# Patent

## Title
Method for producing an L-amino acid using a bacterium of the family Enterobacteriaceae having a disrupted putrescine degradation pathway

## Abstract

The present invention provides a method for producing L-amino acids such as L-amino acids belonging to the glutamate family by fermentation using a bacterium of the family Enterobacteriaceae, particularly a bacterium belonging to the genus Escherichia, which has been modified to disrupt the putrescine degradation pathway by, for example, inactivation of one gene or several genes from the puuADRCBE gene cluster.

## Background
This application is a Continuation of, and claims priority under 35 U.S.C. §120 to, International Application No. PCT/JP2014/078157, filed Oct. 16, 2014, and claims priority therethrough under 35 U.S.C. §119 to Russian Patent Application No. 2013147882, filed Oct. 28, 2013, the entireties of which are incorporated by reference herein. Also, the Sequence Listing filed electronically herewith is hereby incorporated by reference (File name: 2016-04-13T_US-523_Seq_List; File size: 53 KB; Date recorded: Apr. 13, 2016).


BACKGROUND OF THE INVENTION
Technical Field
The present invention relates to the microbiological industry, and specifically to a method for producing L-amino acids by fermentation of a bacterium of the family Enterobacteriaceae that has been modified to disrupt putrescine degradation pathway.
Background Art
Conventionally, L-amino acids are industrially produced by fermentation methods utilizing strains of microorganisms obtained from natural sources, or mutants thereof. Typically, the microorganisms are modified to enhance production yields of L-amino acids.
Many techniques to enhance L-amino acid production yields have been reported, including transformation of microorganisms with recombinant DNA (see, for example, U.S. Pat. No. 4,278,765 A) and alteration of regulatory regions such as promoter, leader sequence, and/or attenuator, or others known to the person skilled in the art (see, for example, US20060216796 A1 and WO9615246 A1). Other techniques for enhancing production yields include increasing the activities of enzymes involved in amino acid biosynthesis and/or desensitizing the target enzymes to the feedback inhibition by the resulting L-amino acid (see, for example, WO9516042 A1, EP0685555 A1 or U.S. Pat. Nos. 4,346,170 A, 5,661,012 A, and 6,040,160 A).
Another method for enhancing L-amino acids production yields is to attenuate expression of a gene or several genes which are involved in degradation of the target L-amino acid, genes which divert the precursors of the target L-amino acid from the L-amino acid biosynthetic pathway, genes involved in the redistribution of the carbon, nitrogen, and phosphate fluxes, and genes encoding toxins, etc.
The pathways that result in the production of arginine, ornithine, putrescine, and γ-aminobutyric acid (GABA) catabolism are related (Schneider B. L. et al., Arginine catabolism and the arginine succinyltransferase pathway in Escherichia coli, J. Bacteriol., 1998, 180(16):4278-4286). Arginine and ornithine are degraded via the ammonia-producing arginine succinyltransferase (AST) pathway to glutamate by AST enzymes encoded by the astCADBE operon genes. The AST pathway is necessary for arginine degradation during nitrogen-limited growth, and it contributes to the degradation of other amino acids. Alternatively, to the AST pathway, another arginine catabolic pathway via agmatine to putrescine exists, which utilizes arginine decarboxylase (ADC) in the initial reaction (ADC pathway). The ADC pathway is not used to degrade arginine when it is the sole nitrogen source (Schneider B. L. et al., Putrescine catabolism is a metabolic response to several stresses in Escherichia coli, Mol. Microbiol., 2013, 88(3):537-550). Contrary to the arginine degradation pathway, ornithine is directly converted to putrescine by ornithine decarboxylase (ODC) in the ODC pathway.
Putrescine, which can also be referred to as tetramethylenediamine or 1,4-diaminobutane, is degraded via GABA to succinate for use as a carbon and nitrogen source by either the glutamylated putrescine (GP) pathway or the transaminase pathway. The GP pathway, also referred to as Puu pathway, utilizes the enzymes to completely degrade putrescine to succinate. The puuA, puuB, puuC, puuD and puuE genes, along with puuP and puuR genes encoding a putrescine/H+ symporter PuuP and DNA-binding transcriptional repressor PuuR, are organized in the puu divergon. The puu gene cluster was found in Escherichia coli (E. coli) and closely related enterobacteria (Nemoto N. et al., Mechanism for regulation of the putrescine utilization pathway by the transcription factor PuuR in Escherichia coli K-12, J. Bacteriol., 2012, 194(13):3437-3447). It is postulated that E. coli and related enterobacteria may utilize the GP pathway as an adaptation for survival in the mammalian intestine, an environment in which polyamines exist at relatively high concentrations. PatA, PatD, GabT, and GabD constitute the transaminase pathway of putrescine degradation (Schneider B. L. et al., 2013, 88(3):537-550).
Until now, no data has been reported demonstrating the effect of the disruption of putrescine degradation pathway on L-amino acid production by modified bacterial strains of the family Enterobacteriaceae.
SUMMARY OF THE INVENTION
An aspect of the present invention is to provide a bacterium belonging to the family Enterobacteriaceae, which can belong to the genus Escherichia and, more specifically, to the species E. coli, which has been modified to disrupt a putrescine degradation pathway such as, for example, the glutamylated putrescine pathway or the transaminase pathway.
Another aspect of the present invention is to provide a method for producing L-amino acids such as L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-citrulline, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-ornithine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine using a bacterium of the family Enterobacteriaceae as described hereinafter.
These aims were achieved by the unexpected findings that disruption of a putrescine degradation pathway confers on the microorganism higher productivity of L-amino acids, in particular, but is not limited to the L-amino acids belonging to the glutamate family such as L-arginine and L-ornithine. For example, a putrescine degradation pathway such as the glutamylated putrescine pathway can be disrupted by deregulation of expression of at least one gene encoding an enzyme from the pathway, in particular, inactivation of one gene or several genes from the puuADRCBE gene cluster on the chromosome of a bacterium belonging to the family Enterobacteriaceae, which can belong to the genus Escherichia and, more specifically, to the species E. coli. These findings have resulted in the following non-limiting aspects of the present invention.
An aspect of the present invention is to provide a method for producing an L-amino acid comprising:
(i) cultivating an L-amino acid-producing bacterium of the family Enterobacteriaceae in a culture medium; and
(ii) collecting said L-amino acid in the bacterium or culture medium, or both, wherein the bacterium has been modified to disrupt a putrescine degradation pathway.
It is a further aspect of the present invention to provide the method as described above, wherein the putrescine degradation pathway is a transaminase pathway or a glutamylated putrescine pathway.
It is a further aspect of the present invention to provide the method as described above, wherein the putrescine degradation pathway is disrupted by attenuation of expression of a gene selected from the group consisting of patA, patD, gabT, gabD, puuA, puuB, puuC, puuD, puuE, and combinations thereof.
It is a further aspect of the present invention to provide the method as described above, wherein the putrescine degradation pathway is the glutamylated putrescine pathway, wherein the glutamylated putrescine pathway is disrupted by attenuation of expression of one gene or several genes from the puuADRCBE gene cluster, with the proviso that the puuR gene cannot be the only gene that is attenuated.
It is a further aspect of the present invention to provide the method as described above, wherein the putrescine degradation pathway is disrupted by attenuation of expression of the puuA gene or the entire puuADRCBE gene cluster.
It is a further aspect of the present invention to provide the method as described above, wherein expression of the gene(s) is/are attenuated by inactivation of said gene(s).
It is a further aspect of the present invention to provide the method as described above, wherein the said gene(s) is/are deleted.
It is a further aspect of the present invention to provide the method as described above, wherein the bacterium belongs to the genus Escherichia. 
It is a further aspect of the present invention to provide the method as described above, wherein the bacterium is Escherichia coli. 
It is a further aspect of the present invention to provide the method as described above, wherein the bacterium belongs to the genus Pantoea. 
It is a further aspect of the present invention to provide the method as described above, wherein the bacterium is Pantoea ananatis. 
It is a further aspect of the present invention to provide the method as described above, wherein the L-amino acid is selected from the group consisting of L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-citrulline, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-ornithine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine.
It is a further aspect of the present invention to provide the method as described above, wherein the L-amino acid belongs to the glutamate family and is selected from the group consisting of L-arginine, L-citrulline, L-glutamic acid, L-glutamine, L-ornithine, and L-proline.
It is a further aspect of the present invention to provide the method as described above, wherein the L-amino acid is L-arginine.
It is a further aspect of the present invention to provide the method as described above, wherein the L-amino acid is L-ornithine.


DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention is described in detail below.
1. Bacterium
The phrase “an L-amino acid-producing bacterium” can mean a bacterium of the family Enterobacteriaceae that has an ability to produce, excrete or secrete, and/or cause accumulation of an L-amino acid in a culture medium or the bacterial cells when the bacterium is cultured in the medium.
The phrase “an L-amino acid-producing bacterium” can also mean a bacterium which is able to produce, excrete or secrete, and/or cause accumulation of an L-amino acid in a culture medium in an amount larger than a wild-type or parental strain, such as E. coli K-12, and can mean that the microorganism is able to cause accumulation in a medium of an amount not less than 0.5 g/L, or not less than 1.0 g/L, of the target L-amino acid. The bacterium can produce either one kind of amino acid solely, or a mixture of two or more kinds of amino acids.
The phrase “L-amino acid-producing ability” can mean the ability of the bacterium to produce, excrete or secrete, and/or cause accumulation of the L-amino acid in a medium or the bacterial cells to such a level that the L-amino acid can be collected from the medium or the bacterial cells, when the bacterium is cultured in the medium.
The phrase “L-amino acid” can mean L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-citrulline, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-ornithine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine.
The phrase “aromatic L-amino acid” can include, for example, L-phenylalanine, L-tryptophan, and L-tyrosine. As L-histidine has an aromatic moiety, specifically, an imidazole ring, the phrase “aromatic L-amino acid” can also include, besides the aforementioned aromatic L-amino acids, L-histidine.
The phrase “non-aromatic L-amino acid” can include, for example, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-citrulline, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-ornithine, L-proline, L-serine, L-threonine, and L-valine. As the biosynthetic pathway of aromatic amino acids such as L-phenylalanine, L-tryptophan, and L-tyrosine is different from the biosynthetic pathway of L-histidine, the phrase “non-aromatic L-amino acid” can also include, besides the aforementioned non-aromatic L-amino acids, L-histidine.
An L-amino acid can belong to more than one L-amino acid family. As an example, L-amino acids belonging to the glutamate family include L-arginine, L-glutamic acid, L-glutamine, and L-proline; L-amino acids belonging to the serine family include L-cysteine, glycine, and L-serine; L-amino acids belonging to the aspartate family include L-asparagine, L-aspartic acid, L-isoleucine, L-lysine, L-methionine, and L-threonine; L-amino acids belonging to the pyruvate family include L-alanine, L-isoleucine, L-valine, and L-leucine; and L-amino acids belonging to the aromatic family include L-phenylalanine, L-tryptophan, and L-tyrosine. As some L-amino acids can be an intermediate in the biosynthetic pathway of another L-amino acid, the aforementioned families of amino acids may also include other L-amino acids, for example, non-proteinogenic L-amino acids. For example, L-citrulline and L-ornithine are amino acids from the arginine biosynthetic pathway. Therefore, the glutamate family may include L-arginine, L-citrulline, L-glutamic acid, L-glutamine, L-ornithine, and L-proline.
L-Arginine, L-cysteine, L-glutamic acid, L-histidine, L-isoleucine, L-lysine, L-ornithine, L-phenylalanine, L-proline, L-threonine, L-tryptophan, and L-valine are particular examples. The glutamate family amino acids such as L-arginine, L-citrulline, L-glutamic acid, L-glutamine, L-ornithine, and L-proline are specific examples. L-Arginine and L-ornithine are particular examples.
The phrase “L-amino acid” can mean not only an L-amino acid in a free form, but may also mean a salt or a hydrate of the L-amino acid, or an adduct formed by the L-amino acid and another organic or inorganic compound as described hereinafter. Salts of amino acids include sulfates, hydrochlorides, carbonates, ammonium salts, sodium salts, and potassium salts.
The bacteria belonging to the family Enterobacteriaceae can be from the genera Escherichia and/or Pantoea, and so forth, and can have the ability to produce an L-amino acid. Specifically, those classified into the family Enterobacteriaceae according to the taxonomy used in the NCBI (National Center for Biotechnology Information) database (ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=543) can be used. Examples of strains from the family Enterobacteriaceae that can be modified include a bacterium of the genus Escherichia, Enterobacter or Pantoea. 
Strains of Escherichia bacterium which can be modified to obtain Escherichia bacteria in accordance with the presently disclosed subject matter are not particularly limited, and specifically, those described in the work of Neidhardt et al. can be used (Bachmann, B. J., Derivations and genotypes of some mutant derivatives of E. coli K-12, p. 2460-2488. In F. C. Neidhardt et al. (ed.), E. coli and Salmonella: cellular and molecular biology, 2nd ed. ASM Press, Washington, D.C., 1996). The species E. coli is a particular example. Specific examples of E. coli include E. coli W3110 (ATCC 27325), E. coli MG1655 (ATCC 47076, ATCC 700926), and so forth, which are derived from the prototype wild-type strain, E. coli K-12 strain. These strains are available from, for example, the American Type Culture Collection (P.O. Box 1549, Manassas, Va. 20108, United States of America). That is, registration numbers are given to each of the strains, and the strains can be ordered by using these registration numbers (refer to atcc.org). The registration numbers of the strains are listed in the catalogue of the American Type Culture Collection.
Examples of the Enterobacter bacteria can include Enterobacter agglomerans, Enterobacter aerogenes, and so forth. Examples of the Pantoea bacteria can include Pantoea ananatis, and so forth. Some strains of Enterobacter agglomerans were recently reclassified into Pantoea agglomerans, Pantoea ananatis or Pantoea stewartii on the basis of nucleotide sequence analysis of 16S rRNA, etc. A bacterium belonging to any of the genus Enterobacter or Pantoea may be used so long as it is a bacterium classified into the family Enterobacteriaceae. When a Pantoea ananatis strain is bred by genetic engineering techniques, Pantoea ananatis AJ13355 strain (FERM BP-6614), AJ13356 strain (FERM BP-6615), AJ13601 strain (FERM BP-7207) and derivatives thereof can be used. These strains were identified as Enterobacter agglomerans when they were isolated, and deposited as Enterobacter agglomerans. However, they were recently re-classified as Pantoea ananatis on the basis of nucleotide sequencing of 16S rRNA and so forth as described above.
L-Amino Acid-Producing Bacteria
A bacterium belonging to the family Enterobacteriaceae and modified to disrupt putrescine degradation pathway, which is able to produce an L-amino acid, can be used.
The bacterium may inherently have the L-amino acid-producing ability or may be modified to have an L-amino acid-producing ability by using a mutation method or DNA recombination techniques. The bacterium can be modified to contain the disrupted putrescine degradation pathway in a bacterium which inherently has the ability to produce an L-amino acid. Alternatively, the bacterium can be obtained by imparting the ability to produce an L-amino acid to a bacterium already modified to disrupt putrescine degradation pathway.
L-Arginine-Producing Bacteria
Examples of parental strains which can be used to derive L-arginine-producing bacteria 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 No. 2215783), E. coli strain 382 (VKPM B-7926, EP1170358 A1), an L-arginine-producing strain into which argA gene encoding N-acetylglutamate synthetase is introduced therein (EP1170361 A1), and the like.
Examples of parental strains that can be used to derive L-arginine-producing bacteria can also include strains in which expression of one or more genes encoding an L-arginine biosynthetic enzyme are enhanced. Examples of such genes include genes encoding N-acetyl-γ-glutamylphosphate reductase (argC), ornithine acetyltransferase (argJ), N-acetylglutamate kinase (argB), N-acetylornithine aminotransferase (argD), ornithine carbamoyltransferase (argF), argininosuccinate synthase (argG), argininosuccinate lyase (argH), and carbamoyl phosphate synthetase (carAB).
L-Citrulline-Producing Bacteria
Examples of parental strains that can be used to derive L-citrulline-producing bacteria can include, but are not limited to, strains belonging to the genus Escherichia such as E. coli mutant N-acetylglutamate synthase strains 237/pMADS11, 237/pMADS12, and 237/pMADS13 (Russian Patent No. 2215783, European Patent No. 1170361 B1, U.S. Pat. No. 6,790,647 B2). E. coli strains 333 (VKPM B-8084) and 374 (VKPM B-8086), both of which harbor harbor mutant feedback-resistant carbamoyl phosphate synthetase (Russian Patent RU2264459 C2), E. coli strains in which α-ketoglutarate synthase activity is increased, and ferredoxin NADP+ reductase, pyruvate synthase or α-ketoglutarate dehydrogenase activities are additionally modified (EP 2133417 A1), and P. ananantis strain NA1sucAsdhA, in which succinate dehydrogenase and α-ketoglutarate dehydrogenase activities are decreased (US Patent Application No 2009286290), and the like, are further examples.
As L-citrulline is an intermediate of L-arginine biosynthetic pathway, examples of parent strains, which can be used to derive L-citrulline-producing bacteria, include strains, in which expression of one or more genes encoding an L-arginine biosynthetic enzyme is enhanced. Examples of such genes include, but are not limited to, genes encoding N-acetylglutamate synthase (argA), N-acetylglutamate kinase (argB), N-acetylglutamyl phosphate reductase (argC), acetylornithine transaminase (argD), acetylornithine deacetylase (argE), ornithine carbamoyltransferase (argF/I), and carbamoyl phosphate synthetase (carAB), and combinations thereof.
L-Citrulline-producing bacterium can be also easily obtained from any L-arginine-producing bacterium, for example E. coli 382 stain (VKPM B-7926), by inactivation of argininosuccinate synthase encoded by argG gene.
L-Cysteine-Producing Bacteria
Examples of parental strains which can be used to derive L-cysteine-producing bacteria 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 encoding feedback-resistant serine acetyltransferases (U.S. Pat. No. 6,218,168, Russian patent No. 2279477), E. coli W3110 having overexpressed 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 (JP11155571 A2), E. coli W3110 with increased activity of a positive transcriptional regulator for cysteine regulon encoded by the cysB gene (WO0127307 A1), E. coli JM15(ydeD) (U.S. Pat. No. 6,218,168), and the like.
L-Glutamic Acid-Producing Bacteria
Examples of parental strains that can be used to derive L-glutamic acid-producing bacteria can include, but are not limited to, strains belonging to the genus Escherichia such as E. coli VL334thrC+ (EP 1172433). The 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 K-12 (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 parental strains, which can be used to derive the L-glutamic acid-producing bacteria 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 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, ipdA), 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 bisphosphate aldolase (fbp), phosphofructokinase (pfkA, pfkB), 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 include those disclosed in EP1078989 A2, EP955368 A2, and EP952221 A2.
Examples of parental strains, which can be used to derive the L-glutamic acid-producing bacteria, can also 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 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 deficient in the α-ketoglutarate dehydrogenase activity or having a 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 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 α-ketoglutarate dehydrogenase.
Other examples of L-glutamic acid-producing bacterium include those that belong to the genus Escherichia and have resistance to an aspartic acid antimetabolite. These strains can also be deficient in the α-ketoglutarate dehydrogenase activity and can 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 include mutant strains belonging to the genus Pantoea that 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 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, Incorporated Administrative Agency, National Institute of Technology and Evaluation, International Patent Organism Depositary (NITE-IPOD), #120, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba-ken 292-0818, 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 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 and so forth. Although AJ13356 was deposited at the aforementioned depository as Enterobacter agglomerans, for the purposes of this specification, they are described as Pantoea ananatis. 
L-Histidine-Producing Bacteria
Examples of parental strains which can be used to derive L-histidine-producing bacteria 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 AI80/pFM201 (U.S. Pat. No. 6,258,554), and the like.
Examples of parental strains that can be used to derive L-histidine-producing bacteria can also 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-AMP cyclohydrolase/phosphoribosyl-ATP pyrophosphatase (hisIE), phosphoribosylformimino-5-aminoimidazole carboxamide ribotide isomerase (hisA), amidotransferase (hisH), histidinol phosphate aminotransferase (hisC), histidinol phosphatase (hisB), histidinol dehydrogenase (hisD), and so forth.
