp with Phe or Tyr, substitution of Tyr with His, Phe, or Trp, and substitution of Val with Met, Ile, or Leu.
Data comparing the primary sequences of glycerol kinase from Escherichia coli, Shigella sonnei, Yersinia pestis, Pseudomonas aeruginosa, and Bacillus subtilis show a high level of homology among these proteins (see FIG. 1). From this point of view, substitutions or deletions of the amino acid residues which are identical (marked by asterisk) in all the above-mentioned proteins can be crucial for their function. It is possible to replace similar (indicated by a colon) amino acids residues with the similar amino acid residues without deterioration of the protein activity. But modifications of other non-conserved amino acid residues can not lead to alteration of the activity of glycerol kinase.
In accordance with the presently disclosed subject matter, “the region corresponding to the positions from 233 to 235 of the amino acid sequence of a wild type glycerol kinase shown in SEQ ID NO: 4” means the region of glycerol kinase of interest which corresponds to the positions from 233 to 235 in SEQ ID NO: 4 in the alignment of the amino acid sequences of the objective glycerol kinase and SEQ ID NO: 4. The position of an amino acid residue can change. For example, if an amino acid residue is inserted at the N-terminus portion, the amino acid residue at position 233 can become position 234. In such a case, the amino acid residue at the original position 233 can be designated as the amino acid residue at the position 233 in accordance with the presently disclosed subject matter.
To determine the positions from 233 to 235 of the glycerol kinase of interest, the amino acid sequence of SEQ ID NO: 4 can be aligned with the amino acid sequence of a glycerol kinase of interest, and the L-amino acids at positions from 233 to 235 in the glycerol kinase of interest can be determined.
The DNA which encodes substantially the same protein as shown in 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: 32, and SEQ ID NO: 34 described above can be obtained, for example, by modifying the nucleotide sequence of DNA shown 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: 31, and SEQ ID NO: 33, respectively, for example, by means of site-directed mutagenesis so that the nucleotide sequence responsible for one or more amino acid residues at a specified site can be deleted, substituted, inserted, or added. DNA modified as described above can be obtained by any mutation treatment(s) known to one skilled in the art. Such treatments can include hydroxylamine treatment of the DNA encoding proteins in accordance with the presently disclosed subject matter, or treatment of the bacterium containing the DNA with UV irradiation or a reagent such as N-methyl-N′-nitro-N-nitrosoguanidine or nitrous acid.
A DNA encoding substantially the same protein as shown in 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: 32, and SEQ ID NO: 34 can be obtained by expressing DNA having a mutation as described above in an appropriate cell, and investigating the activity of any expressed product.
The substitution, deletion, insertion, or addition of nucleotides as described above also can include mutations which naturally occur (mutant or variant), for example, due to variety in the species or genus of bacterium, and which contains the corresponding enzyme.
The phrase “increasing/enhancing the expression of the gene” means that the expression of the gene is increased compared to that of a non-modified strain, for example, a wild-type strain. Examples of such modifications can include increasing the copy number of expressed gene(s) per cell, increasing the expression level of the gene(s), etc.
The quantity of the copy number of an expressed gene can be measured, for example, by restricting the chromosomal DNA followed by Southern blotting using a probe based on the gene sequence, fluorescence in situ hybridization (FISH), and the like. The level of gene expression can be determined by measuring the amount of sRNA transcribed from the gene using various known methods including Northern blotting, quantitative RT-PCR, and the like. The amount of the protein encoded by the gene can be measured by known methods including SDS-PAGE followed by immunoblotting assay (Western blotting analysis), and the like.
Hereinafter, a method for enhancing the activity of glycerol kinase, the glycerol facilitator, glycerol 3-phosphate dehydrogenase, triose phosphate isomerase (hereinafter referred to as glycerol utilization protein) is disclosed.
When using the gene from Escherichia coli, the gene encoding glycerol utilization protein can be obtained by PCR (polymerase chain reaction; White, T. J. et al., Trends Genet. 5, 185 (1989)) using primers based on the nucleotide sequence 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: 31, and SEQ ID NO: 33. The gene encoding the glycerol utilization protein from another bacterium also can be obtained by PCR from the chromosomal DNA or genomic DNA library of the bacterium using, as primers, oligonucleotides prepared based on the known sequences of the gene of the bacterium or of the gene of another kind of bacterium, or hybridization using an oligonucleotide prepared based on the sequence as a probe. A chromosomal DNA can be prepared from a bacterium that serves as a DNA donor by the method of Saito and Miura (see H. Saito and K. Miura, Biochem. Biophys. Acta, 72, 619 (1963), Experiment Manual for Biotechnology, edited by The Society for Biotechnology, Japan, p 97-98, Baifukan Co., Ltd., 1992) or the like.
Then, a recombinant DNA can be prepared by ligating the gene which has been amplified by PCR to a vector DNA which is capable of functioning in the host bacterium. Examples of the vector capable of functioning in the host bacterium can include vectors autonomously replicable in the host bacterium.
Examples of a vector which is autonomously replicable in Escherichia coli can include pUC19, pUC18, pHSG299, pHSG399, pHSG398, pACYC184, (pHSG and pACYC are available from Takara Bio Inc.), RSF1010 (Gene vol. 75(2), p 271-288, 1989), pBR322, pMW219, pMW119 (pMW is available from Nippon Gene Co., Ltd.), pSTV28, and pSTV29 (Takara Bio Inc.). A phage DNA vector also can be used.
To ligate the gene to the above-mentioned vector, the vector can be digested with a restriction enzyme corresponding to a recognition site in the terminus of a DNA fragment containing the gene. Ligation can be performed generally using a ligase such as T4 DNA ligase. Methods of digesting and ligating DNA, preparation of a chromosomal DNA, PCR, preparation of a plasmid DNA, transformation, design of oligonucleotides to be used as primers are methods well known to a person skilled in the art. These methods are described in Sambrook, J., Fritsch, E. F., and Maniatis, T., “Molecular Cloning A Laboratory Manual, Second Edition”, Cold Spring Harbor Laboratory Press, (1989), and the like.
The thus-prepared recombinant DNA can be introduced into a bacterium in accordance with a conventional transformation method. Examples of the method can include electroporation (Gliesche, C. G., Can. J. Microbiol., 43, 2, 197-201 (1997)). Also, it is possible to increase the DNA permeability by treating recipient cells with calcium chloride, which has been reported with Escherichia coli K-12 (Mandel, M. and Higa, A., J. Mol. Biol., 53, 159 (1970), and introduce a DNA into a competent cell prepared from a cell at proliferation stage, which has been reported with Bacillus subtilis (Duncan, C. H., Wilson, G. A and Young, F. E, Gene, 1, 153 (1977)).
The copy number of the gene also can be increased by introducing multiple copies of the genes encoding glycerol utilization protein into the chromosomal DNA of a bacterium. Introduction of multiple copies of the genes into the chromosomal DNA of a bacterium can be attained by homologous recombination using a target sequence present on the chromosomal DNA in multiple copies. Such a sequence present on a chromosomal DNA in multiple copies may be a repetitive DNA or an inverted repeat present on the edge of a transposing element. Alternatively, as disclosed in JP 2-109985 A, multiple copies of a gene can be introduced into the chromosomal DNA by inserting the genes into a transposon, and transferring it so that multiple copies of the gene are integrated into the chromosomal DNA. Integration of these genes into the chromosome can be confirmed by Southern hybridization using a portion of the genes as a probe.
Furthermore, expression of the gene can be enhanced by, as described in WO 00/18935, substituting an expression regulatory sequence such as a promoter of the genes on a chromosomal DNA or of the genes on a plasmid with a stronger promoter, amplifying a regulator that increases expression of the genes, or deleting or attenuating a regulator that decreases expression of the genes. Examples of known strong promoters include the lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, and tet promoter.
Meanwhile, a promoter of a gene can be made stronger so that expression of the genes is enhanced by introducing nucleotide substitution into the promoter. Examples of a method of evaluating the strength of a promoter and examples of strong promoters are described in Goldstein et al. (Prokaryotic promoters in biotechnology. Biotechnol. Annu. Rev., 1995, 1, 105-128) or the like. In addition, it is known that a spacer sequence between the ribosome binding site (RBS) and translation initiation codon, especially, several nucleotides just upstream of the initiation codon, has a great influence on translation efficiency. Therefore, this sequence can be modified.
In addition, to enhance the activity of a protein encoded by the gene encoding glycerol utilization protein, a mutation that increases the enzymatic activity can be introduced into the genes. Examples of such a mutation can include a mutation in a promoter sequence to increase the transcription level of encoding glycerol utilization protein, and a mutation in the coding region of these genes to increase the specific activities of the glycerol utilization protein.
Furthermore, the glpR gene can encode a repressor of the glp regulon that negatively regulates expression of the gene encodes the glycerol utilization protein, so expression of the gene encoding the glycerol utilization protein can be enhanced by attenuation of expression of the glpR gene.
Methods for preparation of plasmid DNA, digestion and ligation of DNA, transformation, selection of an oligonucleotide as a primer, and the like can be ordinary methods well-known to one skilled in the art. These methods are described, for instance, in Sambrook, J., Fritsch, E. F., and Maniatis, T., “Molecular Cloning: A Laboratory Manual, Second Edition”, Cold Spring Harbor Laboratory Press (1989).
L-Amino Acid-Producing Bacteria
Bacteria which are able to produce either an aromatic or a non-aromatic L-amino acids can be used as a bacterium in accordance with the disclosed subject matter which is modified to have enhanced ability to utilize glycerol.
Bacteria in accordance with the disclosed subject matter can be obtained by imparting an enhanced ability to utilize glycerol to a bacterium which inherently has the ability to produce L-amino acids. Alternatively, bacteria in accordance with the disclosed subject matter can be obtained by imparting the ability to produce L-amino acids to a bacterium already having enhanced ability to utilize glycerol.
L-Threonine-Producing Bacteria
Examples of parent strains which can be used to derive the L-threonine-producing bacteria in accordance with the disclosed subject matter can include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli TDH-6/pVIC40 (VKPM B-3996) (U.S. Pat. No. 5,175,107, U.S. Pat. No. 5,705,371), E. coli 472T23/pYN7 (ATCC 98081) (U.S. Pat. No. 5,631,157), E. coli NRRL-21593 (U.S. Pat. No. 5,939,307), E. coli FERM BP-3756 (U.S. Pat. No. 5,474,918), E. coli FERM BP-3519 and FERM BP-3520 (U.S. Pat. No. 5,376,538), E. coli MG442 (Gusyatiner et al., Genetika (in Russian), 14, 947-956 (1978)), E. coli VL643 and VL2055 (EP 1149911 A), and the like.
The strain TDH-6 is deficient in the thrC gene, as well as being sucrose-assimilative, and the ilvA gene has a leaky mutation. This strain also has a mutation in the rhtA gene, which imparts resistance to high concentrations of threonine or homoserine. The strain B-3996 contains the plasmid pVIC40 which was obtained by inserting a thrA*BC operon which includes a mutant thrA gene into a RSF1010-derived vector. This mutant thrA gene encodes aspartokinase homoserine dehydrogenase I which has substantially desensitized feedback inhibition by threonine. The strain B-3996 was deposited on Nov. 19, 1987 in the All-Union Scientific Center of Antibiotics (Russia, 117105 Moscow, Nagatinskaya Street 3-A) under the accession number RIA 1867. The strain also was deposited in the Russian National Collection of Industrial Microorganisms (VKPM)(Russia, 117545 Moscow, 1 Dorozhny proezd, 1) on Apr. 7, 1987 under the accession number VKPM B-3996.
E. coli VKPM B-5318 (EP 0593792B) also can be used as a parent strain for deriving L-threonine-producing bacteria. The strain B-5318 is prototrophic with regard to isoleucine, and a temperature-sensitive lambda-phage C1 repressor and PR promoter replaces the regulatory region of the threonine operon in plasmid pVIC40. The strain VKPM B-5318 was deposited in the Russian National Collection of Industrial Microorganisms (VKPM) on May 3, 1990 under accession number of VKPM B-5318.
Preferably, bacteria in accordance with the disclosed subject matter can be additionally modified to enhance expression of one or more of the following genes:

    
    
        the mutant thrA gene which codes for aspartokinase homoserine dehydrogenase I resistant to feed back inhibition by threonine;
        the thrB gene which codes for homoserine kinase;
        the thrC gene which codes for threonine synthase;
        the rhtA gene which codes for a putative transmembrane protein;
        the asd gene which codes for aspartate-β-semialdehyde dehydrogenase; and
        the aspC gene which codes for aspartate aminotransferase (aspartate transaminase);
    
    


The thrA gene which encodes aspartokinase homoserine dehydrogenase I of Escherichia coli has been elucidated (nucleotide positions 337 to 2799, GenBank accession NC—000913.2, gi: 49175990). The thrA gene is located between the thrL and thrB genes on the chromosome of E. coli K-12. The thrB gene which encodes homoserine kinase of Escherichia coli has been elucidated (nucleotide positions 2801 to 3733, GenBank accession NC—000913.2, gi: 49175990). The thrB gene is located between the thrA and thrC genes on the chromosome of E. coli K-12. The thrC gene which encodes threonine synthase of Escherichia coli has been elucidated (nucleotide positions 3734 to 5020, GenBank accession NC—000913.2, gi: 49175990). The thrC gene is located between the thrB gene and the yaaX open reading frame on the chromosome of E. coli K-12. All three genes functions as a single threonine operon. To enhance expression of the threonine operon, the attenuator region which affects the transcription can be removed from the operon (WO2005/049808, WO2003/097839).
A mutant thrA gene which codes for aspartokinase homoserine dehydrogenase I resistant to feed back inhibition by threonine, as well as, the thrB and thrC genes can be obtained as one operon from well-known plasmid pVIC40 which is present in the threonine producing E. coli strain VKPM B-3996. Plasmid pVIC40 is described in detail in U.S. Pat. No. 5,705,371.
The rhtA gene exists at 18 min on the E. coli chromosome close to the glnHPQ operon, which encodes components of the glutamine transport system. The rhtA gene is identical to ORF1 (ybiF gene, nucleotide positions 764 to 1651, GenBank accession number AAA218541, gi:440181) and located between the pexB and ompX genes. The unit expressing a protein encoded by the ORF1 has been designated the rhtA gene (rht: resistance to homoserine and threonine). Also, it was revealed that the rhtA23 mutation is an A-for-G substitution at position −1 with respect to the ATG start codon (ABSTRACTS of the 17th International Congress of Biochemistry and Molecular Biology in conjugation with Annual Meeting of the American Society for Biochemistry and Molecular Biology, San Francisco, Calif. Aug. 24-29, 1997, abstract No. 457, EP 1013765 A).
The asd gene of E. coli has already been elucidated (nucleotide positions 3572511 to 3571408, GenBank accession NC—000913.1, gi:16131307), and can be obtained by PCR (polymerase chain reaction; refer to White, T. J. et al., Trends Genet., 5, 185 (1989)) utilizing primers prepared based on the nucleotide sequence of the gene. The asd genes of other microorganisms can be obtained in a similar manner.
Also, the aspC gene of E. coli has already been elucidated (nucleotide positions 983742 to 984932, GenBank accession NC—000913.1, gi:16128895), and can be obtained by PCR. The aspC genes of other microorganisms can be obtained in a similar manner.
Another example of a parent strain which can be used to derive the L-threonine producing bacteria in accordance with the disclosed subject matter can include the E. coli strain MG1655 Δtdh::rhtA* (WO2006/078051).
Another example of a parent strain which can be used to derive the L-threonine producing bacteria in accordance with the disclosed subject matter can include the E. coli strain MG1655 Δtdh::rhtA*/PVIC40.
L-Lysine-Producing Bacteria
Examples of L-lysine-producing bacteria belonging to the genus Escherichia can include mutants having resistance to an L-lysine analogue. The L-lysine analogue can inhibit growth of bacteria belonging to the genus Escherichia, but this inhibition can be fully or partially desensitized when L-lysine is present in the medium. Examples of the L-lysine analogue can include, but are not limited to, oxalysine, lysine hydroxamate, S-(2-aminoethyl)-L-cysteine (AEC), γ-methyllysine, α-chlorocaprolactam, etc. Mutants having resistance to these lysine analogues can be obtained by subjecting bacteria belonging to the genus Escherichia to a conventional artificial mutagenesis treatment. Specific examples of bacterial strains useful for producing L-lysine can include Escherichia coli AJ11442 (FERM BP-1543, NRRL B-12185; see U.S. Pat. No. 4,346,170) and Escherichia coli VL611. In these microorganisms, feedback inhibition of aspartokinase by L-lysine is desensitized.
The strain WC196 can be used as an L-lysine producing bacterium of Escherichia coli. This bacterial strain was bred by conferring AEC resistance to the strain W3110, which was derived from Escherichia coli K-12. The resulting strain was designated Escherichia coli AJ13069 strain and was deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology (currently National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary, Tsukuba Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan) on Dec. 6, 1994 and received an accession number of FERM P-14690. Then, it was converted to an international deposit under the provisions of the Budapest Treaty on Sep. 29, 1995, and received an accession number of FERM BP-5252 (U.S. Pat. No. 5,827,698).
Examples of parent strains which can be used to derive L-lysine-producing bacteria in accordance with the disclosed subject matter also can include strains in which expression of one or more genes encoding an L-lysine biosynthetic enzyme are enhanced. Examples of such genes can include, but are not limited to, genes encoding dihydrodipicolinate synthase (dapA), aspartokinase (lysC), dihydrodipicolinate reductase (dapB), diaminopimelate decarboxylase (lysA), diaminopimelate dehydrogenase (ddh)(U.S. Pat. No. 6,040,160), phosphoenolpyrvate carboxylase (ppc), aspartate semialdehyde dehydrogenease (asd), and aspartase (aspA)(EP 1253195 A). In addition, the parent strains can have an increased level of expression of the gene involved in energy efficiency (cyo)(EP 1170376 A), the gene encoding nicotinamide nucleotide transhydrogenase (pntAB)(U.S. Pat. No. 5,830,716), the ybjE gene (WO2005/073390), or combinations thereof.
Examples of parent strains which can be used to derive L-lysine-producing bacteria in accordance with the disclosed subject matter also can include strains having decreased or no activity of an enzyme that catalyzes a reaction which results in the generation of a compound other than L-lysine, by causing a deviation from the biosynthetic pathway of L-lysine. Examples of the such enzymes can include homoserine dehydrogenase, lysine decarboxylase (U.S. Pat. No. 5,827,698), and the malic enzyme (WO2005/010175).
L-Cysteine-Producing Bacteria
Examples of parent strains which can be used to derive L-cysteine-producing bacteria in accordance with the disclosed subject matter can include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli JM15 which is transformed with different cysE alleles coding for feedback-resistant serine acetyltransferases (U.S. Pat. No. 6,218,168, Russian patent application 2003121601); E. coli W3110 having over-expressed genes which encode proteins suitable for secreting substances toxic for cells (U.S. Pat. No. 5,972,663); E. coli strains having lowered cysteine desulfohydrase activity (JP11155571A2); E. coli W3110 with increased activity of a positive transcriptional regulator for cysteine regulon encoded by the cysB gene (WO0127307A1), and the like.
L-Leucine-Producing Bacteria
Examples of parent strains which can be used to derive L-leucine-producing bacteria in accordance with the disclosed subject matter can include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli strains resistant to leucine (for example, the strain 57 (VKPM B-7386, U.S. Pat. No. 6,124,121)) or leucine analogs including β-2-thienylalanine, 3-hydroxyleucine, 4-azaleucine, 5,5,5-trifluoroleucine (JP 62-34397 B and JP 8-70879 A); E. coli strains obtained by the gene engineering method described in WO96/06926; E. coli H-9068 (JP 8-70879 A), and the like.
Bacteria in accordance with the disclosed subject matter can be improved by enhancing the expression of one or more genes involved in L-leucine biosynthesis. Examples can include genes of the leuABCD operon, which are preferably represented by a mutant leuA gene coding for isopropylmalate synthase not subject to feedback inhibition by L-leucine (U.S. Pat. No. 6,403,342). In addition, bacteria in accordance with the disclosed subject matter can be improved by enhancing the expression of one or more genes coding for proteins which excrete L-amino acid from the bacterial cell. Examples of such genes include the b2682 and b2683 genes (ygaZH genes)(EP 1239041 A2).
L-Histidine-Producing Bacteria
Examples of parent strains which can be used to derive L-histidine-producing bacteria in accordance with the disclosed subject matter can include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli strain 24 (VKPM B-5945, RU2003677); E. coli strain 80 (VKPM B-7270, RU2119536); E. coli NRRL B-12116-B12121 (U.S. Pat. No. 4,388,405); E. coli H-9342 (FERM BP-6675) and H-9343 (FERM BP-6676)(U.S. Pat. No. 6,344,347); E. coli H-9341 (FERM BP-6674)(EP1085087); E. coli A180/pFM201 (U.S. Pat. No. 6,258,554) and the like.
Examples of parent strains which can be used to derive L-histidine-producing bacteria in accordance with the disclosed subject matter also can include strains in which expression of one or more genes encoding an L-histidine biosynthetic enzyme are enhanced. Examples of such genes include genes encoding ATP phosphoribosyltransferase (hisG), phosphoribosyl AMP cyclohydrolase (hisI), phosphoribosyl-ATP pyrophosphohydrolase (hislE), phosphoribosylformimino-5-aminoimidazole carboxamide ribotide isomerase (hisA), amidotransferase (hisH), histidinol phosphate aminotransferase (hisC), histidinol phosphatase (hisB), histidinol dehydrogenase (hisD), etc.
It is known that the L-histidine biosynthetic enzymes encoded by hisG and hisBHAFI are inhibited by L-histidine, and therefore the L-histidine-producing ability also can be efficiently enhanced by introducing a mutation conferring resistance to the feedback inhibition into ATP phosphoribosyltransferase (Russian Patent Nos. 2003677 and 2119536).
Specific examples of strains having an L-histidine-producing ability can include E. coli FERM P-5038 and 5048 which have been introduced with a vector carrying a DNA encoding an L-histidine-biosynthetic enzyme (JP 56-005099 A), E. coli strains introduced with rht, a gene for an amino acid-export (EP1016710A), E. coli 80 strain imparted with sulfaguanidine, DL-1,2,4-triazole-3-alanine, and streptomycin-resistance (VKPM B-7270, Russian Patent No. 2119536), etc.
L-Glutamic Acid-Producing Bacteria
Examples of parent strains which can be used to derive L-glutamic acid-producing bacteria in accordance with the disclosed subject matter can include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli VL334thrC+ (EP 1172433). E. coli VL334 (VKPM B-1641) is an L-isoleucine and L-threonine auxotrophic strain having mutations in thrC and ilvA genes (U.S. Pat. No. 4,278,765). A wild-type allele of the thrC gene was transferred by the method of general transduction using a bacteriophage P1 grown on the wild-type E. coli strain K12 (VKPM B-7) cells. As a result, an L-isoleucine auxotrophic strain VL334thrC+ (VKPM B-8961), which is able to produce L-glutamic acid, was obtained.
Examples of parent strains which can be used to derive the L-glutamic acid-producing bacteria in accordance with the disclosed subject matter can include, but are not limited to, strains in which expression of one or more genes encoding an L-glutamic acid biosynthetic enzyme are enhanced. Examples of such genes can include genes encoding glutamate dehydrogenase (gdhA), glutamine synthetase (glnA), glutamate synthetase (gltAB), isocitrate dehydrogenase (icdA), aconitate hydratase (acnA, acnB), citrate synthase (gltA), phosphoenolpyruvate carboxylase (ppc), pyruvate carboxylase (pyc), pyruvate dehydrogenase (aceEF, lpdA), pyruvate kinase (pykA, pykF), phosphoenolpyruvate synthase (ppsA), enolase (eno), phosphoglyceromutase (pgmA, pgmI), phosphoglycerate kinase (pgk), glyceraldehyde-3-phophate dehydrogenase (gapA), triose phosphate isomerase (tpiA), fructose 1,6-bisphosphatase (Jbp), phosphofructokinase (pjkA, pjkB), and glucose phosphate isomerase (pgi).
Examples of strains modified so that expression of the citrate synthetase gene, the phosphoenolpyruvate carboxylase gene, and/or the glutamate dehydrogenase gene is/are enhanced can include those disclosed in EP1078989A, EP955368A, and EP952221A.