It is known that the L-histidine biosynthetic enzymes encoded by hisG and hisBHAFI are inhibited by L-histidine, and therefore L-histidine-producing ability can also be efficiently enhanced by introducing a mutation into ATP phosphoribosyltransferase to confer resistance to feedback inhibition (Russian Patent Nos. 2003677 and 2119536).
Specific examples of strains having an L-histidine-producing ability include E. coli FERM-P 5038 and 5048 which have been transformed with a vector carrying a DNA encoding an L-histidine-biosynthetic enzyme (JP 56-005099 A), E. coli strains transformed 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, RU2119536), and E. coli MG1655+hisGr hisL′_ΔpurR (RU2119536 and Doroshenko V. G. et al., The directed modification of Escherichia coli MG1655 to obtain histidine-producing mutants, Prikl. Biochim. Mikrobiol. (Russian), 2013, 49(2):149-154.), and so forth.
L-Isoleucine-Producing Bacteria
Examples of parental strains which can be used to derive L-isoleucine-producing bacteria 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, can also be used as parental strains (JP 2-458 A, EP0356739 A1, and U.S. Pat. No. 5,998,178).
L-Leucine-Producing Bacteria
Examples of parental strains which can be used to derive L-leucine-producing bacteria 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.
The bacterium can be improved by enhancing the expression of one or more genes involved in L-leucine biosynthesis. Examples include genes of the leuABCD operon, which can be represented by a mutant leuA gene encoding isopropylmalate synthase freed from feedback inhibition by L-leucine (U.S. Pat. No. 6,403,342). In addition, the bacterium can be improved by enhancing the expression of one or more genes encoding proteins that excrete L-amino acid from the bacterial cell. Examples of such genes include b2682 and b2683 (ygaZH genes) (EP1239041 A2).
L-Lysine-Producing Bacteria
Examples of L-lysine-producing bacteria belonging to the family Escherichia include mutants having resistance to an L-lysine analogue. The L-lysine analogue inhibits growth of bacteria belonging to the genus Escherichia, but this inhibition is fully or partially desensitized when L-lysine is present in the medium. Examples of the L-lysine analogue include, but are not limited to oxalysine, lysine hydroxamate, S-(2-aminoethyl)-L-cysteine (AEC), γ-methyllysine, α-chlorocaprolactam, and so forth. 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 include E. coli AJ11442 (FERM BP-1543, NRRL B-12185; see U.S. Pat. No. 4,346,170) and E. coli VL611. In these microorganisms, feedback inhibition of aspartokinase by L-lysine is desensitized.
Examples of parental strains which can be used to derive L-lysine-producing bacteria also include, but are not limited to strains in which expression of one or more genes encoding an L-lysine biosynthetic enzyme are enhanced. Examples of such genes 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) (EP1253195 A1). In addition, the parental strains may have an increased level of expression of the gene involved in energy efficiency (cyo) (EP1170376 A1), the gene encoding nicotinamide nucleotide transhydrogenase (pntAB) (U.S. Pat. No. 5,830,716), the ybjE gene (WO2005/073390), or combinations thereof.
L-Amino acid-producing bacteria may have reduced or no activity of an enzyme that catalyzes a reaction which causes a branching off from the L-amino acid biosynthesis pathway and results in the production of another compound. Also, the bacteria may have reduced or no activity of an enzyme that negatively acts on L-amino acid synthesis or accumulation. Examples of such enzymes involved in L-lysine production include homoserine dehydrogenase, lysine decarboxylase (cadA, ldcC), malic enzyme, and so forth, and strains in which activities of these enzymes are decreased or deleted are disclosed in WO95/23864, WO96/17930, WO2005/010175, and so forth.
Expression of both the cadA and ldcC genes encoding lysine decarboxylase can be decreased in order to decrease or delete the lysine decarboxylase activity. Expression of the both genes can be decreased by, for example, the method described in WO2006/078039.
Examples of L-lysine-producing bacteria can include the E. coli WC196ΔcadAΔldcC/pCABD2 strain (WO2006/078039). The strain was constructed by introducing the plasmid pCABD2 containing lysine biosynthesis genes (U.S. Pat. No. 6,040,160) into the WC196 strain having disrupted cadA and ldcC genes which encode lysine decarboxylase.
The WC196 strain was bred from the W3110 strain, which was derived from E. coli K-12 by replacing the wild-type lysC gene on the chromosome of the W3110 strain with a mutant lysC gene encoding a mutant aspartokinase III in which threonine at position 352 was replaced with isoleucine, resulting in desensitization of the feedback inhibition by L-lysine (U.S. Pat. No. 5,661,012), and conferring AEC resistance to the resulting strain (U.S. Pat. No. 5,827,698). The WC196 strain was designated E. coli AJ13069, deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology (currently, Incorporated Administrative Agency, National Institute of Technology and Evaluation, International Patent Organism Depositary (NITE-IPOD), #120, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba-ken 292-0818, JAPAN) on Dec. 6, 1994, and assigned 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 assigned an accession number of FERM BP-5252 (U.S. Pat. No. 5,827,698).
The WC196ΔcadAΔldcC strain itself is also an exemplary L-lysine-producing bacterium. The WC196ΔcadΔAldcC was designated AJ110692 and deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology (currently, Incorporated Administrative Agency, National Institute of Technology and Evaluation, International Patent Organism Depositary (NITE-IPOD), #120, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba-ken 292-0818, JAPAN) on Oct. 7, 2008 as an international deposit under an accession number of FERM BP-11027.
L-Methionine-Producing Bacteria
Examples of L-methionine-producing bacteria and parent strains which can be used to derive L-methionine-producing bacteria include, but are not limited to Escherichia bacteria strains such as strains AJ11539 (NRRL B-12399), AJ11540 (NRRL B-12400), AJ11541 (NRRL B-12401), AJ 11542 (NRRL B-12402) (patent GB2075055); strains 218 (VKPM B-8125) (patent RU2209248) and 73 (VKPM B-8126) (patent RU2215782) resistant to norleucine, the L-methionine analog, or the like. The strain E. coli 73 has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russian Federation, 117545 Moscow, 1st Dorozhny Proezd, 1) on May 14, 2001 under accession number VKPM B-8126, and was converted to an international deposit under the Budapest Treaty on Feb. 1, 2002. Furthermore, a methionine repressor-deficient strain and recombinant strains transformed with genes encoding proteins involved in L-methionine biosynthesis such as homoserine transsuccinylase and cystathionine γ-synthase (JP 2000-139471 A) can also be used as parent strains.
L-Ornithine-Producing Bacteria
L-ornithine-producing bacterium can be easily obtained from any L-arginine-producing bacterium, for example E. coli 382 stain (VKPM B-7926), by inactivation of ornithine carbamoyltransferase encoded by both argF and argI genes. Methods for inactivation of ornithine carbamoyltransferase are described herein.
L-Phenylalanine-Producing Bacteria
Examples of parental strains which can be used to derive L-phenylalanine-producing bacteria 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, 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) may be used as a parental strain (EP488424 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 may also be used (U.S. patent applications 2003/0148473 A1 and 2003/0157667 A1).
L-Proline-Producing Bacteria
Examples of parental strains that can be used to derive L-proline-producing bacteria 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 (EP1172433 A1). The bacterium can be improved by enhancing the expression of one or more genes involved in L-proline biosynthesis. Examples of genes that can be used in L-proline-producing bacteria include the proB gene encoding glutamate kinase with desensitized feedback inhibition by L-proline (DE3127361 A1). In addition, the bacterium can be improved by enhancing the expression of one or more genes encoding proteins responsible for excreting L-amino acids from the bacterial cell. Such genes can be exemplified by b2682 and b2683 (ygaZH genes) (EP1239041 A2).
Examples of bacteria belonging to the genus Escherichia, which have an activity to produce L-proline include the following E. coli strains: NRRL B-12403 and NRRL B-12404 (GB Patent 2075056), VKPM B-8012 (Russian Patent application No. 2000124295), plasmid mutants described in DE3127361 A1, plasmid mutants described by Bloom F. R. et al. in “The 15th Miami winter symposium”, 1983, p. 34, and the like.
L-Threonine-Producing Bacteria
Examples of parental strains which can be used to derive L-threonine-producing bacteria 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 (Russian), 1978, 14:947-956), E. coli VL643 and VL2055 (EP1149911 A2), 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 (Russian Federation, 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) (Russian Federation, 117545 Moscow, 1st Dorozhny proezd, 1) on Apr. 7, 1987 under the accession number B-3996.
E. coli VKPM B-5318 (EP0593792 A1) may also be used as a parental 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 replace 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 the accession number of VKPM B-5318.
The bacterium can be additionally modified to enhance expression of one or more of the following genes:
the mutant thrA gene which encodes aspartokinase homoserine dehydrogenase I resistant to feedback inhibition by threonine;
the thrB gene which encodes homoserine kinase;
the thrC gene which encodes threonine synthase;
the rhtA gene which encodes a putative transmembrane protein of the threonine and homoserine efflux system;
the asd gene which encodes aspartate-β-semialdehyde dehydrogenase; and
the aspC gene which encodes aspartate aminotransferase (aspartate transaminase);
The thrA gene which encodes aspartokinase I and homoserine dehydrogenase I of E. coli has been elucidated (KEGG entry No. b0002; GenBank accession No. NC_000913.2; nucleotide positions: 337 to 2,799; Gene ID: 945803). 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 E. coli has been elucidated (KEGG entry No. b0003; GenBank accession No. NC_000913.2; nucleotide positions: 2,801 to 3,733; Gene ID: 947498). 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 E. coli has been elucidated (KEGG entry No. b0004; GenBank accession No. NC_000913.2; nucleotide positions: 3,734 to 5,020; Gene ID: 945198). The thrC gene is located between the thrB and yaaX genes on the chromosome of E. coli K-12. All three genes function as a single threonine operon thrABC. To enhance expression of the threonine operon, the attenuator region which affects transcription is desirably removed from the operon (WO2005049808 A1, WO2003097839 A1).
The mutant thrA gene which encodes aspartokinase I and homoserine dehydrogenase I resistant to feedback inhibition by L-threonine, as well as, the thrB and thrC genes can be obtained as one operon from the well-known plasmid pVIC40 which is present in the L-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 which encodes a protein of the threonine and homoserine efflux system (an inner membrane transporter) of E. coli has been elucidated (KEGG entry No. b0813; GenBank accession No. NC_000913.2; nucleotide positions: 848,433 to 849,320, complement; Gene ID: 947045). The rhtA gene is located between the dps and ompX genes on the chromosome of E. coli K-12 close to the glnHPQ operon, which encodes components of the glutamine transport system. The rhtA gene is identical to the ybiF gene (KEGG entry No. B0813).
The asd gene which encodes aspartate-β-semialdehyde dehydrogenase of E. coli has been elucidated (KEGG entry No. b3433; GenBank accession No. NC_000913.2; nucleotide positions: 3,571,798 to 3,572,901, complement; Gene ID: 947939). The asd gene is located between the glgB and gntU gene on the same strand (yhgN gene on the opposite strand) on the chromosome of E. coli K-12.
Also, the aspC gene which encodes aspartate aminotransferase of E. coli has been elucidated (KEGG entry No. b0928; GenBank accession No. NC_000913.2; nucleotide positions: 983,742 to 984,932, complement; Gene ID: 945553). The aspC gene is located between the ycbL gene on the opposite strand and the ompF gene on the same strand on the chromosome of E. coli K-12.
L-Tryptophan-Producing Bacteria
Examples of parental strains which can be used to derive the L-tryptophan-producing bacteria 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 free from feedback inhibition by serine and a trpE allele encoding anthranilate synthase free from 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 may 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 may also be used (U.S. patent applications 2003/0148473 A1 and 2003/0157667 A1).