Examples of parent strains which can be used to derive the L-glutamic acid-producing bacteria in accordance with the disclosed subject matter also can include strains having decreased or eliminated activity of an enzyme that catalyzes synthesis of a compound other than L-glutamic acid by branching off from an L-glutamic acid biosynthesis pathway. Examples of such enzymes can include isocitrate lyase (aceA), α-ketoglutarate dehydrogenase (sucA), phosphotransacetylase (pta), acetate kinase (ack), acetohydroxy acid synthase (ilvG), acetolactate synthase (ilvI), formate acetyltransferase (pfl), lactate dehydrogenase (ldh), and glutamate decarboxylase (gadAB). Bacteria belonging to the genus Escherichia which are deficient in α-ketoglutarate dehydrogenase activity or having reduced α-ketoglutarate dehydrogenase activity and methods for obtaining them are described in U.S. Pat. Nos. 5,378,616 and 5,573,945. Specifically, these strains can include the following:
E. coli W3110sucA::KmR 
E. coli AJ12624 (FERM BP-3853)
E. coli AJ12628 (FERM BP-3854)
E. coli AJ12949 (FERM BP-4881)
E. coli W3110sucA::KmR is a strain obtained by disrupting the α-ketoglutarate dehydrogenase gene (hereinafter referred to as “sucA gene”) of E. coli W3110. This strain is completely deficient in the α-ketoglutarate dehydrogenase.
Other examples of L-glutamic acid-producing bacterium can include those which belong to the genus Escherichia and have resistance to an aspartic acid antimetabolite. These strains also can be deficient in the α-ketoglutarate dehydrogenase activity and include, for example, E. coli AJ13199 (FERM BP-5807)(U.S. Pat. No. 5,908,768), FFRM P-12379, which additionally has a low L-glutamic acid decomposing ability (U.S. Pat. No. 5,393,671); AJ13138 (FERM BP-5565)(U.S. Pat. No. 6,110,714), and the like.
Examples of L-glutamic acid-producing bacteria can include mutant strains belonging to the genus Pantoea which are deficient in the α-ketoglutarate dehydrogenase activity or have a decreased α-ketoglutarate dehydrogenase activity, and can be obtained as described above. Such strains can include Pantoea ananatis AJ13356. (U.S. Pat. No. 6,331,419). Pantoea ananatis AJ13356 was deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (currently, National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary, Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan) on Feb. 19, 1998 under an accession number of FERM P-16645. It was then converted to an international deposit under the provisions of Budapest Treaty on Jan. 11, 1999 and received an accession number of FERM BP-6615. Pantoea ananatis AJ13356 is deficient in the α-ketoglutarate dehydrogenase activity as a result of disruption of the αKGDH-E1 subunit gene (sucA). The above strain was identified as Enterobacter agglomerans when it was isolated and deposited as the Enterobacter agglomerans AJ13356. However, it was recently re-classified as Pantoea ananatis on the basis of nucleotide sequencing of 16S rRNA, etc. Although AJ13356 was deposited at the aforementioned depository as Enterobacter agglomerans, for the purposes of this specification, they are described as Pantoea ananatis. 
L-Phenylalanine-Producing Bacteria
Examples of parent strains which can be used to derive L-phenylalanine-producing bacteria in accordance with the disclosed subject matter can include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli AJ12739 (tyrA::Tn10, tyrR)(VKPM B-8197); E. coli HW1089 (ATCC 55371) harboring the mutant pheA34 gene (U.S. Pat. No. 5,354,672); E. coli MWEC101-b (KR8903681); E. coli NRRL B-12141, NRRL B-12145, NRRL B-12146 and NRRL B-12147 (U.S. Pat. No. 4,407,952). Also, as a parent strain, E. coli K-12 [W3110 (tyrA)/pPHAB (FERM BP-3566), E. coli K-12 [W3110 (tyrA)/pPHAD] (FERM BP-12659), E. coli K-12 [W3110 (tyrA)/pPHATerm] (FERM BP-12662) and E. coli K-12 [W3110 (tyrA)/pBR-aroG4, pACMAB] named as AJ 12604 (FERM BP-3579) can be used (EP 488-424 B1). Furthermore, L-phenylalanine producing bacteria belonging to the genus Escherichia with an enhanced activity of the protein encoded by the yedA gene or the yddG gene also can be used (U.S. patent applications 2003/0148473 A1 and 2003/0157667 A1).
L-Tryptophan-Producing Bacteria
Examples of parent strains which can be used to derive the L-tryptophan-producing bacteria in accordance with the disclosed subject matter can include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli JP4735/pMU3028 (DSM10122) and JP6015/pMU91 (DSM10123) deficient in the tryptophanyl-tRNA synthetase encoded by mutant trpS gene (U.S. Pat. No. 5,756,345); E. coli SV164 (pGH5) having a serA allele encoding phosphoglycerate dehydrogenase not subject to feedback inhibition by serine and a trpE allele encoding anthranilate synthase not subject to feedback inhibition by tryptophan (U.S. Pat. No. 6,180,373); E. coli AGX17 (pGX44)(NRRL B-12263) and AGX6(pGX50)aroP (NRRL B-12264) deficient in the enzyme tryptophanase (U.S. Pat. No. 4,371,614); E. coli AGX17/pGX50,pACKG4-pps in which a phosphoenolpyruvate-producing ability is enhanced (WO9708333, U.S. Pat. No. 6,319,696), and the like can be used. L-tryptophan-producing bacteria belonging to the genus Escherichia with an enhanced activity of the identified protein encoded by and the yedA gene or the yddG gene also can be used (U.S. patent applications 2003/0148473 A1 and 2003/0157667 A1).
Examples of parent strains which can be used to derive the L-tryptophan-producing bacteria in accordance with the disclosed subject matter also can include strains in which one or more activities of the enzymes anthranilate synthase, phosphoglycerate dehydrogenase, and tryptophan synthase are enhanced. The anthranilate synthase and phosphoglycerate dehydrogenase are both subject to feedback inhibition by L-tryptophan and L-serine, so that a mutation desensitizing the feedback inhibition can be introduced into these enzymes. Specific examples of strains having such a mutation can include a E. coli SV164 which harbors desensitized anthranilate synthase and a transformant strain obtained by introducing into the E. coli SV164 the plasmid pGH5 (WO 94/08031), which contains a mutant serA gene encoding feedback-desensitized phosphoglycerate dehydrogenase.
Examples of parent strains which can be used to derive the L-tryptophan-producing bacteria in accordance with the disclosed subject matter also can include strains into which the tryptophan operon which contains a gene encoding desensitized anthranilate synthase has been introduced (JP 57-71397 A, JP 62-244382 A, U.S. Pat. No. 4,371,614). Moreover, L-tryptophan-producing ability can be imparted by enhancing expression of a gene which encodes tryptophan synthase, among tryptophan operons (trpBA). The tryptophan synthase consists of a and β subunits which are encoded by the trpA and trpB genes, respectively. In addition, L-tryptophan-producing ability can be improved by enhancing expression of the isocitrate lyase-malate synthase operon (WO2005/103275).
L-Proline-Producing Bacteria
Examples of parent strains which can be used to derive L-proline-producing bacteria oin accordance with the disclosed subject matter can include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli 702ilvA (VKPM B-8012) which is deficient in the ilvA gene and is able to produce L-proline (EP 1172433). Bacteria in accordance with the disclosed subject matter can be improved by enhancing the expression of one or more genes involved in L-proline biosynthesis. Examples of such genes for L-proline producing bacteria which are preferred can include the proB gene coding for glutamate kinase which is desensitized to feedback inhibition by L-proline (DE Patent 3127361). In addition, bacteria in accordance with the disclosed subject matter can be improved by enhancing the expression of one or more genes coding for proteins excreting L-amino acid from bacterial cell. Such genes can be exemplified by b2682 and b2683 genes (ygaZH genes)(EP1239041 A2).
Examples of bacteria belonging to the genus Escherichia, which have an activity to produce L-proline can include the following E. coli strains: NRRL B-12403 and NRRL B-12404 (GB Patent 2075056), VKPM B-8012 (Russian patent application 2000124295), plasmid mutants described in DE Patent 3127361, plasmid mutants described by Bloom F. R. et al (The 15th Miami winter symposium, 1983, p. 34), and the like.
L-Arginine-Producing Bacteria
Examples of parent strains which can be used to derive L-arginine-producing bacteria in accordance with the disclosed subject matter can include, but are not limited to, strains belonging to the genus Escherichia, such as E. coli strain 237 (VKPM B-7925)(U.S. Patent Application 2002/058315 A1) and its derivative strains harboring mutant N-acetylglutamate synthase (Russian Patent Application No. 2001112869), E. coli strain 382 (VKPM B-7926) (EP1170358A1), an arginine-producing strain into which argA gene encoding N-acetylglutamate synthetase is introduced therein (EPI 170361A1), and the like.
Examples of parent strains which can be used to derive L-arginine producing bacteria in accordance with the disclosed subject matter also can include strains in which expression of one or more genes encoding an L-arginine biosynthetic enzyme are enhanced. Examples of such genes can include genes encoding N-acetylglutamyl phosphate reductase (argC), ornithine acetyl transferase (argJ), N-acetylglutamate kinase (argB), acetylornithine transaminase (argD), ornithine carbamoyl transferase (argF), argininosuccinic acid synthetase (argG), argininosuccinic acid lyase (argH), and carbamoyl phosphate synthetase (carAB).
L-Valine-Producing Bacteria
Example of parent strains which can be used to derive L-valine-producing bacteria in accordance with the disclosed subject matter can include, but are not limited to, strains which have been modified to overexpress the ilvGMEDA operon (U.S. Pat. No. 5,998,178). The region of the ilvGMEDA operon which is required for attenuation can be removed so that expression of the operon is not attenuated by the L-valine that is produced. Furthermore, the ilvA gene in the operon can be disrupted so that threonine deaminase activity is decreased.
Examples of parent strains which can be used to derive L-valine-producing bacteria in accordance with the disclosed subject matter can include also mutants having a mutation of amino-acyl t-RNA synthetase (U.S. Pat. No. 5,658,766). For example, E. coli VL1970, which has a mutation in the ileS gene encoding isoleucine tRNA synthetase, can be used. E. coli VL1970 has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM)(Russia, 117545 Moscow, 1 Dorozhny Proezd, 1) on Jun. 24, 1988 under accession number VKPM B-4411.
Furthermore, mutants requiring lipoic acid for growth and/or lacking H+-ATPase can be also used as parent strains (WO96/06926).
L-Isoleucine-Producing Bacteria
Examples of parent strains which can be used to derive L-isoleucine producing bacteria in accordance with the disclosed subject matter can include, but are not limited to, mutants having resistance to 6-dimethylaminopurine (JP 5-304969 A), mutants having resistance to an isoleucine analogue such as thiaisoleucine and isoleucine hydroxamate, and mutants additionally having resistance to DL-ethionine and/or arginine hydroxamate (JP 5-130882 A). In addition, recombinant strains transformed with genes encoding proteins involved in L-isoleucine biosynthesis, such as threonine deaminase and acetohydroxate synthase, also can be used as parent strains (JP 2-458 A, FR 0356739, and U.S. Pat. No. 5,998,178).
2. Method of the Present Invention
A method in accordance with the disclosed subject matter can produce an L-amino acid by cultivating bacteria in accordance with the disclosed subject matter in a culture medium containing glycerol to produce and excrete the L-amino acid into the medium, and collecting the L-amino acid from the medium.
In accordance with the disclosed subject matter, the cultivation, collection, and purification of an L-amino acid from the medium, and the like, can be performed in a manner similar to conventional fermentation methods wherein an amino acid can be produced using a bacterium.
A medium used for culture can be either a synthetic or natural medium, so long as the medium includes a carbon source and a nitrogen source and minerals and, if necessary, appropriate amounts of nutrients which the bacterium requires for growth.
As a carbon source, glycerol can be used. Glycerol can be used individually or as a mixture with carbohydrate(s) or sugar(s). Examples of carbohydrates and sugars can include glucose, sucrose, lactose, fructose, maltose, starch. Additionally, cellulose, oils and fats (such as soybean oil, sunflower oil, peanut oil, and coconut fat), fatty acids (such as palmitic acid, stearic acid and linoleic acid), and alcohols (such as, ethanol and methanol) can be used in combination with the above mentioned carbohydrates and sugars. The ratio of glycerol in a total carbon source can be in range of more than 50%, a range of more than 60%, a range of more than 70%, a range of more than 80%, or a range of more than 90%. Alternatively, the ratio of glycerol can be 100%. In an exemplary embodiment in accordance with the disclosed subject matter, the glycerol can be obtained as by-product of biodiesel production (Mu Y, et al, Biotechnol Lett., 28, 1755-91759 (2006), Haas M J, et al; Bioresour Technol. 97, 4, 671-8678 (2006)).