Examples of parental strains which can be used to derive the L-tryptophan-producing bacteria can also include strains in which one or more activities of the enzymes selected from 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 may be introduced into these enzymes. Specific examples of strains having such a mutation include an 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 parental strains which can be used to derive the L-tryptophan-producing bacteria can also 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 may be imparted by enhancing expression of a gene which encodes tryptophan synthase, among tryptophan operons (trpBA). The tryptophan synthase consists of α and β subunits which are encoded by the trpA and trpB genes, respectively. In addition, L-tryptophan-producing ability may be improved by enhancing expression of the isocitrate lyase-malate synthase operon (WO2005/103275).
L-Valine-Producing Bacteria
Examples of parental strains which can be used to derive L-valine-producing bacteria can include, but are not limited to strains which have been modified to overexpress the ilvGMEDA operon (U.S. Pat. No. 5,998,178). It is desirable to remove the region of the ilvGMEDA operon which is required for attenuation so that expression of the operon is not attenuated by the L-valine that is produced. Furthermore, the ilvA gene in the operon is desirably disrupted so that threonine deaminase activity is decreased.
Examples of parental strains that can be used to derive L-valine-producing bacteria can also include mutants having a mutation of aminoacyl-tRNA 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 was deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russian Federation, 117545 Moscow, 1st Dorozhny Proezd, 1) on Jun. 24, 1988 under the accession number VKPM B-4411.
Furthermore, mutants requiring lipoic acid for growth and/or lacking H+-ATPase can also be used as parental strains (WO96/06926).
Examples of L-valine-producing strain include E. coli strain H-81 (VKPM B-8066), NRRL B-12287 and NRRL B-12288 (U.S. Pat. No. 4,391,907), VKPM B-4411 (U.S. Pat. No. 5,658,766), VKPM B-7707 (European patent application EP1016710A2), or the like.
The bacterium of the present invention belonging to the family Enterobacteriaceae can be modified to disrupt a putrescine degradation pathway, which can be either the transaminase pathway or glutamylated putrescine pathway. Exemplary and without limiting the scope of the present invention, a putrescine degradation pathway can be disrupted by attenuation of expression of at least one gene encoding an enzyme from the pathway, in particular, inactivation of one gene or several genes from the puuADRCBE gene cluster, and/or one gene or several genes such as the patA, patD, gabT, or gabD genes on the chromosome of a bacterium belonging to the family Enterobacteriaceae such as a bacterium of the species E. coli. 
The phrase “a putrescine degradation pathway” refers to one of the following pathways for degradation of putrescine such as “the transaminase pathway” or “the glutamylated putrescine pathway”, which degrade putrescine to succinate via γ-aminobutyric acid (GABA) as explained hereinafter.
The phrase “the transaminase pathway” for degradation of putrescine refers to a set of consecutive enzymatic reactions catalyzed by putrescine transaminase (PatA), γ-aminobutyraldehyde dehydrogenase (PatD), γ-aminobutyrate transaminase (GabT), and succinic semialdehyde dehydrogenase (GabD), in which putrescine is converted into succinic acid (succinate) via γ-aminobutyraldehyde (ABAL), GABA and succinic semialdehyde (SSA) as described in Schneider B. L. et al., 2013, 88(3):537-550. The genes encoding enzymes from the transaminase pathway of E. coli have been elucidated such as patA (synonyms oat, pat, ygjG; KEGG, Kyoto Encyclopedia of Genes and Genomes, entry No. b3073; encodes PatA), patD (synonyms prr, ydcW; KEGG, entry No. b1444; encodes PatD), gabT (KEGG, entry No. b2662; encodes GabT), and gabD (KEGG, entry No. b2661; encodes GabD).
The phrase “the glutamylated putrescine pathway” for degradation of putrescine refers to a set of consecutive enzymatic reactions catalyzed by γ-glutamylputrescine synthase (PuuA), γ-glutamylputrescine oxidase (PuuB), γ-glutamyl-γ-aminobutyraldehyde dehydrogenase (PuuC), γ-glutamyl-γ-aminobutyrate hydrolase (PuuD), 4-aminobutyrate aminotransferase (PuuE), in which putrescine is converted into succinic acid (succinate) via γ-glutamyl putrescine, γ-glutamyl ABAL, γ-glutamyl GABA, GABA and SSA as described in Schneider B. L. et al., 2013, 88(3):537-550. The putrescine-binding repressor PuuR represses the glutamylated putrescine pathway. Therefore, attenuation of expression of the puuR gene alone may result in enhancing putrescine degradation pathway. Thus, attenuation of the puuR gene alone is not encompassed in disruption of the putrescine degradation pathway.
The genes encoding enzymes from the glutamylated putrescine pathway of E. coli have been elucidated such as puuA, puuB, puuC, puuD, puuE and puuR as described hereinafter. These genes are organized into the puuADRCBE gene cluster on the chromosome of E. coli strain K-12.
The puuA gene (synonym ycjK) encodes γ-glutamylputrescine synthase PuuA (KEGG, entry No. b1297; Protein Knowledgebase, UniProtKB/Swiss-Prot, accession No. P78061). The puuA gene (GenBank accession No. NC_000913.2; nucleotide positions: 1357514 to 1358932, complement; Gene ID: 946202) is located between the puuP gene on the same strand and the puuD gene on the opposite strand on the chromosome of E. coli strain K-12. The nucleotide sequence of the puuA gene and the amino acid sequence of the PuuA protein encoded by the puuA gene are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
The puuD gene (synonym ycjL) encodes γ-glutamyl-γ-aminobutyrate hydrolase PuuD (KEGG, entry No. b1298; Protein Knowledgebase, UniProtKB/Swiss-Prot, accession No. P76038). The puuD gene (GenBank accession No. NC_000913.2; nucleotide positions: 1359144 to 1359908, complement; Gene ID: 945882) is located between the puuR gene on the same strand and the pupP gene on the opposite strand on the chromosome of E. coli strain K-12. The nucleotide sequence of the puuD gene and the amino acid sequence of the PuuD protein encoded by the puuD gene are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.
The puuR gene (synonym ycjC) encodes the DNA-binding transcriptional repressor for the puu divergon PuuR (KEGG, entry No. b1299; Protein Knowledgebase, UniProtKB/Swiss-Prot, accession No. P0A9U6). The puuD gene (GenBank accession No. NC_000913.2; nucleotide positions: 1359935 to 1360492, complement; Gene ID: 945886) is located between the puuD and puuC genes on the same strand on the chromosome of E. coli strain K-12. The nucleotide sequence of the puuR gene and the amino acid sequence of the PuuR protein encoded by the puuR gene are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.
The puuC gene (synonym aldH) encodes γ-glutamyl-γ-aminobutyraldehyde dehydrogenase PuuC (KEGG, entry No. b1300; Protein Knowledgebase, UniProtKB/Swiss-Prot, accession No. P23883). The puuC gene (GenBank accession No. NC_000913.2; nucleotide positions: 1360767 to 1362254, complement; Gene ID: 947003) is located between the puuR and puuB genes on the same strand on the chromosome of E. coli strain K-12. The nucleotide sequence of the puuC gene and the amino acid sequence of the PuuC protein encoded by the puuC gene are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively.
The puuB gene (synonyms ycjA, ordL) encodes γ-glutamylputrescine oxidase PuuB (KEGG, entry No. b1301; Protein Knowledgebase, UniProtKB/Swiss-Prot, accession No. P37906). The puuB gene (GenBank accession No. NC_000913.2; nucleotide positions: 1362256 to 1363536, complement; Gene ID: 945072) is located between the puuC and puuB genes on the same strand on the chromosome of E. coli strain K-12. The nucleotide sequence of the puuB gene and the amino acid sequence of the PuuB protein encoded by the puuB gene are shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively.
The puuE gene (synonym goaG) encodes the 4-aminobutyrate aminotransferase PuuE (KEGG, entry No. b1302; Protein Knowledgebase, UniProtKB/Swiss-Prot, accession No. P50457). The puuB gene (GenBank accession No. NC_000913.2; nucleotide positions: 1363574 to 1364839, complement; Gene ID: 945446) is located between the puuB on the same strand and the pspF gene on the opposite strand on the chromosome of E. coli strain K-12. The nucleotide sequence of the puuE gene and the amino acid sequence of the PuuE protein encoded by the puuE gene are shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively.
A “pathway,” which can be also referred to as “a biochemical pathway” or “a metabolic pathway,” can mean a set of anabolic or catabolic (bio)chemical reactions which act collectively to convert one biomolecule species into another one. Thus, the “pathway” can include a series of (bio)chemical reactions connected by their intermediates, that is, one or more products of one reaction is/are the substrate(s) for subsequent one or more reactions, and so forth. The meaning of the phrase “pathway” is usually apparent to the person skilled in the art.
The phrase “a bacterium modified to disrupt a putrescine degradation pathway” can mean that the bacterium has been modified in such a way that in the modified bacterium the carbon flux through one or more (bio)chemical reactions of the pathway is lower or even absent as compared with a bacterium in which the putrescine degradation pathway is not disrupted such as in a non-modified bacterium, for example, a wild-type or parental strain such as E. coli K-12. In the bacterium, which has been modified to disrupt the putrescine degradation pathway, the carbon flux through one or more (bio)chemical reactions of the pathway can be 99% or less, 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, or 0%, as compared with the carbon flux in the pathway of a non-modified bacterium.
The phrase “carbon flux” can mean the rate of turnover of carbon-containing molecules through a metabolic pathway. For example, the carbon flux of an amino acid such as, for example, L-arginine or L-ornithine can be calculated to determine carbon distribution from the L-amino acid in a putrescine degradation pathway such as the transaminase pathway or glutamylated putrescine pathway (Szyperski T. Biosynthetically directed fractional 13C-labeling of proteinogenic amino acids. An efficient analytical tool to investigate intermediary metabolism, Eur. J. Biochem., 1995, 232:433-448; Stephanopoulos, G., Aristidou, A. A., Nielsen, J. Metabolic engineering: principles and methodologies, Academic Press, 1st ed., (1998); Matsuoka Y. and Shimizu K. 13C-Metabolic flux analysis and metabolic regulation, Chemical Biology, Prof. Deniz Ekinci (Ed.), DOI: 10.5772/35121, intechopen.com/books/chemical-biology/13c-metabolic-flux-analysis-and-metabolic-regulation).
The phrase “a bacterium modified to disrupt putrescine degradation pathway” can also mean that the bacterium has been modified in such a way that in the modified bacterium, the putrescine degradation pathway is attenuated.
The phrase “in the modified bacterium, the putrescine degradation pathway is attenuated” can mean that the bacterium has been modified in such a way that in the modified bacterium the expression of at least one gene encoding an enzyme from the putrescine degradation pathway is attenuated. For example, expression of one gene or several genes selected from patA, patD, gabT and gabD, encoding enzymes from the transaminase pathway, can be attenuated. In another example, expression of one or several of the puuA, puuB, puuC, puuD, puuE and puuR genes, encoding enzymes from the glutamylated putrescine pathway, can be attenuated. In yet another example, expression of one or several of the patA, patD, gabT, gabD, puuA, puuB, puuC, puuD, puuE and puuR genes may be attenuated. A combination of genes, expression of which can be attenuated to disrupt the putrescine degradation pathway, is not specifically limited so long as the attenuation of one gene or several genes results in disruption of the pathway. That is, expression of one or several of the patA, patD, gabT, gabD, puuA, puuB, puuC, puuD, and puuE genes can be attenuated in any combination to disrupt the putrescine degradation pathway so long as the bacterium has L-amino acid-producing ability. For example, expression of one gene such as, for example, puuA can be attenuated, or all of the puuA, puuB, puuC, puuD, puuE and puuR genes, constituting the entire puuADRCBE gene cluster, can be attenuated to disrupt the putrescine degradation pathway. However, attenuation of the puuR gene is not essential for disruption of the putrescine degradation pathway.
The phrase “several genes” can mean two or more genes such as, for example, three, four, five, six, seven, eight, nine or ten genes, or more. Therefore, the phrase “one or several genes” can mean a single gene such as, for example, the puuA gene, or an operon such as, for example, the puuCBE operon, or even an entire gene cluster such as, for example, the puuADRCBE gene cluster as described herein. It is, therefore, acceptable that the phrases “one or several genes” and “at least one gene” are equivalent.
For the purposes of the present invention, explanations given hereinafter as to proteins and genes encoding thereof from the glutamylated putrescine pathway can be applied mutatis mutandis to proteins and genes encoding thereof from the transaminase pathway. Furthermore, for the purposes of the present invention, explanations given hereinafter as to the puuADRCBE gene cluster can be applied mutatis mutandis to one or several of the patA, patD, gabT, gabD, puuA, puuB, puuC, puuD, and puuE genes, or a combination thereof.
The phrase “a bacterium modified to attenuate expression of at least one gene from the puuADRCBE gene cluster” can mean that the bacterium has been modified in such a way that in the modified bacterium, the expression of at least one gene from the puuADRCBE gene cluster is decreased as compared to a bacterium which contains a non-modified puuADRCBE gene cluster, for example, a wild-type or parental strain.
The phrase “attenuation” or “attenuated” can mean the alteration or modification of at least one gene in a bacterium that encodes an enzyme from the putrescine degradation pathway, such that an amount or activity of one or more enzymes in the bacterium is decreased or absent, thereby decreasing or completely eliminating the activity of the gene products.
The phrase “at least one gene from the puuADRCBE gene cluster gene is inactivated” can mean that at least one of the genes such as puuA, puuB, puuC, puuD, puuE and puuR from the modified gene cluster encodes a completely inactive or non-functional protein, as compared with a bacterium which contains the non-modified gene or non-modified puuADRCBE gene cluster.
It is also possible that at least one of the modified DNA regions from the puuADRCBE gene cluster is unable to naturally express the gene due to deletion of a part of the gene or deletion of the entire gene, replacement of one base or more to cause an amino acid substitution in the protein encoded by the gene (missense mutation), introduction of a stop codon (nonsense mutation), deletion of one or two bases to cause a reading frame shift of the gene, insertion of a drug-resistance gene and/or transcription termination signal, or modification of an adjacent region of the gene, including sequences controlling gene expression such as promoter(s), enhancer(s), attenuator(s), ribosome-binding site(s) (RBS), etc. Inactivation of at least one gene from the puuADRCBE gene cluster can also be performed, for example, by conventional methods such as a mutagenesis treatment using UV irradiation or nitrosoguanidine (N-methyl-N′-nitro-N-nitrosoguanidine), site-directed mutagenesis, gene disruption using homologous recombination, and/or insertion-deletion mutagenesis (Yu D. et al., Proc. Natl. Acad. Sci. USA, 2000, 97(11):5978-5983; Datsenko K. A. and Wanner B. L., Proc. Natl. Acad. Sci. USA, 2000, 97(12):6640-6645; Zhang Y. et al., Nature Genet., 1998, 20:123-128) based on “Red/ET-driven integration” or “λRed/ET-mediated integration”.
The phrase “the expression of at least one gene from the puuADRCBE gene cluster is attenuated” can mean that an amount of the protein(s) encoded by modified gene(s) from the puuADRCBE gene cluster such as puuA, puuB, puuC, puuD, puuE and puuR in the modified bacterium, in which expression of the gene(s) is/are attenuated, is/and educed as compared with a non-modified bacterium, for example, a wild-type or parental strain such as E. coli K-12.
The phrase “at least one gene from the puuADRCBE gene cluster is attenuated” can also mean that the modified bacterium contains one or more regions operably linked to one or more genes from the puuADRCBE gene cluster, including sequences controlling expression of genes from the puuADRCBE gene cluster such as promoters, enhancers, attenuators and transcription termination signals, ribosome-binding sites (RBS), and other expression control elements, which are modified resulting in a decrease in the expression level of at least one gene from the puuADRCBE gene cluster; and other examples (see, for example, WO95/34672; Carrier T. A. and Keasling J. D., Biotechnol. Prog., 1999, 15:58-64). The phrase “operably linked to the gene” is a specific example of the phrase “operably linked to one or more genes,” and can mean that the regulatory region(s) is/are linked to the nucleotide sequence of the nucleic acid molecule or gene in a manner which allows for expression (e.g., enhanced, increased, constitutive, basal, antiterminated, attenuated, deregulated, decreased or repressed expression) of the nucleotide sequence, specifically, expression of a gene product encoded by the nucleotide sequence.
Expression of at least one gene from the puuADRCBE gene cluster can be attenuated by replacing an expression control sequence of the gene, such as a promoter on the chromosomal DNA, with a weaker one. The strength of a promoter is defined by the frequency of initiation acts of RNA synthesis. Examples of methods for evaluating the strength of promoters and strong promoters are described in Goldstein M. A. et al. (Goldstein M. A. and Doi R. H., Prokaryotic promoters in biotechnology, Biotechnol. Annu. Rev., 1995, 1:105-128), and so forth. Furthermore, it is also possible to introduce a nucleotide substitution for several nucleotides in a promoter region of a target gene and thereby modify the promoter to be weakened as disclosed in International Patent Publication WO00/18935. Furthermore, it is known that substitution of several nucleotides in the Shine-Dalgarno (SD) sequence, and/or in the spacer between the SD sequence and the start codon, and/or a sequence immediately upstream and/or downstream from the start codon in the ribosome-binding site (RBS) greatly affects the translation efficiency of mRNA. This modification of the RBS may be combined with decreasing transcription of at least one gene from the puuADRCBE gene cluster.
Expression of at least one gene from the puuADRCBE gene cluster can also be attenuated by insertion of a transposon or an insertion sequence (IS) into the coding region of the gene (U.S. Pat. No. 5,175,107) or in the region controlling gene expression, or by conventional methods such as mutagenesis with ultraviolet irradiation (UV) irradiation or nitrosoguanidine (N-methyl-N′-nitro-N-nitrosoguanidine). Furthermore, the incorporation of a site-specific mutation can be conducted by known chromosomal editing methods based, for example, on λRed/ET-mediated recombination.
The copy number, presence or absence of a gene from the puuADRCBE gene cluster or the entire cluster 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 mRNA transcribed from the gene using various well-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), or mass spectrometry analysis of the protein samples, and the like.
Methods for manipulation with recombinant molecules of DNA and molecular cloning such as preparation of plasmid DNA, digestion, ligation and transformation of DNA, selection of an oligonucleotide as a primer, incorporation of mutations, and the like may be ordinary methods well-known to the person skilled in the art. These methods are described, for example, in Sambrook J., Fritsch E. F. and Maniatis T., “Molecular Cloning: A Laboratory Manual”, 2nd ed., Cold Spring Harbor Laboratory Press (1989) or Green M. R. and Sambrook J. R., “Molecular Cloning: A Laboratory Manual”, 4th ed., Cold Spring Harbor Laboratory Press (2012); Bernard R. Glick, Jack J. Pasternak and Cheryl L. Patten, “Molecular Biotechnology: principles and applications of recombinant DNA”, 4th ed., Washington, D.C., ASM Press (2009).
As explained above, the genes encoding enzymes from the transaminase pathway (patA, patD, gabT and gabD) and the glutamylated putrescine pathway (puuA, puuB, puuC, puuD, puuE and puuR) of E. coli have been elucidated. For the purposes of the present invention, explanations given hereinafter as to the genes from the puuADRCBE gene cluster such as puuA, puuD, puuR, puuC, puuB and puuE can be applied mutatis mutandis to the patA, patD, gabT and gabD genes.
Since there may be some differences in DNA sequences between the genera, species or strains of the family Enterobacteriaceae, the puuA, puuD, puuR, puuC, puuB and puuE genes are not limited to the genes shown in SEQ ID NOs: 1, 3, 5, 7, 9 and 11, respectively, but may include genes which are variant nucleotide sequences of or homologous to SEQ ID NOs: 1, 3, 5, 7, 9 and 11, and which encode variants of the PuuA, PuuD, PuuR, PuuC, PuuB and PuuE proteins, respectively.
The phrase “a variant protein” can mean a protein which has one or several changes in the sequence compared with SEQ ID NO: 2, 4, 6, 8, 10 or 12, whether they are substitutions, deletions, insertions, and/or additions of one or several amino acid residues, but still maintains an activity or function similar to that of the PuuA, PuuD, PuuR, PuuC, PuuB or PuuE protein, respectively, or the three-dimensional structure of the PuuA, PuuD, PuuR, PuuC, PuuB or PuuE protein is not significantly changed relative to the wild-type or non-modified protein. 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 residues. It can be, but is not strictly limited to, 1 to 30, in another example 1 to 15, in another example 1 to 10, and in another example 1 to 5, in SEQ ID NO: 2, 4, 6, 8, 10 or 12. This is because some amino acids have high homology to one another so that the activity or function is not affected by such a change, or the three-dimensional structure of PuuA, PuuD, PuuR, PuuC, PuuB or PuuE protein is not significantly changed relative to the wild-type or non-modified protein. Therefore, the protein variants encoded by the puuA, puuD, puuR, puuC, puuB and puuE genes may have a homology, defined as the parameter “identity” when using the computer program BLAST, of not less than 80%, not less than 90%, not less than 95%, or not less than 98% with respect to the entire amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10 or 12, respectively, as long as the activity or function of the PuuA, PuuD, PuuR, PuuC, PuuB and PuuE proteins is maintained, or the three-dimensional structure of the PuuA, PuuD, PuuR, PuuC, PuuB and PuuE is not significantly changed relative to the wild-type or non-modified proteins.
The exemplary substitution, deletion, insertion, and/or addition of one or several amino acid residues can be a conservative mutation(s). The representative conservative mutation is a conservative substitution. The conservative substitution can be, but is not limited to, a substitution, wherein substitution takes place mutually among Phe, Trp and Tyr, if the substitution site is an aromatic amino acid; among Ala, Leu, Ile and Val, if the substitution site is a hydrophobic amino acid; between Glu, Asp, Gln, Asn, Ser, His and Thr, if the substitution site is a hydrophilic amino acid; between Gln and Asn, if the substitution site is a polar amino acid; among Lys, Arg and His, if the substitution site is a basic amino acid; between Asp and Glu, if the substitution site is an acidic amino acid; and between Ser and Thr, if the substitution site is an amino acid having hydroxyl group. Examples of conservative substitutions include substitution of Ser or Thr for Ala, substitution of Gln, His or Lys for Arg, substitution of Glu, Gln, Lys, His or Asp for Asn, substitution Asn, Glu or Gln for Asp, substitution of Ser or Ala for Cys, substitution Asn, Glu, Lys, His, Asp or Arg for Gln, substitution Asn, Gln, Lys or Asp for Glu, substitution of Pro for Gly, substitution Asn, Lys, Gln, Arg or Tyr for His, substitution of Leu, Met, Val or Phe for Ile, substitution of Ile, Met, Val or Phe for Leu, substitution Asn, Glu, Gln, His or Arg for Lys, substitution of Ile, Leu, Val or Phe for Met, substitution of Trp, Tyr, Met, Ile or Leu for Phe, substitution of Thr or Ala for Ser, substitution of Ser or Ala for Thr, substitution of Phe or Tyr for Trp, substitution of His, Phe or Trp for Tyr, and substitution of Met, Ile or Leu for Val.
The exemplary substitution, deletion, insertion, and/or addition of one or several amino acid residues can also be a non-conservative mutation(s) provided that the mutation(s) is/are compensated by one or more secondary mutations in the different position(s) of amino acids sequence so that the activity or function of the variant protein is maintained and similar to that of the PuuA, PuuD, PuuR, PuuC, PuuB or PuuE protein, or the three-dimensional structure of PuuA, PuuD, PuuR, PuuC, PuuB or PuuE is not significantly changed relative to the wild-type or non-modified protein.
To evaluate the degree of protein or DNA homology, several calculation methods can be used, such as BLAST search, FASTA search and ClustalW method. The BLAST (Basic Local Alignment Search Tool, ncbi.nlm.nih.gov/BLAST/) search is the heuristic search algorithm employed by the programs blastp, blastn, blastx, megablast, tblastn, and tblastx; these programs ascribe significance to their findings using the statistical methods of Samuel K. and Altschul S. F. (“Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes” Proc. Natl. Acad. Sci. USA, 1990, 87:2264-2268; “Applications and statistics for multiple high-scoring segments in molecular sequences”. Proc. Natl. Acad. Sci. USA, 1993, 90:5873-5877). The computer program BLAST calculates three parameters: score, identity and similarity. The FASTA search method is described by Pearson W. R. (“Rapid and sensitive sequence comparison with FASTP and FASTA”, Methods Enzymol., 1990, 183:63-98). The ClustalW method is described by Thompson J. D. et al. (“CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice”, Nucleic Acids Res., 1994, 22:4673-4680).
Moreover, the puuA, puuD, puuR, puuC, puuB and puuE genes can be variant nucleotide sequences. The phrase “a variant nucleotide sequence” can mean a nucleotide sequence which encodes the PuuA, PuuD, PuuR, PuuC, PuuB or PuuE protein using any synonymous amino acid codons according to the standard genetic code table (see, e.g., Lewin B., “Genes VIII”, 2004, Pearson Education, Inc., Upper Saddle River, N.J. 07458), which can also be referred to as “a variant protein” of the PuuA, PuuD, PuuR, PuuC, PuuB or PuuE protein. The puuA, puuD, puuR, puuC, puuB and puuE genes can be variant nucleotide sequences due to degeneracy of genetic code.