In a method in accordance with the disclosed subject matter, a batch culture, fed-batch culture, and/or a continuous culture can be employed. And, the glycerol in the medium can be regulated to be at a predetermined level in the initial medium, or the feed medium or by using these techniques in combination. The glycerol can be used for both the initial medium and the feed medium, or the carbon source in the feed medium can be different from that used in the initial medium.
As the nitrogen source, various ammonium salts such as ammonia and ammonium sulfate, other nitrogen compounds such as amines, a natural nitrogen source such as peptone, soybean-hydrolysate, and digested fermentative microorganism can be used. As minerals, potassium monophosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, calcium chloride, and the like can be used. As vitamins, thiamine, yeast extract, and the like, can be used.
The cultivation can be performed under aerobic conditions, such as a shaking culture, and a stirring culture with aeration, at a temperature of 20 to 40° C., preferably 30 to 38° C. The pH of the culture is usually between 5 and 9, preferably between 6.5 and 7.2. The pH of the culture can be adjusted with ammonia, calcium carbonate, various acids, various bases, and buffers. Usually, a 1 to 5-day cultivation leads to accumulation of the target L-amino acid in the liquid medium.
After cultivation, solids such as cells can be removed from the liquid medium by centrifugation or membrane filtration, and then the L-amino acid can be collected and purified by ion-exchange, concentration, and/or crystallization methods.
EXAMPLES
The present invention will be explained below in more detail with reference to the following non-limiting Examples.
Reference Example 1
Construction of a Strain Containing Mutant glpK Gene Coding for Glycerol Kinase with the Known Mutation
1. Deletion of the glpK Gene
According to the approach described by Yu D. et al., a small deletion (34 bp) in the gene glpK around the site of interest was introduced by the Red-dependent recombination procedure. A strain having a deletion of the glpK gene was constructed by the Red-driven integration. The DNA fragment containing the CmR marker encoded by the cat gene was obtained by PCR, using primers P3 (SEQ ID NO: 15) and P4 (SEQ ID NO: 16) and plasmid pMW118-attL-Cm-attR as a template. Primer P3 contains both a region complementary to the 36-nt region of the glpK gene upstream the region of deletion and a region complementary to the attL region. Primer P4 contains both a region complementary to the 35-nt region of the glpK downstream of the region of deletion gene, and a region complementary to the attR region. Conditions for PCR were as follows: denaturation step for 3 min at 95° C.; profile for two first cycles: 1 min at 95° C., 30 sec at 50° C., 40 sec at 72° C.; profile for the last 25 cycles: 30 sec at 95° C., 30 sec at 54° C., 40 sec at 72° C.; final step: 5 min at 72° C.
A 1699-bp PCR product (FIG. 2) was obtained and purified in agarose gel and was used for electroporation of the E. coli strain MG1655, which contains the plasmid pKD46. The plasmid pKD46 (Datsenko, K. A. and Wanner, B. L., Proc. Natl. Acad. Sci. USA, 97: 6640-6645 (2000)) includes a 2,154 nucleotide DNA fragment of phage λ (nucleotide positions 31088 to 33241, GenBank accession no. J02459), and contains genes of the λ Red homologous recombination system (γ, β, exo genes) under the control of the arabinose-inducible ParaB promoter. The plasmid pKD46 is necessary for integration of the PCR product into the chromosome of strain MG1655. The MG1655 strain is available from the American Type Culture Collection (P.O. Box 1549, Manassas, Va. 20108, 1, United States of America; ATCC700926, ATCC47076).
Electrocompetent cells were prepared as follows: E. coli MG1655/pKD46 was grown overnight at 30° C. in LB medium containing ampicillin (100 mg/l), and the culture was diluted 100 times with 5 ml of SOB medium (Sambrook et al, “Molecular Cloning: A Laboratory Manual, Second Edition”, Cold Spring Harbor Laboratory Press, 1989) containing ampicillin and L-arabinose (1 mM). The cells were grown with aeration at 30° C. to an OD600 of ≈0.6 and then were made electrocompetent by concentrating 100-fold and washing three times with ice-cold deionized H2O. Electroporation was performed using 70 μl of cells and ≈100 ng of the PCR product. Cells after electroporation were incubated with 1 ml of SOC medium (Sambrook et al, “Molecular Cloning: A Laboratory Manual, Second Edition”, Cold Spring Harbor Laboratory Press, 1989) at 37° C. for 2.5 hours and then were plated onto L-agar containing chloramphenicol (30 μg/ml) and grown at 37° C. to select CmR recombinants.
Electroporation was performed using 70 μl of cells and ≈100 ng of the PCR product. Cells after electroporation were incubated with 1 ml of SOC medium at 37° C. for 2.5 hours and then were plated onto L-agar containing chloramphenicol (30 μg/ml) and grown at 37° C. to select CmR recombinants.
2. Verification of the glpK Gene Deletion by PCR
The mutants having the glpK gene deleted and marked with the Cm resistance gene were verified by PCR. Locus-specific primers P1 (SEQ ID NO: 13) and P2 (SEQ ID NO: 14) were used in PCR for the verification. Conditions for PCR verification were as follows: denaturation step for 3 min at 94° C.; profile for 30 cycles: 30 sec at 94° C., 30 sec at 54° C., 1 min at 72° C.; final step: 7 min at 72° C. The PCR product obtained in the reaction with the cells of parental glpK+ strain MG1655 as a template, was  ̃0.4 kbp in length. The PCR product obtained in the reaction with the cells of mutant strain as the template was  ̃2.1 kbp in length (FIG. 3). The mutant strain was named MG1655 AglpK::cat-2. This strain was not able to grow on minimal Adams medium with agar containing glycerol (0.5%) as the sole carbon source and formed white-color colonies on MacConkey agar containing glycerol (0.5%).
3. Construction of strains containing mutant glpK gene coding for glycerol kinase with Gly231Asp substitution
A strain containing the glpK gene coding for glycerol kinase with Gly231Asp substitution was constructed by using the Red-driven integration. To introduce G231D substitution the special oligonucleotides with overlapping termini, primers P5 (SEQ ID NO: 17) and P6(SEQ ID NO: 18) were synthesized. Primers P5 and P6 were used for electroporation of the E. coli strain MG1655ΔglpK::cat-2, which contains the plasmid pKD46. Electrocompetent cells were prepared as described in Example 1. Electroporation was performed using 70 μl of cells and ≈100 ng of P5 and P6 oligonucleotides. Cells after electroporation were incubated with 1 ml of SOC medium at 37° C. for 2.5 hours and then were plated onto minimal Adams medium with agar containing glycerol (0.5%) as the sole carbon source and grown at 37° C. to select recombinants. Several colonies, which were able to grow on glycerol, were selected. All these clones were CmS and formed red-color colonies on MacConkey agar containing glycerol(0.5%). Then, to eliminate the pKD46 plasmid, two passages on L-agar at 42° C. were performed and the obtained colonies were tested for sensitivity to ampicillin.
4. Verification of the glpK Gene Mutation Causing Gly231Asp Substitution in Glycerol Kinase
The selected recombinants with deletion of the glpK gene were verified by PCR. Locus-specific primers P1 (SEQ ID NO: 13) and P2 (SEQ ID NO: 14) were used in PCR for the verification. Conditions for PCR verification were as follows: denaturation step for 3 min at 94° C.; profile for 30 cycles: 30 sec at 94° C., 30 sec at 54° C., 1 min at 72° C.; final step: 7 min at 72° C. The obtained PCR product was  ̃0.4 kbp in length, and that confirmed the presence of the full-length glpK gene in selected recombinants. One of them was chosen for sequence analysis and the presence of the Gly231Asp mutation in glpK gene was confirmed. The mutant strain was named MG1655 glpK*-231.
Example 1
Construction of Strains Containing the Mutant glpK Gene Coding for Glycerol Kinase with the Gly234Asp Substitution
1. Introduction of the glpK gene coding for glycerol kinase with Gly234Asp substitution into the glpK gene-deleted strain
A strain containing the glpK gene coding for glycerol kinase with Gly234Asp substitution was constructed by using the Red-driven integration. To introduce the G234D substitution of the special oligonucleotides with overlapping termini, primers P7 (SEQ ID NO: 19) and P8 (SEQ ID NO: 20) were synthesized. Primers P7 and P8 were used for electroporation of the E. coli strain MG1655ΔglpK::cat-2, which contains the plasmid pKD46. Electrocompetent cells were prepared as described in Example 1. Electroporation was performed using 70 μl of cells and ≈100 ng of P7 and P8 oligonucleotides. Cells after electroporation were incubated with 1 ml of SOC medium at 37° C. for 2.5 hours and then were plated onto minimal medium Adams with agar containing glycerol (0.5%) as the sole carbon source and grown at 37° C. to select recombinants. Several colonies, which were able to grow on glycerol, were selected. All these clones were CmS and formed red-color colonies on MacConkey agar containing glycerol(0.5%). Then, to eliminate the pKD46 plasmid, two passages on L-agar at 42° C. were performed and the obtained colonies were tested for sensitivity to ampicillin.
2. Verification of the glpK Gene Mutation Causing Gly234Asp Substitution in Glycerol Kinase
The selected recombinants with deletion of the glpK gene were verified by PCR. Locus-specific primers P1 (SEQ ID NO: 13) and P2 (SEQ ID NO: 14) were used in PCR for the verification. Conditions for PCR verification were as follows: denaturation step for 3 min at 94° C.; profile for 30 cycles: 30 sec at 94° C., 30 sec at 54° C., 1 min at 72° C.; final step: 7 min at 72° C. The obtained PCR product was  ̃0.4 kbp in length, which confirmed presence of full-length glpK gene in selected recombinants. One of them was chosen for sequence analysis and the presence of Gly234Asp mutation in glpK gene was confirmed. The mutant strain was named MG1655 glpK*-234.
Example 2
Construction of Strains Containing Mutant glpK Gene Coding for Glycerol Kinase with Random Substitutions in Positions 231-235
1. Introduction of the glpK gene encoding glycerol kinase having random substitutions into the glpK gene-deleted strain
Strains containing the glpK gene coding for glycerol kinase with random substitutions in positions 231-235 were constructed by using the Red-driven integration. To introduce random substitutions in positions 231-234 the special oligonucleotides with overlapping termini, primers P9 (SEQ ID NO: 21) and P10 (SEQ ID NO: 22) were synthesized. Primer P9 has 70 nucleotides and has a region with 7 random nucleotides, depicted in SEQ ID NO: 21 by the letters “h” (for A or C or T), “d” (for A or G or T) and “v” (for A or C or G). Primer P10 has 70 nucleotides and has a region with 7 random nucleotides, depicted in SEQ ID NO: 22 by the letters “h” (for A or C or T), “d” (for A or G or T) and “b” (for C or G or T). Primers P9 and P10 were used for electroporation of the E. coli strain MG1655ΔglpK::cat-2, which contains the plasmid pKD46. Electrocompetent cells were prepared as described in Reference Example 1. Electroporation was performed using 70 μl of cells and ≈100 ng of P9 and P10 oligonucleotides. Cells after electroporation were incubated with 1 ml of SOC medium at 37° C. for 2.5 hours and then were plated onto minimal Adams medium with agar containing glycerol (0.5%) as the sole carbon source and grown at 37° C. to select recombinants. Several colonies, which were able to grow on glycerol, were selected. All these clones were CmS and formed red-color colonies on MacConkey agar containing glycerol (0.5%). Then, to eliminate the pKD46 plasmid, two passages on L-agar at 42° C. were performed and the obtained colonies were tested for sensitivity to ampicillin.
2. Sequencing the glpK Gene Mutations Causing Random Substitutions in Positions 231-235 in Glycerol Kinase
The selected recombinants with deletion of the glpK gene were verified by PCR. Locus-specific primers P1 (SEQ ID NO:) and P2 (SEQ ID NO: 14) were used in PCR for the verification. Conditions for PCR verification were as follows: denaturation step for 3 min at 94° C.; profile for 30 cycles: 30 sec at 94° C., 30 sec at 54° C., 1 min at 72° C.; final step: 7 min at 72° C. The obtained PCR product was  ̃0.4 kbp in length, which confirmed the presence of the full-length glpK gene in selected recombinants. Then selected recombinants were grown in minimal Adams medium containing glycerol (0.5%). Several of them, which utilized glycerol more effectively than other strains, were chosen for sequence analysis. Sequences of 231-235 region of mutated glycerol kinase genes with SEQ ID numbers and corresponding amino acid sequences are shown in Table 1. The general name of strains having substitutions in positions 231-235 in glycerol kinase is MG1655 glpK*-random.












TABLE 1







SEQ ID NOs
Corresponding
SEQ





for nucleotide
amino acid
ID


Strain
Nucleotide sequence
sequence
sequence
NOs







MG1655
GGC-GGC-AAA-GGC-GGC
3 (691-705)
Gly-Gly-Lys-Gly-Gly
48



 


glpK-234
GGC-GGC-AAA-GAC-GGC
27
Gly-Gly-Lys-Asp-Gly
49


 


RM-1
GGC-GGC-AGA-ATA-CTA
28
Gly-Gly-Arg-Ile-Leu
50


 


RM-3
GGC-GGC-AGA-CTT-GAA
29
Gly-Gly-Arg-Leu-Glu
51


 


RM-10
GGC-GGC-AAA-ACG-TTT
30
Gly-Gly-Lys-Thr-Phe
52


 


RM-18
GGC-GGC-AGA-ATA-CCT
39
Gly-Gly-Arg-Ile-Pro
53


 


RM-19
GGC-GGC-ACA-TTG-TTA
40
Gly-Gly-Thr-Leu-Leu
54


 


RM-20
CGG-GAA-GCT-CGA-GCC
41
Arg-Glu-Ala-Arg-Ala
55


 


RM-39
CGA-GCA-CAC-CTT-GCC
42
Arg-Ala-His-Leu-Ala
56


 


glpK-231
GAC-GGC-AAA-GGC-GGC
47
Asp-Gly-Lys-Gly-Gly
57









Example 3
Production of L-Threonine by E. coli Strain MG1655 Δtdh::rhtA*glpK*-234/pVIC40
To test the effect of increased activity of the glycerol kinase on threonine production, a DNA fragment from the chromosome of the above-described E. coli MG1655 glpK*-234 was transferred to the threonine-producing E. coli strain MG1655 Δtdh::rhtA*/pVIC40 by P1 transduction (Miller, J. H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, N.Y.) to obtain strain MG1655 Δtdh::rhtA* glpK*-234/pVIC40. Construction of the E. coli strain MG1655 Δtdh::rhtA*/pVIC40 is described below in the Reference example 2.
Both E. coli strains, MG1655 Δtdh::rhtA*/pVIC40 and MG1655 Δtdh::rhtA* glpK*-234/pVIC40, were grown for 18-24 hours at 37° C. on L-agar plates. To obtain a seed culture, the strains were grown on a rotary shaker (250 rpm) at 32° C. for 18 hours in 20×200-mm test tubes containing 2 ml of L-broth supplemented with 4% glycerol. Then, the fermentation medium was inoculated with 0.21 ml (10%) of seed material. The fermentation was performed in 2 ml of minimal medium for fermentation in 20×200-mm test tubes. Cells were grown for 72 hours at 34° C. with shaking at 250 rpm.
After cultivation, the amount of L-threonine which had accumulated in the medium, was determined by paper chromatography using the following mobile phase: butanol−acetic acid−water=4:1:1 (v/v). A solution of ninhydrin (2%) in acetone was used as a visualizing reagent. A spot containing L-threonine was cut out, L-threonine was eluted with 0.5% water solution of CdCl2, and the amount of L-threonine was estimated spectrophotometrically at 540 nm. The results of five independent test tube fermentations are shown in Table 2. As follows from Table 2, MG1655 Δtdh::rhtA* glpK*-234/pVIC40 produced a higher amount of L-threonine, as compared with MG1655 Δtdh::rhtA*/pVIC40.
The composition of the fermentation medium (g/l) was as follows:

















Glycerol
60.0




(NH4)2SO4
31.2



KH2PO4
1.9



MgSO4 7H2O
0.63



Thiamine HCl
0.0002



Mameno(soybean hydrolysate)
1.36
ml



CaCO3
20.0










Magnesium sulfate were sterilized separately. CaCO3 was sterilized by dry-heat at 180° C. for 2 hours. The pH was adjusted to 7.0.









TABLE 2







Amount of


Strain
OD540
L-threonine, g/l







MG1655 Δtdh::rhtA*/pVIC40
56.5 ± 1.8
5.8 ± 0.6


MG1655 Δtdh::rhtA* glpK*-234/pVIC40
65.7 ± 2.1
7.2 ± 0.3









Example 4
Production of L-Threonine by E. coli Strain MG1655 Δtdh::rhtA* glpK*-RM18/pVIC40 and MG1655 Δtdh::rhtA* glpK*-RM39/pVIC40
To test the effect of increased activity of the glycerol kinase on threonine production, DNA fragments from the chromosome of the above-described E. coli MG1655 glpK*-RM18, E. coli MG1655 glpK*-RM39 and E. coli MG1655 glpK*-231 were transferred to the threonine-producing E. coli strain MG1655 Δtdh::rhtA*/PVIC40 by P1 transduction (Miller, J. H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, N.Y.) to obtain strains MG1655 Δtdh::rhtA* glpK*-RM18/pVIC40, MG1655 Δtdh::rhtA* glpK*-RM39/pVIC40 and MG1655 Δtdh::rhtA* glpK*-231/pVIC40. Construction of the E. coli strain MG1655 Δtdh::rhtA*/PVIC40 is described below in the Reference example 2.
E. coli strains, MG1655 Δtdh::rhtA*/pVIC40, MG1655 Δtdh::rhtA* glpK*-231/pVIC40, MG1655 Δtdh::rhtA* glpK*-RM18/pVIC40 and MG1655 Δtdh::rhtA* glpK*-RM39/pVIC40, were grown for 18-24 hours at 37° C. on L-agar plates. To obtain a seed culture, the strains were grown on a rotary shaker (250 rpm) at 32° C. for 18 hours in 20×200-mm test tubes containing 2 ml of L-broth supplemented with 4% glycerol. Then, the fermentation medium was inoculated with 0.21 ml (10%) of seed material. The fermentation was performed in 2 ml of minimal medium for fermentation in 20×200-mm test tubes. Cells were grown for 72 hours at 34° C. with shaking at 250 rpm.
After cultivation, the amount of L-threonine which had accumulated in the medium, was determined by paper chromatography using the following mobile phase: butanol−acetic acid−water=4:1:1 (v/v). A solution of ninhydrin (2%) in acetone was used as a visualizing reagent. A spot containing L-threonine was cut out, L-threonine was eluted with 0.5% water solution of CdCl2, and the amount of L-threonine was estimated spectrophotometrically at 540 nm. The results of five independent test tube fermentations are shown in Table 3. As follows from Table 3, MG1655 Δtdh::rhtA* glpK*-RM 18/pVIC40 and MG1655 Δtdh::rhtA* glpK*-RM 39/pVIC40 produced a higher amount of L-threonine, as compared with MG1655 Δtdh::rhtA*/pVIC40 and MG1655 Δtdh::rhtA* glpK*-231/pVIC40.
The composition of the fermentation medium (g/l) was as follows:

















Glycerol
60.0




(NH4)2SO4
31.2



KH2PO4
1.9



MgSO4 7H2O
0.63



Thiamine HCl
0.0002



Mameno
1.36
ml



CaCO3
20.0










Magnesium sulfate was sterilized separately. CaCO3 was sterilized by dry-heat at 180° C. for 2 hours. The pH was adjusted to 7.0.










TABLE 3









48 hours
72 hours











Strains
OD540
Thr, g/l
OD540
Thr, g/l





MGΔtdh-rhtA*(pVIC40)
40.3 ± 0.1
 7.6 ± 0.2
39.4 ± 0.2
 8.7 ± 0.3


MGΔtdh-rhtA*(pVIC40)
19.8 ± 0.1
 5.8 ± 0.2
35.5 ± 0.3
13.4 ± 0.5


glpK-RM18


MGΔtdh-rhtA*(pVIC40)
35.2 ± 0.4
10.1 ± 0.4
36.3 ± 0.3
12.4 ± 0.3


glpK-RM39


MGΔtdh-rhtA*(pVIC40)
31.1 ± 0.2
10.3 ± 0.3
35.2 ± 0.3
11.7 ± 0.4


glpK-231









Example 5
Production of L-Threonine by E. coli Strains B-3996 glpK*-231, and B-3996 glpK*-Random
To test the effect of enhanced ability to utilize glycerol on threonine production, DNA fragments from the chromosome of the above-described E. coli strains MG1655 glpK*-231 and MG1655 glpK*-random can be transferred to the threonine-producing E. coli strain VKPM B-3996 by P1 transduction (Miller, J. H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, N.Y.) to obtain the strains B-3996 glpK*-231 and B-3996 glpK*-random, respectively.
E. coli strains, B-3996, B-3996 glpK*-231, and B-3996 glpK*-random, can be grown for 18-24 hours at 37° C. on L-agar plates. To obtain a seed culture, the strains can be grown on a rotary shaker (250 rpm) at 32° C. for 18 hours in 20×200-mm test tubes containing 2 ml of L-broth supplemented with 4% glucose. Then, the fermentation medium can be inoculated with 0.21 ml (10%) of seed material. The fermentation can be performed in 2 ml of minimal medium for fermentation in 20×200-mm test tubes. Cells can be grown for 65 hours at 32° C. with shaking at 250 rpm.
After cultivation, the amount of L-threonine, which had accumulated in the medium, can be determined by paper chromatography using the following mobile phase: butanol−acetic acid−water=4:1:1 (v/v). A solution of ninhydrin (2%) in acetone can be used as a visualizing reagent. A spot containing L-threonine can be cut out, L-threonine can be eluted with 0.5% water solution of CdCl2, and the amount of L-threonine can be estimated spectrophotometrically at 540 nm.
The composition of the fermentation medium (g/l) is as follows:
