The phrase “a variant nucleotide sequence” can also mean, but is not limited to a nucleotide sequence which hybridizes under stringent conditions with the nucleotide sequence complementary to the sequence shown in SEQ ID NO: 1, 3, 5, 7, 9 or 11, or a probe which can be prepared from the nucleotide sequence under stringent conditions provided that it encodes active or functional protein. “Stringent conditions” include those under which a specific hybrid, for example, a hybrid having homology, defined as the parameter “identity” when using the computer program BLAST, of not less than 70%, not less than 80%, not less than 90%, not less than 95%, not less than 96%, not less than 97%, not less than 98%, or not less than 99% 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 in another example, two or three times, at a salt concentration of 1×SSC (standard sodium citrate or standard sodium chloride), 0.1% SDS (sodium dodecyl sulphate), or in another example, 0.1×SSC, 0.1% SDS at 60° C. or 65° C. Duration of washing depends on the type of membrane used for blotting and, as a rule, can be what is recommended by the manufacturer. For example, the recommended duration of washing for the Amersham HybondTM-N+ positively charged nylon membrane (GE Healthcare) under stringent conditions is 15 minutes. The washing step can be performed 2 to 3 times. As the probe, a part of the sequences complementary to the sequences shown in SEQ ID NO: 1, 3, 5, 7, 9 or 11 may also be used. Such a probe can be produced by PCR using oligonucleotides as primers prepared on the basis of the sequences shown in SEQ ID NO: 1, 3, 5, 7, 9 or 11 and a DNA-fragment containing the nucleotide sequence as template. The length of the probe is recommended to be >50 bp; it can be suitably selected depending on the hybridization conditions, and is usually 100 bp to 1 kbp. For example, when a DNA-fragment having a length of about 300 by is used as the probe, the washing conditions after hybridization can be exemplified by 2×SSC, 0.1% SDS at 50° C., 60° C. or 65° C.
As the genes encoding the PuuA, PuuD, PuuR, PuuC, PuuB and PuuE proteins of the species E. coli have already been elucidated (see above), the variant nucleotide sequences encoding variant proteins of PuuA, PuuD, PuuR, PuuC, PuuB and PuuE proteins can be obtained by PCR (polymerase chain reaction; refer to White T. J. et al., The polymerase chain reaction, Trends Genet., 1989, 5:185-189) utilizing primers prepared based on the nucleotide sequences of the puuA, puuD, puuR, puuC, puuB and puuE genes; or the site-directed mutagenesis method by treating DNA containing the wild-type puuA, puuD, puuR, puuC, puuB and puuE gene in vitro, for example, with hydroxylamine, or a method for treating a microorganism, for example, a bacterium belonging to the family Enterobacteriaceae harboring the wild-type puuA, puuD, puuR, puuC, puuB and puuE genes with ultraviolet (UV) irradiation or a mutating agent such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) and nitrous acid usually used for the such treatment; or chemically synthesized as full-length gene structures. Genes encoding the PuuA, PuuD, PuuR, PuuC, PuuB and PuuE proteins or its variant proteins of other microorganisms can be obtained in a similar manner.
The phrase “a wild-type protein” can mean a native protein naturally produced by a wild-type or parent bacterial strain of the family Enterobacteriaceae, for example, by the wild-type E. coli MG1655 strain. A wild-type protein can be encoded by the “wild-type gene”, or the “non-modified gene” naturally occurring in the genome of a wild-type bacterium.
The bacterium as described herein can be obtained by attenuating expression of at least one of the genes of the putrescine degradation pathway in a bacterium inherently having an ability to produce an L-amino acid. Alternatively, the bacterium as described herein can be obtained by imparting the ability to produce an L-amino acid to a bacterium in which expression of at least one of the genes or the putrescine degradation pathway is already attenuated.
The bacterium can have, in addition to the properties already mentioned, other specific properties such as various nutrient requirements, drug resistance, drug sensitivity, and drug dependence, without departing from the scope of the present invention.
2. Method
A method of the present invention includes the method for producing an L-amino acid such as L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-citrulline, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-ornithine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine, or a mixture thereof. More specifically, a method of the present invention includes the method for producing an L-amino acid of the glutamate family such as L-arginine, L-citrulline, L-glutamic acid, L-glutamine, L-proline, and L-ornithine, or a mixture thereof. Furthermore, a method of the present invention includes the method for producing L-arginine.
The method for producing an L-amino acid can include the steps of cultivating the bacterium in a culture medium to allow the L-amino acid to be produced, excreted, and/or accumulated in the culture medium, and collecting the L-amino acid from the culture medium and/or the bacterial cells. Collected amino acid can be further purified. The L-amino acid can be produced in a salt or a hydrate form thereof, or a combination thereof. For example, sodium, potassium, ammonium, and the like salts of the L-amino acid can be produced by the method. The L-amino acid can be produced in an adduct form thereof with, for example, another organic or inorganic compound. Specifically, a monochlorhydrate salt of an L-amino acid can be produced by the method such as monochlorhydrate salt of L-lysine (L-lysine-HCl) or monochlorhydrate salt of L-arginine (L-arginine-HCl).
The cultivation of the bacterium, and collection and purification of L-amino acid, or a salt or hydrate thereof, from the medium and the like may be performed in a manner similar to conventional fermentation methods wherein L-amino acid is produced using a microorganism. The culture medium for production of the L-amino acid can be either a synthetic or natural medium such as a typical medium that contains a carbon source, a nitrogen source, a sulphur source, inorganic ions, and other organic and inorganic components as required. As the carbon source, saccharides such as glucose, lactose, galactose, fructose, sucrose, arabinose, maltose, xylose, trehalose, ribose, and hydrolyzates of starches; alcohols such as glycerol, mannitol, and sorbitol; organic acids such as gluconic acid, fumaric acid, citric acid, malic acid, and succinic acid; and the like can be used. As the nitrogen source, inorganic ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate; organic nitrogen such as of soy bean hydrolyzates; ammonia gas; aqueous ammonia; and the like can be used. The sulphur source can include ammonium sulphate, magnesium sulphate, ferrous sulphate, manganese sulphate, and the like. Vitamins such as vitamin B1, required substances, for example, organic nutrients such as nucleic acids such as adenine and RNA, or yeast extract, and the like may be present in appropriate, even if trace, amounts. Other than these, small amounts of calcium phosphate, iron ions, manganese ions, and the like may be added, if necessary.
Cultivation can be performed under aerobic conditions for 16 to 72 h, or for 16 to 65 h; the culture temperature during cultivation can be controlled within 30 to 45° C., or within 30 to 37° C.; and the pH can be adjusted between 5 and 8, or between 6.5 and 7.5. The pH can be adjusted by using an inorganic or organic acidic or alkaline substance, as well as ammonia gas.
After cultivation, solids such as cells and cell debris can be removed from the liquid medium by centrifugation or membrane filtration, and then the target L-amino acid or a salt thereof can be recovered from the fermentation liquor by any combination of conventional techniques such as concentration, ion-exchange chromatography, and crystallization.
The collected target L-amino acid composition may contain microbial cells, medium components, moisture, and by-product metabolites of the microorganism in addition to the target substance. Purity of the collected target substance is 50% or higher, preferably 85% or higher, particularly preferably 95% or higher (U.S. Pat. No. 5,431,933, Japanese Patent No. 1214636, U.S. Pat. Nos. 4,956,471, 4,777,051, 4,946,654, 5,840,358, 6,238,714, U.S. Patent Published Application No. 2005/0025878).
EXAMPLES
The present invention will be more specifically explained below with reference to the following non-limiting Examples.
Example 1. Construction of E. coli Strain Having Inactivated the puuADRCBE Gene Cluster
An E. coli strain having inactivated the puuADRCBE gene cluster was constructed by the method initially developed by Datsenko K. A. and Wanner B. L. called “λRed/ET-mediated integration” (Datsenko K. A. and Wanner B. L., Proc. Natl. Acad. Sci. USA, 2000, 97(12):6640-6645). A DNA-fragment containing the λattL-Cm-λattR cassette was obtained by PCR using primers P1 (SEQ ID NO: 13) and P2 (SEQ ID NO: 14) and the pMW118-λattL-Cm-λattR plasmid (WO2005010175 A1) as the template. Primer P1 contains both a region complementary to the region located at the 5′-end of the puuA gene and a region complementary to the attR region. Primer P2 contains both a region complementary to the region located at the 3′-end of the puuE gene and a region complementary to the attL region. The 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., 1 min 40 sec at 72° C.; final step: 5 min at 72° C.
The PCR-product obtained (about 1.6 kbp) was purified by electrophoresis in agarose gel and used for electroporation of the E. coli MG1655 strain containing the pKD46 plasmid having a temperature-sensitive replication origin. The pKD46 plasmid (Datsenko K. A. and Wanner B. L., Proc. Natl. Acad. Sci. USA, 2000, 97(12):6640-6645) includes a 2,154 nucleotides DNA-fragment of phage λ (nucleotide positions from 31088 to 33241, GenBank accession No.: J02459), and contains genes of the λRed homologous recombination system (γ, βexo genes) under the control of the arabinose-inducible ParaB promoter. The pKD46 plasmid is necessary for integration of a PCR-product into chromosome of the E. coli MG1655 strain. The E. coli MG1655 strain containing the recombinant plasmid pKD46 can be obtained from the E. coli Genetic Stock Center, Yale University, New Haven, USA (Accession No. CGSC7669).
Electrocompetent cells were prepared as follows: the E. coli MG1655/pKD46 strain was grown at 30° C. overnight in LB-medium (Luria-Bertani broth, also referred to as lysogenic broth; Sambrook J. and Russell D. W., Molecular Cloning: A Laboratory Manual (3rd ed.), Cold Spring Harbor Laboratory Press, 2001) containing ampicillin (100 mg/L); then the culture was diluted 100 times with 5 mL of SOB-medium (Sambrook J. and Russell D. W., Molecular Cloning: A Laboratory Manual (3rd ed.), Cold Spring Harbor Laboratory Press, 2001) containing ampicillin (100 mg/L) and L-arabinose (1 mM). The cells were grown with aeration (250 rpm) at 30° C. to OD600 of  ̃0.6. Electrocompetent cells were made by concentrating 100-fold and washing three times with ice-cold deionized H2O. Electroporation was performed using 70 μL of cells and about 100 ng of the PCR-product. Electroporated cells were incubated with 1 mL of SOC-medium (Sambrook J. and Russell D. W., Molecular Cloning: A Laboratory Manual (3rd ed.), Cold Spring Harbor Laboratory Press, 2001) at 37° C. for 2.5 hours, plated onto L-agar containing chloramphenicol (25 mg/L), and grown at 37° C. to select CmR-recombinants. To eliminate the pKD46 plasmid, two passages on L-agar supplemented with chloramphenicol (25 mg/L) at 42° C. were performed, and the obtained colonies were tested for sensitivity to ampicillin. Thus the E. coli MG1655ΔpuuADRCBE::Cm strain was obtained.
Example 2. Production of L-Arginine by E. coli 382ilvA+ΔpuuADRCBE
Clones of E. coli 382ilvA+ were selected as good-growing colonies on minimal agar plates. The strain 382ilvA+ was obtained from the L-arginine-producing strain 382 (VKPM B-7926, EP1170358 A1) by P1-transduction of the wild-type ilvA gene from E. coli K-12 strain. The strain 382 was deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russian Federation, 117545 Moscow, 1st Dorozhny proezd, 1) on Apr. 10, 2000 under the accession number VKPM B-7926 and then converted to a deposit under the Budapest Treaty on May 18, 2001. To test the effect from inactivation of the puuADRCBE gene cluster on L-arginine production, a DNA-fragment from the chromosome of the obtained E. coli MG1655ΔpuuADRCBE::Cm was transferred to the L-arginine-producing E. coli strain 382ilvA+ by P1-transduction (Miller, J. H. (1972) <<Experiments in Molecular Genetics>>, Cold Spring Harbor Lab. Press, Plainview, N.Y.) to obtain the E. coli 382ilvA+ΔpuuADRCBE::Cm strain.
Both E. coli strains 382ilvA+ and 382ilvA+ΔpuuADRCBE::Cm were each cultivated with shaking (220 rpm) at 37° C. for 18 hours in 3 mL of nutrient broth. Then 0.3 mL of the obtained cultures was 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 to an OD540 of  ̃38 until glucose consumption.
After the cultivation, the amount of L-arginine which accumulated in the medium was determined by paper chromatography using a mobile phase consisting of butan-1-ol: 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-arginine was cut out, L-arginine was eluted with a 0.5% water solution of CdCl2, and the amount of L-arginine was estimated spectrophotometrically at 540 nm.
The composition of the fermentation medium (g/L) was as follows:
