Glycerol
60.0



(NH4)2SO4
22.0



NaCl
0.8



KH2PO4
2.0



MgSO4 7H2O
0.8



FeSO4 7H2O
0.02



MnSO4 5H2O
0.02



Thiamine HCl
0.0002



Yeast extract
1.0



CaCO3
30.0










Glucose and magnesium sulfate are sterilized separately. CaCO3 is sterilized by dry-heat at 180° C. for 2 hours. The pH is adjusted to 7.0. The antibiotic is introduced into the medium after sterilization.
Example 6
Construction of a strain containing mutant glpK gene and an inactivated glpR gene
1. Deletion of the glpR gene
A strain having the mutation in the position 231-235 and deletion of the glpR gene is constructed by the Red-driven integration. The DNA fragment containing the CmR marker encoded by the cat gene is obtained by PCR, using primers P11 (SEQ ID NO: 23) and P12 (SEQ ID NO: 24) and plasmid pMW118-attL-Cm-attR as a template. Primer P11 contains both a region complementary to the 36-nt region located at the 3′ end of the glpR gene and a region complementary to the attL region. Primer P12 contains both a region complementary to the 35-nt region located at the 5′ end of the glpR gene and a region complementary to the attR region. Conditions for PCR are as follows: denaturation step for 3 min at 95° C.; profile for two first cycles: 1 min at 95° C., 30 sec at 50° C., 40 sec at 72° C.; profile for the last 25 cycles: 30 sec at 95° C., 30 sec at 54° C., 40 sec at 72° C.; final step: 5 min at 72° C.
A 1699-bp PCR product (FIG. 2) is obtained and purified in agarose gel and is used for electroporation of the E. coli strain MG1655, which contains the plasmid pKD46.
Electrocompetent cells are prepared as described in Reference Example 1. Electroporation is performed using 70 μl of cells and ≈100 ng of the PCR product. Cells after electroporation are incubated with 1 ml of SOC medium at 37° C. for 2.5 hours and then are plated onto L-agar containing chloramphenicol (30 μg/ml) and grown at 37° C. to select CmR recombinants. Then, to eliminate the pKD46 plasmid, two passages on L-agar with Cm at 42° C. is performed and the obtained colonies are tested for sensitivity to ampicillin.
2. Verification of the glpR Gene Deletion by PCR
The mutants having the glpR gene deleted and marked with the Cm resistance gene are verified by PCR. Locus-specific primers P13 (SEQ ID NO: 25) and P14 (SEQ ID NO: 26) are used in PCR for the verification. Conditions for PCR verification are as follows: denaturation step for 3 min at 94° C.; profile for 30 cycles: 30 sec at 94° C., 30 sec at 54° C., 1 min at 72° C.; final step: 7 min at 72° C. The PCR product obtained in the reaction with the cells of parental glpR+ strain MG1655 as a template, is  ̃1.1 kbp in length. The PCR product obtained in the reaction with the cells of mutant strain as the template is  ̃2.1 kbp in length (FIG. 3). The mutant strain is named MG1655 ΔglpR glpK*-234.
3. Utilization of glycerol by E. coli MG1655 ΔglpR glpK*-234
Overnight cultures of E. coli strains MG1655 and MG1655ΔglpR glpK*-234 are grown at 37° C. in LB broth, and the cultures are washed with NaCl(9%) and diluted 100 times with 5 ml of minimal Adams medium containing glycerol (0.5%) in 20×200-mm test tubes. The tubes with cultures are incubated at 37° C. with shaking at 240 rpm. Periodically aliquots of cultures are taken and optical density of cells is measured spectrophotometrically at 540 nm.
Example 7
Production of L-threonine by E. coli strain B-3996 glpK*-234-ΔglpR
To test the effect of the combination glpK mutation and deletion of the glpR gene on threonine production, DNA fragments from the chromosome of the above-described E. coli MG1655 glpK*ΔglpR can be transferred to the threonine-producing E. coli strain VKPM B-3996 glpK*-234 by P1 transduction to obtain the strain B-3996 glpK*-234-ΔglpR. The strain B-3996 was deposited on Nov. 19, 1987 in the All-Union Scientific Center of Antibiotics (Russia, 117105 Moscow, Nagatinskaya Street, 3-A) under the accession number RIA 1867. The strain was also deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1 Dorozhny proezd, 1) under the accession number B-3996.
Both E. coli strains, B-3996 and B-3996-glpK*-234, and B-3996 glpK*-234-ΔglpR are grown for 18-24 hours at 37° C. on L-agar plates. To obtain a seed culture, the strains are grown on a rotary shaker (250 rpm) at 32° C. for 18 hours in 20×200-mm test tubes containing 2 ml of L-broth supplemented with 4% glycerol. Then, the fermentation medium is inoculated with 0.21 ml (10%) of seed material. The fermentation is performed in 2 ml of minimal medium for fermentation in 20×200-mm test tubes. Cells are grown for 72 hours at 34° C. with shaking at 250 rpm.
After cultivation, the amount of L-threonine which accumulates in the medium, is determined by paper chromatography using the following mobile phase: butanol−acetic acid−water=4:1:1 (v/v). A solution of ninhydrin (2%) in acetone is used as a visualizing reagent. A spot containing L-threonine is cut out, L-threonine is eluted with 0.5% water solution of CdCl2, and the amount of L-threonine is estimated spectrophotometrically at 540 nm. The composition of the fermentation medium (g/l) is as follows:
















(NH4)2SO4
22.0



NaCl
0.8



MgSO4 7H2O
0.8



Fe SO4
0.02



Mn SO4
0.02



K2HPO4
2.0



Thiamine HCl
0.0002



Yeast extract
1.0



CaCO3
30.0



Glycerol
80.0










Magnesium sulfate is sterilized separately. CaCO3 is sterilized by dry-heat at 180° C. for 2 hours. The pH is adjusted to 7.0.
Example 8
Production of L-Lysine by E. coli Strain AJ11442 glpK*-234 or AJ11442 glpK*-Random or AJ11442 glpK*ΔglpR
To test the effect of enhanced ability to utilize glycerol on lysine production, DNA fragments from the chromosome of the above-described E. coli strain MG1655 glpK*-231, MG1655 glpK*-234, MG1655 glpK*-random, or MG1655 ΔglpR can be transferred to the lysine-producing E. coli strain AJ11442 by P1 transduction (Miller, J. H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, N.Y.) to obtain the strain AJ11442 glpK*-231, AJ11442 glpK*-234, AJ11442 glpK*-random, or AJ11442 glpK*-ΔglpR, respectively. The strain AJ14442 was deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology (currently National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary, Tsukuba Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan) on May 1, 1981 and received an accession number of FERM P-5084. Then, it was converted to an international deposit under the provisions of the Budapest Treaty on Oct. 29, 1987, and received an accession number of FERM BP-1543.
E. coli strains, AJ11442, AJ11442 glpK*-231, AJ11442 glpK*-234, AJ11442 glpK*-random, and AJ11442 glpK*-ΔglpR, can be cultured in L-medium at 37° C., and 0.3 ml of the obtained culture can be inoculated into 20 ml of the fermentation medium containing the required drugs in a 500-ml flask. The cultivation can be carried out at 37° C. for 16 h by using a reciprocal shaker at the agitation speed of 115 rpm. After the cultivation, the amounts of L-lysine and residual glucose in the medium can be measured by a known method (Biotech-analyzer AS210 manufactured by Sakura Seiki Co.). Then, the yield of L-lysine can be calculated relative to consumed glucose for each of the strains.
The composition of the fermentation medium (g/l) is as follows:
















Glycerol
60.0



(NH4)2SO4
24



K2HPO4
1.0



MgSO4 7H2O
1.0



FeSO4 7H2O
0.01



MnSO4 5H2O
0.01



Yeast extract
2.0










The pH is adjusted to 7.0 by KOH and the medium is autoclaved at 115° C. for 10 min. Glucose and MgSO4 7H2O are sterilized separately. CaCO3 is dry-heat sterilized at 180° C. for 2 hours and added to the medium for a final concentration of 30 μl.
Example 9
Production of L-cysteine by E. coli strains JM15(ydeD) glpK*-234, JM15(ydeD) glpK*-random or JM15(ydeD) glpK*-ΔglpR
To test the effect of enhanced ability to utilize glycerol on L-cysteine production, DNA fragments from the chromosome of the above-described E. coli strain MG1655 glpK*-231, MG1655 glpK*-234, MG1655 glpK*-random or MG1655 glpK*-ΔglpR can be transferred to the E. coli L-cysteine-producing strain JM15(ydeD) by P1 transduction (Miller, J. H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, N.Y.) to obtain the strains JM15(ydeD) glpK*-231, JM15(ydeD) glpK*-234, JM15(ydeD) glpK*-random and JM15(ydeD) glpK*-ΔglpR, respectively.
E. coli strain JM15(ydeD) is a derivative of E. coli strain JM15 (U.S. Pat. No. 6,218,168) which can be transformed with DNA having the ydeD gene, which codes for a membrane protein, and is not involved in a biosynthetic pathway of any L-amino acid (U.S. Pat. No. 5,972,663). The strain JM15 (CGSC# 5042) can be obtained from The Coli Genetic Stock Collection at the E. coli Genetic Resource Center, MCD Biology Department, Yale University (http://cgsc.biology.yale.edu/).
Fermentation conditions for evaluation of L-cysteine production were described in detail in Example 6 of U.S. Pat. No. 6,218,168.
Example 10
Production of L-leucine by E. coli strains 57 glpK*-234 or 57 glpK*-random or E. coli strains 57 glpK*-ΔglpR
To test the effect of the enhanced ability to utilize glycerol on L-leucine production, DNA fragments from the chromosome of the above-described E. coli strain MG1655 glpK*-231, MG1655 glpK*-234, MG1655 glpK*-random, or MG1655 glpK*-ΔglpR can be transferred to the E. coli L-leucine-producing strain 57 (VKPM B-7386, U.S. Pat. No. 6,124,121) by P1 transduction (Miller, J. H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, N.Y.) to obtain the strain 57 glpK*-231, 57 glpK*-234, 57 glpK*-random, or 57 ΔglpR. The strain 57 has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM)(Russia, 117545 Moscow, 1 Dorozhny proezd, 1) on May 19, 1997 under accession number VKPM B-7386, respectively.
E. coli strains, 57, 57 glpK*-231, 57 glpK*-234, 57 glpK*-random, and 57 glpK*-ΔglpR, can be cultured for 18-24 hours at 37° C. on L-agar plates. To obtain a seed culture, the strains can be grown on a rotary shaker (250 rpm) at 32° C. for 18 hours in 20×200-mm test tubes containing 2 ml of L-broth supplemented with 4% glycerol. Then, the fermentation medium can be inoculated with 0.21 ml of seed material (10%). The fermentation can be performed in 2 ml of a minimal fermentation medium in 20×200-mm test tubes. Cells can be grown for 48-72 hours at 32° C. with shaking at 250 rpm. The amount of L-leucine can be measured by paper chromatography (liquid phase composition: butanol−acetic acid−water=4:1:1).
The composition of the fermentation medium (g/l)(pH 7.2) is as follows:
