Glucose
48.0



(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 were sterilized separately. CaCO3 was dry-heat sterilized at 180° C. for 2 hours. The pH was adjusted to 7.0 with KOH solution.
The results of eight independent test tube fermentations (as average values) are shown in Table 1. As it can be seen from the Table 1, the modified E. coli 382ilvA+ΔpuuADRCBE::Cm strain was able to produce a higher amount of L-arginine (Arg) as compared with the parent E. coli 382ilvA+ strain.
The E. coli 382ilvA+ΔpuuA::Km strain having inactivated the puuA gene is described in Example 5.







TABLE 1







Production of L-arginine.










Strain
Arg, g/L














E. coli 382ilvA+ (control)
6.4



E. coli 382ilvA+ΔpuuADRCBE::Cm
7.6



E. coli 382ilvA+ΔpuuA::Km
7.5










Example 3. Construction of E. coli L-Ornithine-Producing Strain
An E. coli L-ornithine-producing strain was obtained from E. coli L-arginine-producing strain 382ilvA+ by inactivation of ornithine carbamoyltransferase encoded by both argF and argI genes.
3.1. Inactivation of the argF Gene
A strain in which the argF gene is deleted was constructed by the method initially developed by Datsenko K. A. and Wanner B. L. called “λRed/ET-mediated integration” (Datsenko K. A. and Wanner B. L., Proc. Natl. Acad. Sci. USA, 2000, 97(12):6640-6645). A DNA-fragment containing the kanamycin resistance marker (KmR) was obtained by PCR using primers P3 (SEQ ID NO: 15) and P4 (SEQ ID NO: 16) and the pMW118-attL-Km-attR plasmid as the template (WO2011043485 A1). Primer P3 contains both a region complementary to the region located at the 5′-end of the argF gene and a region complementary to the attR region. Primer P4 contains both a region complementary to the region located at the 3′-end of the argF gene and a region complementary to the attL region. The 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.
The PCR-product obtained (about 1.6 kbp) was purified by electrophoresis in agarose gel and integrated into the chromosome of the E. coli MG1655 strain by the λRed-mediated integration as described above (Example 1) to replace the native argF gene. Thus the E. coli MG1655ΔargF::Km strain was obtained.
3.2. Inactivation of the argI Gene
A strain in which the argI gene is deleted was constructed by the method initially developed by Datsenko K. A. and Wanner B. L. called “λRed/ET-mediated integration” (Datsenko K. A. and Wanner B. L., Proc. Natl. Acad. Sci. USA, 2000, 97(12):6640-6645). A DNA-fragment containing the λattL-Cm-λattR cassette was obtained by PCR using primers P5 (SEQ ID NO: 17) and P6 (SEQ ID NO: 18) and the pMW118-attL-Cm-attR plasmid as the template (WO2005010175 A1). Primer P5 contains both a region complementary to the region located at the 5′-end of the argI gene and a region complementary to the attR region. Primer P6 contains both a region complementary to the region located at the 3′-end of the argI gene and a region complementary to the attL region. The 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.
The PCR-product obtained (about 1.6 kbp) was purified in agarose gel and integrated into the chromosome of the E. coli MG1655 strain by the λRed-mediated integration as described above (Example 1) to replace the native argI gene. Thus the E. coli MG1655ΔargI:Cm strain was obtained.
3.3. Construction of E. coli Strain Having Inactivated the argI and argF Genes
To obtain an E. coli L-ornithine-producing strain, DNA-fragments from the chromosomes of the obtained E. coli MG1655ΔargF::Km (Example 3.1) and E. coli MG1655ΔargI:Cm (Example 3.2) strains were consecutively transferred to the L-arginine-producing E. coli strain 382ilvA+ by P1-transductions to obtain the E. coli 382ilvA+ΔargI:Cm ΔargF::Km strain.
The CmR and KmR-markers were eliminated simultaneously using the pMW-Int/Xis helper plasmid (WO2005010175 A1) which was electroporated into the selected plasmid-less integrants using electroporation as described above (Example 1) for electroporation of the PCR-generated DNA-fragment. After electroporation, cells were plated on L-agar containing 0.5% glucose and ampicillin (150 mg/L), and incubated at 30° C. overnight to induce synthesis of the Int/Xis proteins. The grown clones were replica-plated on L-agar with and without a mixture of chloramphenicol and kanamycin to select the CmS and KmS (chloramphenicol and kanamycin-sensitive) variants. Thus the E. coli 382ilvA+ΔargI ΔargF strain was obtained.
Example 4. Production of L-Ornithine by E. coli 382ilvA+ΔargI ΔargF ΔpuuADRCBE::Cm
To test the effect from inactivation of the puuADRCBE gene cluster on L-ornithine production, a DNA-fragment from the chromosome of the obtained E. coli MG1655ΔpuuADRCBE::Cm (Example 1) was transferred to the L-ornithine-producing E. coli strain 382ilvA+ΔargI ΔargF (Example 3) by P1-transduction to obtain the E. coli 382ilvA+ΔargI ΔargF ΔpuuADRCBE::Cm strain.
The L-ornithine production was evaluated as described above for L-arginine production (Example 2). The results of six independent test tube fermentations (as average values) are shown in Table 2. As it can be seen from the Table 2, the modified E. coli 382ilvA+ΔargI ΔargF ΔpuuADRCBE::Cm strain was able to produce a higher amount of L-ornithine (Orn) as compared with the parent E. coli 382ilvA+ΔargI ΔargF strain.