Glycerol
60.0



(NH4)2SO4
25.0



K2HPO4
2.0



MgSO4 7H2O
1.0



Thiamine
0.01



CaCO3
25.0










Glucose and CaCO3 are sterilized separately.
Example 11
Production of L-histidine by E. coli strains 80 glpK*-234 or 80 glpK*-random or 80 glpK*-ΔglpR
To test the effect of the enhanced ability to utilize glycerol on L-histidine production, DNA fragments from the chromosome of the above-described E. coli strain MG1655 glpK*-231, MG1655 glpK*-234, MG1655 glpK*-random, or MG1655-glpK*-ΔglpR can be transferred to the histidine-producing E. coli strain 80 by P1 transduction (Miller, J. H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, N.Y.) to obtain the strain 80 glpK*-231, 80 glpK*-234, 80 glpK*-random, or 80 ΔglpR, respectively. The strain 80 has been described in Russian patent 2119536 and deposited in the Russian National Collection of Industrial Microorganisms (Russia, 117545 Moscow, 1 Dorozhny proezd, 1) on Oct. 15, 1999 under accession number VKPM B-7270 and then converted to a deposit under the Budapest Treaty on Jul. 12, 2004.
E. coli strains, 80, 80 glpK*-231, 80 glpK*-234, 80 glpK*-random, and 80 glpK*-ΔglpR, can be cultured in L-broth supplemented with 4% glycerol for 6 h at 29° C. Then, 0.1 ml of obtained culture can be inoculated into 2 ml of fermentation medium in a 20×200-mm test tube and cultivated for 65 hours at 29° C. with shaking on a rotary shaker (350 rpm). After cultivation, the amount of histidine which accumulates in the medium can be determined by paper chromatography. The paper can be developed with a mobile phase consisting of n-butanol:acetic acid:water=4:1:1 (v/v). A solution of ninhydrin (0.5%) in acetone can be used as a visualizing reagent.
The composition of the fermentation medium (g/l) is as follows (pH 6.0):
















Glycerol
60.0



Mameno (soybean hydrolysate)
0.2 of as total nitrogen



-proline
1.0



(NH4)2SO4
25.0



KH2PO4
2.0



MgSO4 7H20
1.0



FeSO4 7H20
0.01



MnSO4
0.01



Thiamine
0.001



Betaine
2.0



CaCO3
60.0










Glucose, proline, betaine and CaCO3 are sterilized separately. The pH is adjusted to 6.0 before sterilization.
Example 12
Production of L-glutamate by E. coli strains VL334thrC+glpK*-234 or VL334thrC+ glpK*-random or VL334thrC+ glpK*-ΔglpR
To test the effect of enhanced ability to utilize glycerol on L-glutamate production, DNA fragments from the chromosome of the above-described E. coli strain MG1655 glpK*-231, MG1655 glpK*-234, MG1655 glpK*-random, and MG1655 glpK*-ΔglpR can be transferred to the E. coli L-glutamate-producing strain VL334thrC+(EP 1172433) by P1 transduction (Miller, J. H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, N.Y.) to obtain the strain VL334thrC+glpK*-231, VL334thrC+glpK*-234, VL334thrC+glpK*-random, or VL334thrC+ ΔglpR, respectively. The strain VL334thrC+ has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM)(Russia, 117545 Moscow, 1 Dorozhny proezd, 1) on Dec. 6, 2004 under the accession number B-8961 and then converted to a deposit under the Budapest Treaty on Dec. 8, 2004.
E. coli strains, VL334thrC+, VL334thrC+glpK*-231, VL334thrC+glpK*-234, VL334thrC+glpK*-random, and VL334thrC+glpK*-ΔglpR, can be grown for 18-24 hours at 37° C. on L-agar plates. Then, one loop of the cells can be transferred into test tubes containing 2 ml of fermentation medium. The fermentation medium contains glycerol (60 μl), ammonium sulfate (25 μl), KH2PO4 (2 g/l), MgSO4 (1 μl), thiamine (0.1 mg/ml), L-isoleucine (70 μg/ml), and CaCO3 (25 μl). The pH is adjusted to 7.2. Glyserol and CaCO3 are sterilized separately. Cultivation can be carried out at 30° C. for 3 days with shaking. After the cultivation, the amount of L-glutamic acid which is produced can be determined by paper chromatography (liquid phase composition of butanol-acetic acid-water=4:1:1) with subsequent staining by ninhydrin (1% solution in acetone) and further elution of the compounds in 50% ethanol with 0.5% CdCl2.
Example 13
Production of L-phenylalanine by E. coli strains AJ12739 glpK*-234, AJ12739 glpK*-random or AJ12739 glpK*-ΔglpR
To test the effect of enhanced ability to utilize glycerol on L-phenylalanine production, DNA fragments from the chromosome of the above-described E. coli strain MG1655 glpK*-231, MG1655 glpK*-234, MG1655 glpK*-random, or MG1655 glpK*-ΔglpR can be transferred to the phenylalanine-producing E. coli strain AJ12739 by P1 transduction (Miller, J. H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, N.Y.) to obtain the strain AJ12739 glpK*-231, AJ12739 glpK*-234, AJ12739 glpK*-random, or AJ12739 AglpR, respectively. The strain AJ12739 has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM)(Russia, 117545 Moscow, 1 Dorozhny proezd, 1) on Nov. 6, 2001 under accession no. VKPM B-8197 and then converted to a deposit under the Budapest Treaty on Aug. 23, 2002.
E. coli strains, AJ12739, AJ12739 glpK*-231, AJ12739 glpK*-234, AJ12739 glpK*-random, and AJ12739 glpK*-ΔglpR, can be cultivated at 37° C. for 18 hours in a nutrient broth supplemented with 4% glycerol, and 0.3 ml of the obtained culture can each be inoculated into 3 ml of a fermentation medium in a 20×200-mm test tube and cultivated at 37° C. for 48 hours with shaking on a rotary shaker. After cultivation, the amount of phenylalanine which accumulates in the medium can be determined by TLC. The 10×15-cm TLC plates coated with 0.11-mm layers of Sorbfil silica gel containing no fluorescent indicator (Stock Company Sorbpolymer, Krasnodar, Russia) can be used. The Sorbfil plates can be developed with a mobile phase consisting of propan-2-ol:ethylacetate:25% aqueous ammonia:water=40:40:7:16 (v/v). A solution of ninhydrin (2%) in acetone can be used as a visualizing reagent.
The composition of the fermentation medium (g/l) is as follows:
















Glycerol
60.0



(NH4)2SO4
16.0



K2HPO4
0.1



MgSO4 7H2O
1.0



FeSO4 7H2O
0.01



MnSO4 5H2O
0.01



Thiamine HCl
0.0002



Yeast extract
2.0



Tyrosine
0.125



CaCO3
20.0










Glucose and magnesium sulfate are sterilized separately. CaCO3 is dry-heat sterilized at 180° C. for 2 hours. The pH is adjusted to 7.0.
Example 14
Production of L-tryptophan by E. coli strains SV164(pGH5) glpK*-234 or SV164(pGH5) glpK*-random or SV164(pGH5) glpK*-ΔglpR
To test the effect of enhanced ability to utilize glycerol on L-tryptophan production, DNA fragments from the chromosome of the above-described E. coli strain MG1655 glpK*-231, MG1655 glpK*-234, MG1655 glpK*-random, or MG1655 glpK*-ΔglpR can be transferred to the tryptophan-producing E. coli strain SV164 (pGH5) by P1 transduction (Miller, J. H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, N.Y.) to obtain the strain SV164(pGH5) glpK*-231, SV164(pGH5) glpK*-234, SV164(pGH5) glpK*-random, or SV164(pGH5) ΔglpR, respectively. The strain SV164 has the trpE allele encoding anthranilate synthase free from feedback inhibition by tryptophan. The plasmid pGH5 harbors a mutant serA gene encoding phosphoglycerate dehydrogenase free from feedback inhibition by serine. The strain SV164 (pGH5) was described in detail in U.S. Pat. No. 6,180,373 or European patent 0662143.
E. coli strains, SV164(pGH5), SV164(pGH5) glpK*-231, SV164(pGH5) glpK*-234, SV164(pGH5) glpK*-random, and SV164(pGH5) glpK*-ΔglpR, can be cultivated with shaking at 32° C. for 18 hours in 3 ml of nutrient broth supplemented with tetracycline (10 mg/ml, marker of pGH5 plasmid) and 4% glycerol. The obtained cultures (0.3 ml each) can be inoculated into 3 ml of a fermentation medium containing tetracycline (10 mg/ml) in 20×200-mm test tubes, and cultivated at 32° C. for 72 hours with a rotary shaker at 250 rpm. After cultivation, the amount of tryptophan which accumulates in the medium can be determined by TLC as described in Example 12.
The fermentation medium components are listed in Table 4, but should be sterilized in separate groups (A, B, C, D, E, F, and G), as shown, to avoid adverse interactions during sterilization.









TABLE 4





Solutions
Component
Final concentration, g/l

















A
KH2PO4
0.28



NaCl
0.14



(NH4)2SO4
16



L-Methionine
0.08



L-Phenylalanine
0.28



L-Tyrosine
0.28



Mameno (total N)
0.07


B
Glucose
40.0



MgSO4•7H2O
0.03


C
FeSO4•7H2O
0.03


D
Na2MoO4•2H2O
0.00015



H3BO3
0.0025



CoCl2•6H2O
0.00007



CuSO4•5H2O
0.00025



MnCl2•4H2O
0.0016



ZnSO4•7H2O
0.0003


E
Thiamine HCl
0.001


F
CaCO3
30.0


G
Pyridoxine
0.03





The pH of solution A is adjusted to 7.1 with NH4OH.






Example 15
Production of L-proline by E. coli strains 702ilvA glpK*-234 or 702ilvA glpK*-random or 702ilvA glpK*-ΔglpR
To test the effect of enhanced ability to utilize glycerol on L-proline production, DNA fragments from the chromosome of the above-described E. coli strain MG1655 glpK*-231, MG1655 glpK*-234, MG1655 glpK*-random, or MG1655_glpK*-ΔglpR can be transferred to the proline-producing E. coli strain 702ilvA by P1 transduction (Miller, J. H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, N.Y.) to obtain the strain 702ilvA glpK*-231, 702ilvA glpK*-234, 702ilvA glpK*-random, or 702ilvA ΔglpR, respectively. The strain 702ilvA has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM)(Russia, 117545 Moscow, 1 Dorozhny proezd, 1) on Jul. 18, 2000 under accession number VKPM B-8012 and then converted to a deposit under the Budapest Treaty on May 18, 2001.
E. coli strains, 702ilvA, 702ilvA glpK*-231, 702ilvA glpK*-234, 702ilvA glpK*-random, and 702ilvA glpK*-ΔglpR, can be grown for 18-24 hours at 37° C. on L-agar plates. Then, these strains can be cultivated under the same conditions as in Example 11.
Example 16
Production of L-arginine by E. coli strains 382 glpK*-234 or 382 glpK*-random or 382-glpK*-ΔglpR
To test the effect of enhanced ability to utilize glycerol on L-arginine production, DNA fragments from the chromosome of the above-described E. coli strain MG1655 glpK*-231, MG1655 glpK*-234, MG1655 glpK*-random, or MG1655 glpK*-ΔglpR can be transferred to the arginine-producing E. coli strain 382 by P1 transduction (Miller, J. H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, N.Y.) to obtain the strain 382 glpK*-231, 382 glpK*-234, 382 glpK*-random, or 382 ΔglpR, respectively. The strain 382 has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1 Dorozhny proezd, 1) on Apr. 10, 2000 under accession number VKPM B-7926 and then converted to a deposit under the Budapest Treaty on May 18, 2001.
E. coli strains, 382, 382 glpK*-231, 382 glpK*-234, 382 glpK*-random, and 382 glpK*-ΔglpR, can be cultivated with shaking at 37° C. for 18 hours in 3 ml of nutrient broth supplemented with 4% glycerol, and 0.3 ml of the obtained cultures can be inoculated into 2 ml of a fermentation medium in 20×200-mm test tubes and cultivated at 32° C. for 48 hours on a rotary shaker.
After the cultivation, the amount of L-arginine which had accumulated in the medium can be determined by paper chromatography using the following mobile phase:butanol:acetic acid:water=4:1:1 (v/v). A solution of ninhydrin (2%) in acetone can be used as a visualizing reagent. A spot containing L-arginine can be cut out, L-arginine can be eluted with 0.5% water solution of CdCl2, and the amount of L-arginine can be estimated spectrophotometrically at 540 nm.
The composition of the fermentation medium (g/l) is as follows:
