TABLE 2







Production of L-ornithine.










Strain 
Orn, g/L







E. coli 382ilvA+ΔargI ΔargF (control)
3.0



E. coli 382ilvA+ΔargI ΔargF ΔpuuADRCBE::Cm
3.7










Example 5. Construction of E. coli Strain Having Inactivated the puuA Gene
A strain in which the puuA gene is deleted was constructed by the method initially developed by Datsenko K. A. and Wanner B. L. called “λRed/ET-mediated integration” (Datsenko K. A. and Wanner B. L., Proc. Natl. Acad. Sci. USA, 2000, 97(12):6640-6645). A DNA-fragment containing the λattL-Km-λattR cassette was obtained by PCR using primers P7 (SEQ ID NO: 19) and P8 (SEQ ID NO: 20) and the pMW118-attL-Km-attR plasmid as the template (WO2011043485 A1). Primer P7 contains both a region complementary to the region located at the 5′-end of the puuA gene and a region complementary to the attR region. Primer P8 contains both a region complementary to the region located at the 3′-end of the puuA gene and a region complementary to the attL region. The 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 59° C., 1 min 40 sec at 72° C.; final step: 5 min at 72° C.
The PCR-product obtained (about 1.6 kbp) was purified in agarose gel and integrated into the chromosome of the E. coli MG1655 strain by the λRed-mediated integration as described above (Example 1) to replace the native puuA gene. Thus the E. coli MG1655ΔpuuA::Km strain was obtained.
Example 6. Production of L-Arginine by E. coli 382ilvA+ΔpuuA::Km
To test the effect of inactivation of the puuA gene on L-arginine production, a DNA-fragment from the chromosome of the obtained E. coli MG1655ΔpuuA::Km was transferred to the L-arginine-producing E. coli strain 382ilvA+ by P1-transduction to obtain the E. coli 382ilvA+ΔpuuA::Km strain.
The L-arginine production was evaluated as described in Example 2. The results of eight independent test tube fermentations (as average values) are shown in Table 1. As it can be seen from the Table 1, the modified E. coli 382ilvA+ΔpuuA::Km strain was able to accumulate a higher amount of L-arginine as compared with the parent E. coli 382ilvA+ strain.
Example 7. Production of L-Citrulline by E. coli 382ΔargG ΔpuuADRCBE::Cm
To test the effect from inactivation of the puuADRCBE gene cluster on L-citrulline production, a DNA-fragment from the chromosome of the above-described E. coli MG1655ΔpuuADRCBE::Cm strain is transferred to the citrulline producing E. coli strain 382ΔargG by P1-transduction to obtain the strain 382ΔargG ΔpuuADRCBE::Cm. The strain 382ΔargG is obtained by deletion of argG gene on the chromosome of the L-arginine-producing strain 382 (VKPM B-7926, EP1170358 A1) by the method initially developed by Datsenko K. A. and Wanner B. L. called “λRed/ET-mediated integration” (Datsenko K. A. and Wanner B. L., Proc. Natl. Acad. Sci. USA, 2000, 97(12):6640-6645). According to this procedure, the PCR-primers homologous to both the region adjacent to the argG gene and the gene which confers antibiotic resistance in the template plasmid are constructed. The plasmid pMW118-λattL-Cm-λattR (WO2005010175 A1) is used as the template in the PCR-reaction.
E. coli strains 382ΔargG and 382ΔargG ΔpuuADRCBE::Cm are separately cultivated with shaking at 37° C. for 18 h in 3 mL of nutrient broth, and 0.3 mL of the obtained cultures are inoculated into 2 mL of a fermentation medium in 20×200-mm test tubes and cultivated at 32° C. for 48 h on a rotary shaker.
After the cultivation, the amount of L-citrulline which accumulates in the medium is determined by paper chromatography using a mobile phase consisting of butan-1-ol:acetic acid:water=4:1:1 (v/v). A solution of ninhydrin (2%) in acetone is used as a visualizing reagent. A spot containing citrulline is cut out, L-citrulline is eluted with 0.5% water solution of CdCl2, and the amount of L-citrulline is estimated spectrophotometrically at 540 nm.
The composition of the fermentation medium (g/L) is as follows:
















Glucose
48.0



(NH4)2SO4
35.0



KH2PO4
2.0



MgSO4•7H2O
1.0



Thiamine-HCl
0.0002



Yeast extract 
1.0



L-Isoleucine
0.1



L-Arginine
0.1



CaCO3
5.0










Glucose and magnesium sulfate are sterilized separately. CaCO3 is dry-heat sterilized at 180° C. for 2 h. The pH is adjusted to 7.0.
Example 8. Production of L-Glutamic Acid by E. coli VL334thrC+ΔpuuADRCBE::Cm
To test the effect from inactivation of the puuADRCBE gene cluster on L-glutamic acid production, a DNA-fragment from the chromosome of the above-described E. coli MG1655ΔpuuADRCBE::Cm strain is transferred to the glutamate-producing E. coli strain VL334thrC+ (EP1172433 A1) by P1-transduction to obtain the strain VL334thrC+ΔpuuADRCBE::Cm. The strain VL334thrC+ was deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russian Federation, 117545 Moscow, 1 Dorozhny proezd, 1) on Dec. 6, 2004 under the accession number VKPM B-8961 and then converted to a deposit under the Budapest Treaty on Dec. 8, 2004.
E. coli strains VL334thrC+ and VL334thrC+ΔpuuADRCBE::Cm are separately cultivated for 18-24 h at 37° C. on L-agar plates. Then, one loop of the cells is transferred into test tubes containing 2 mL of fermentation medium. Cultivation is carried out at 30° C. for 3 days with shaking.
After the cultivation, the amount of L-glutamic acid which accumulates in the medium is determined by paper chromatography using a mobile phase consisting of butan-1-ol:acetic acid:water=4:1:1 (v/v) with subsequent staining by ninhydrin (1% solution in acetone), elution of the compounds in 50% ethanol with 0.5% CdCl2 and further estimation of L-glutamic acid at 540 nm.
The composition of the fermentation medium (g/L) is as follows:
















Glucose
60.0



(NH4)2SO4
25.0



KH2PO4
2.0



MgSO4•7H2O
1.0



Thiamine-HCl
0.1



L-Isoleucine
0.07



CaCO3
25.0










Glucose and CaCO3 are sterilized separately. The pH is adjusted to 7.2.
Example 9. Production of L-Proline by E. coli 702ilvAΔpuuADRCBE::Cm
To test the effect from inactivation of the puuADRCBE gene cluster on L-proline production, a DNA-fragment from the chromosome of the above-described E. coli MG1655ΔpuuADRCBE::Cm strain is transferred to the proline-producing E. coli strain 702ilvA by P1-transduction to obtain the strain 702ilvAΔpuuADRCBE::Cm. The strain 702ilvA was deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russian Federation, 117545 Moscow, 1st Dorozhny proezd, 1) on Jul. 18, 2000 under the accession number VKPM B-8012 and then converted to a deposit under the Budapest Treaty on May 18, 2001.
E. coli strains 702ilvA and 702ilvAΔpuuADRCBE::Cm are separately cultivated for 18-24 h at 37° C. on L-agar plates. Then, these strains are cultivated under the same conditions as in Example 8 (Production of L-glutamic acid).
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 by reference as a part of this application.
INDUSTRIAL APPLICABILITY
According to the present invention, production of L-amino acids such as those belonging to the glutamate family of amino acids by a bacterium of the Enterobacteriaceae family can be improved.

## Claims

1. A method for producing an L-amino acid comprising:
(i) cultivating an L-amino acid-producing bacterium of the family Enterobacteriaceae in a culture medium; and
(ii) collecting said L-amino acid in the culture medium, wherein said bacterium has been modified to disrupt a putrescine degradation pathway,
wherein said bacterium belongs to the genus Escherichia, and
wherein said putrescine degradation pathway is disrupted by a method selected from the group consisting of:
(a) attenuation of expression of a gene selected from the group consisting of patA, patD, gabT, gabD, puuA, puuB, puuC, puuD, puuE, and combinations thereof, by mutating said gene, and
(b) attenuation of expression of at least one gene from the puuADRCBE gene cluster by mutating said gene, with the proviso that the puuR gene cannot be the only gene that is attenuated; and
wherein said L-amino acid is L-arginine or L-omithine.

2. The method according to claim 1, wherein said putrescine degradation pathway is a transaminase pathway or a glutamylated putrescine pathway.

3. The method according to claim 1, wherein said putrescine degradation pathway is disrupted by mutating the puuA gene or the entire puuADRCBE gene cluster.

4. The method according to claim 1, wherein said gene is inactivated.

5. The method according to claim 4, wherein said gene is deleted.

6. The method according to claim 1, wherein said bacterium is Escherichia coli.