Glycerol
60.0



(NH4)2SO4
35.0



KH2PO4
2.0



MgSO4 7H2O
1.0



Thiamine HCl
0.0002



Yeast extract
1.0



L-isoleucine
0.1



CaCO3
5.0










Glucose and magnesium sulfate are sterilized separately. CaCO3 is dry-heat sterilized at 180° C. for 2 hours. The pH is adjusted to 7.0.
Reference example 2. Construction of the E. coli strain MG1655 Δtdh::rhtA*/pVIC40.
The L-threonine producing E. coli strain MG1655 Δtdh, rhtA* (pVIC40) was constructed by inactivation of the native tdh gene in E. coli MG1655 using the cat gene followed by introduction of an rhtA23 mutation which confers resistance to high concentrations of threonine (>40 mg/ml) and homoserine (>5 mg/ml). Then, the resulting strain was transformed with plasmid pVIC40 from E. coli VKPM B-3996. Plasmid pVIC40 is described in detail in U.S. Pat. No. 5,705,371.
To replace the native tdh gene, a DNA fragment carrying the chloramphenicol resistance marker (CmR) encoded by the cat gene was integrated into the chromosome of E. coli MG1655 (ATCC 700926) in place of the native tdh gene by the method described by Datsenko K. A. and Wanner B. L. (Proc. Natl. Acad. Sci. USA, 2000, 97, 6640-6645) which is also called “Red-mediated integration” and/or “Red-driven integration”. The recombinant plasmid pKD46 (Datsenko, K. A., Wanner, B. L., Proc. Natl. Acad. Sci. USA, 2000, 97, 6640-6645) with the thermo sensitive replicon was used as the donor of the phage λ-derived genes responsible for the Red-mediated recombination system. E. coli BW25113 containing the recombinant plasmid pKD46 can be obtained from the E. coli Genetic Stock Center, Yale University, New Haven, USA, the accession number of which is CGSC7630.
A DNA fragment containing a CmR marker encoded by the cat gene was obtained by PCR using the commercially available plasmid pACYC184 (GenBank/EMBL accession number X06403, “Fermentas”, Lithuania) as the template, and primers P15 (SEQ ID NO: 35) and P16 (SEQ ID NO: 36). Primer P15 contains 35 nucleotides homologous to the 5′-region of the tdh gene introduced into the primer for further integration into the bacterial chromosome. Primer P16 contains 32 nucleotides homologous to the 3′-region of the tdh gene introduced into the primer for further integration into the bacterial chromosome.
PCR was provided using the “Gene Amp PCR System 2700” amplificatory (Applied Biosystems). The reaction mixture (total volume—50 μl) consisted of 5 μl of 10×PCR-buffer with 25 mM MgCl2 (“Fermentas”, Lithuania), 200 μM each of dNTP, 25 μmol each of the exploited primers and 1 U of Taq-polymerase (“Fermentas”, Lithuania). Approximately 5 ng of the plasmid DNA was added in the reaction mixture as a template DNA for the PCR amplification. The temperature profile was the following: initial DNA denaturation for 5 min at 95° C., followed by 25 cycles of denaturation at 95° C. for 30 sec, annealing at 55° C. for 30 sec, elongation at 72° C. for 40 sec; and the final elongation for 5 min at 72° C. Then, the amplified DNA fragment was purified by agarose gel-electrophoresis, extracted using “GenElute Spin Columns” (Sigma, USA), and precipitated by ethanol.
The obtained DNA fragment was used for electroporation and Red-mediated integration into the bacterial chromosome of the E. coli MG1655/pKD46. Electrocompetent cells were prepared as described in Reference Example 1.
Electroporation was performed by “Bio-Rad” electroporator (USA)(No. 165-2098, version 2-89) according to the manufacturer's instructions. Shocked cells were added to 1-ml of SOC medium (Sambrook et al, “Molecular Cloning A Laboratory Manual, Second Edition”, Cold Spring Harbor Laboratory Press (1989)), incubated for 2 hours at 37° C., and then were spread onto L-agar containing 25 μg/ml of chloramphenicol. Colonies grown for 24 hours were tested for the presence of CmR marker instead of the native tdh gene by PCR using primers P17 (SEQ ID NO: 37) and P18 (SEQ ID NO: 38). For this purpose, a freshly isolated colony was suspended in 20 μl water and then 1 μl of obtained suspension was used for PCR. The temperature profile follows: initial DNA denaturation for 5 min at 95° C.; then 30 cycles of denaturation at 95° C. for 30 sec, annealing at 55° C. for 30 sec and elongation at 72° C. for 30 sec; the final elongation for 5 min at 72° C. A few CmR colonies tested contained the desired 1104 bp DNA fragment, confirming the presence of CmR marker DNA instead of 1242 bp fragment of tdh gene. One of the obtained strains was cured of the thermosensitive plasmid pKD46 by culturing at 37° C. and the resulting strain was named E. coli MG1655Δtdh.
Then, the rhtA23 mutation from the strain VL614rhtA23 (Livshits V. A. et al, 2003, Res. Microbiol., 154:123-135) was introduced into the obtained strain MG1655 Δtdh resulting in strain MG1655 Δtdh, rhtA*. The rhtA23 is a mutation which confers resistance to high concentrations of threonine (>40 mg/ml) and homoserine (>5 mg/ml). For that purpose the strain MG1655 Δtdh was infected with phage P1vir grown on the donor strain VL614rhtA23. The transductants were selected on M9 minimal medium containing 8 mg/ml homoserine and 0.4% glucose as the sole carbon source.
While the invention has been described in detail with reference to preferred embodiments thereof, it will be apparent to one skilled in the art that various changes can be made, and equivalents employed, without departing from the scope of the invention. All the cited references herein are incorporated as a part of this application by reference.
Explanation of Sequence Listing
SEQ ID NO: 1 nucleotide sequence of glpF
SEQ ID NO: 2 amino acid seqeunce of GlpF
SEQ ID NO: 3 nucleotide sequence of glpK
SEQ ID NO: 4 amino acid seqeunce of glycerol kinase
SEQ ID NO: 5 nucleotide sequence of glpA
SEQ ID NO: 6 amino acid seqeunce of GlpA
SEQ ID NO: 7 nucleotide sequence of glpB
SEQ ID NO: 8 amino acid seqeunce of GlpB
SEQ ID NO: 9 nucleotide sequence of glpC
SEQ ID NO: 10 amino acid seqeunce of GlpC
SEQ ID NO: 11 nucleotide sequence of glpD
SEQ ID NO: 12 amino acid seqeunce of GlpD
SEQ ID NO: 13 primer P1
SEQ ID NO: 14 primer P2
SEQ ID NO: 15 primer P3
SEQ ID NO: 16 primer P4
SEQ ID NO: 17 primer P5
SEQ ID NO: 18 primer P6
SEQ ID NO: 19 primer P7
SEQ ID NO: 20 primer P8
SEQ ID NO: 21 primer P9
SEQ ID NO: 22 primer P10
SEQ ID NO: 23 primer P11
SEQ ID NO: 24 primer P12
SEQ ID NO: 25 primer P13
SEQ ID NO: 26 primer P14
SEQ ID NO: 27 DNA fragment coding 231-235 region of GlpK
SEQ ID NO: 28 DNA fragment coding 231-235 region of GlpK
SEQ ID NO: 29 DNA fragment coding 231-235 region of GlpK
SEQ ID NO: 30 DNA fragment coding 231-235 region of GlpK
SEQ ID NO: 31 nucleotide sequence of glpR
SEQ ID NO: 32 amino acid seqeunce of GlpR
SEQ ID NO: 33 nucleotide sequence of tpiA
SEQ ID NO: 34 amino acid seqeunce of TpiA
SEQ ID NO: 35 primer P15
SEQ ID NO: 36 primer P16
SEQ ID NO: 37 primer P17
SEQ ID NO: 38 primer P18
SEQ ID NO: 39 DNA fragment coding 231-235 region of GlpK
SEQ ID NO: 40 DNA fragment coding 231-235 region of GlpK
SEQ ID NO: 41 DNA fragment coding 231-235 region of GlpK
SEQ ID NO: 42 DNA fragment coding 231-235 region of GlpK
SEQ ID NO: 43 amino acid seqeunce of glycerol kinase from S. Sonnei 
SEQ ID NO: 44 amino acid seqeunce of glycerol kinase from Y. Pestis 
SEQ ID NO: 45 amino acid seqeunce of glycerol kinase from P. Aeruginosa 
SEQ ID NO: 46 amino acid seqeunce of glycerol kinase from B. Subtilis 
SEQ ID NO: 47 DNA fragment coding 231-235 region of GlpK
SEQ ID NO: 48 231-235 region of mutant GlpK
SEQ ID NO: 49 231-235 region of mutant GlpK
SEQ ID NO: 50 231-235 region of mutant GlpK
SEQ ID NO: 51 231-235 region of mutant GlpK
SEQ ID NO: 52 231-235 region of mutant GlpK
SEQ ID NO: 53 231-235 region of mutant GlpK
SEQ ID NO: 54 231-235 region of mutant GlpK
SEQ ID NO: 55 231-235 region of mutant GlpK
SEQ ID NO: 56 231-235 region of mutant GlpK
SEQ ID NO: 47 231-235 region of mutant GlpK
INDUSTRIAL APPLICABILITY
According to the present invention, an L-amino acid-producing bacterium having enhanced ability to utilize glycerol, and a method for producing an L-amino acid by fermentation of glycerol using the bacterium are provided.

## Claims

1. A method for producing an L-amino acid comprising:
a) culturing in a culture medium containing glycerol an L-amino acid producing bacterium of the Enterobacteriaceae family, and
b) collecting the L-amino acid from the culture,

wherein said bacterium comprises glycerol kinase in which feedback inhibition by fructose-1,6-bisphosphate is desensitized, and said glycerol kinase comprises a mutation which results in the replacement of at least one amino acid with another amino acid in the region corresponding to the positions from 233 to 235 of the amino acid sequence of the wild type glycerol kinase shown in SEQ ID NO: 4.

2. The method according to claim 1, wherein said glycerol kinase further comprises a mutation which results in the replacement of at least one amino acid with another amino acid in the region corresponding to the positions from 231 to 232.

3. The method according to claim 1, wherein said glycerol kinase comprises a mutation selected from the group consisting of replacing the Gly residue at position 234 with an Asp residue, replacing Lys-Gly-Gly at the 233 to 235 positions with Arg-Ile-Pro, and replacing Gly-Gly-Lys-Gly-Gly at the 231 to 235 positions with Arg-Ala-His-Leu-Ala.

4. The method according to claim 1, wherein said bacterium is further modified so that activity of a glycerol facilitator and/or glycerol 3-phosphate dehydrogenase is enhanced.

5. The method according to claim 4, wherein the expression of at least one of said glycerol facilitator and said glycerol 3-phosphate dehydrogenase is increased by increasing the copy number of the gene or modifying an expression control sequence of the gene.

6. The method according to claim 1, wherein said bacterium is further modified so that activity of triose phosphate isomerase is enhanced.

7. The method according to claim 6, wherein the expression of said triose phosphate isomerase is increased by increasing the copy number of the gene or modifying an expression control sequence of the gene.

8. The method according to claim 1, wherein said L-amino acid is selected from the group consisting of an aromatic L-amino acid and a non-aromatic L-amino acid.

9. The method according to claim 8, wherein said aromatic L-amino acid is selected from the group consisting of L-phenylalanine, L-tyrosine, and L-tryptophan.

10. The method according to claim 8, wherein said non-aromatic L-amino acid is selected from the group consisting of L-threonine, L-lysine, L-cysteine, L-methionine, L-leucine, L-isoleucine, L-valine, L-histidine, glycine, L-serine, L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, L-proline, and L-arginine.

11. The method according to claim 1, wherein said bacterium belongs to the genus Escheriahia or Pantoea.

12. The method according to claim 1, wherein the glycerol is obtained as from biodiesel production.