# Patent

## Title
Method for producing an L-amino acid using a bacterium of the Enterobacteriaceae family with attenuated expression of the ydiN gene or the ydiB gene or combination thereof

## Abstract

The present invention provides a method for producing an aromatic L-amino acid using a bacterium of the Enterobacteriaceae family, particularly a bacterium belonging to genus Escherichia or Pantoea, which has been modified to attenuate expression of the ydiN gene, the ydiB gene, or both.

## Background
This application is a continuation of PCT/JP2007/066322, filed Aug. 16, 2007, which claims priority under 35 U.S.C. §119 to Russian Patent Application No. 2006129690, filed on Aug. 16, 2006, which are incorporated in their entireties by reference. The Sequence Listing in electronic format filed herewith is also hereby incorporated by reference in its entirety (File Name: US-384_Seq_List; File Size: 39 KB; Date Created: Feb. 17, 2009).


BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to the microbiological industry, and specifically to a method for producing an aromatic L-amino acid using a bacterium of the Enterobacteriaceae family which has been modified to attenuate expression of the ydiN gene, the ydiB gene, or both.
2. Brief Description of the Related Art
Conventionally, L-amino acids are industrially produced by fermentation 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 by transforming microorganisms with recombinant DNA (see, for example, U.S. Pat. No. 4,278,765). Other techniques for enhancing production yields include increasing the activities of enzymes involved in amino acid biosynthesis and/or desensitizing the target enzymes of the feedback inhibition by the resulting L-amino acid (see, for example, WO 95/16042, or U.S. Pat. Nos. 4,346,170, 5,661,012, and 6,040,160).
Another way to enhance L-amino acid production yields is to attenuate expression of a gene or several genes 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 carbon, nitrogen, and phosphate fluxes, genes coding for toxins, etc.
Shikimate dehydrogenase catalyzes the fourth step of the shikimate pathway, which is the essential route for the biosynthesis of aromatic compounds in plants and microorganisms. Escherichia coli expresses two shikimate dehydrogenase paralogs, the NADP-specific AroE and a putative enzyme YdiB. YdiB is characterized as a dual specificity quinate/shikimate dehydrogenase that utilizes either NAD or NADP as a cofactor. The structures of AroE and YdiB with bound cofactors were determined at 1.5 and 2.5 A resolution, respectively. Both enzymes display a similar structure with two alpha/beta domains separated by a wide cleft. Comparison of their dinucleotide-binding domains reveals the molecular basis for cofactor specificity. Independent molecules display conformational flexibility which suggests that a switch between the open and closed conformation occurs upon substrate binding. Sequence analysis and structural comparison led to a proposal for the catalytic machinery and a model for 3-dehydroshikimate recognition. (Michel G., et al., J Biol. Chem. 23; 278(21):19463-72 (2003)).
The Escherichia coli YdiB protein, an orthologue of shikimate 5-dehydrogenase, catalyzes the reduction of 3-dehydroshikimate to shikimate as part of the shikimate pathway, which is absent in mammals but required for the de novo synthesis of aromatic amino acids, quinones, and folate in many other organisms. In this context, the shikimate pathway has been selected as a target for the development of antimicrobial agents. The crystal structure of YdiB shows that the promoter contains two alpha/beta domains connected by two alpha-helices, with the N-terminal domain being novel and the C-terminal domain being a Rossmann fold. The NAD+ cofactor, which co-purified with the enzyme, is bound to the Rossmann domain in an elongated fashion with the nicotinamide ring in the pro-R conformation. Its binding site contains several unusual features, including a cysteine residue opposite to the nicotinamide ring and a clamp-like structure over the ribose of the adenosine moiety formed by phenylalanine and lysine residues. The structure explains the specificity for NAD versus NADP in different members of the shikimate dehydrogenase family on the basis of variations in the amino acid identity of several other residues in the vicinity of this ribose group. A cavity lined by residues that are 100% conserved among all shikimate dehydrogenases is found between the two domains of YdiB, in close proximity to the hydride acceptor site on the nicotinamide ring. Shikimate was modeled into this site in a geometry such that all of its heteroatoms form high quality hydrogen bonds with these invariant residues. Their strong conservation among all the orthologues supports the possibility of developing broad spectrum inhibitors of this enzyme. The nature and disposition of the active site residues suggest a novel reaction mechanism in which an aspartate acts as the general acid/base catalyst during the hydride transfer reaction (Benach J., et. al., J Biol. Chem. 23; 278(21):19176-82 (2003)).
When shikimic acid is produced by genetically modified Escherichia coli, it has previously been found that carbon-rich conditions (e.g. phosphate-limiting) favor production of shikimic acid over shikimate pathway by-products, whereas the situation is the opposite under carbon-(glucose-) limited conditions. Gene expression patterns of the shikimate producing strain W3110.shik1 (W3110 with an aroL deletion and plasmid-overexpressed aroF) and the wild-type strain W3110 grown under carbon- and phosphate-limited (carbon-rich) chemostat conditions (D=0.23 h(−1)) were analyzed. The study suggests that the by-product formation when carbon is limited is explained by a set of upregulated genes coupled to the shikimate pathway. The genes ydiB, aroD, and ydiN were strongly induced only in carbon-limited W3110.shik1. Compared to W3110, the 1 g(2)-fold changes were: 6.25 (ydiB), 3.93 (aroD), and 8.18 (ydiN). In addition, the transcriptome analysis revealed a large change in the gene expression when comparing phosphate-limited conditions to carbon-limited, which to a large part could be explained by anabolic-catabolic uncoupling, which is present under phosphate-limited but not under carbon-limited conditions. Interestingly, there was also a larger difference between the two strains under carbon-limited conditions than under phosphate-limited. The reason for this difference is interpreted as a starvation for aromatic amino acids under carbon-limited conditions, which is relieved under phosphate-limited conditions due to an upregulation of aroK and aroA (Johansson L. and Liden G., J. Biotechnol. In Press, Corrected Proof, Available online 17 May 2006).
But currently, there have been no reports of attenuating expression of the ydiN gene or the ydiB gene or the combination thereof for the purpose of producing L-amino acids.
SUMMARY OF THE INVENTION
Aspects of the present invention include enhancing the productivity of aromatic L-amino acid-producing strains and providing a method for producing L-amino acids using these strains.
The above aspects were achieved by finding that attenuating expression of the ydiN gene, the ydiB gene, or both can enhance production of aromatic L-amino acids, such as L-phenylalanine, L-tyrosine and L-tryptophan.
The present invention provides a bacterium of the Enterobacteriaceae family which has an increased ability to produce aromatic L-amino acids, such as L-phenylalanine, L-tyrosine, and L-tryptophan.
It is an aspect of the present invention to provide an aromatic L-amino acid-producing bacterium of the Enterobacteriaceae family, wherein the bacterium has been modified to attenuate expression of a gene selected from the group consisting of ydiN, ydiB, or both.
It is a further aspect of the present invention to provide the bacterium as described above, wherein said expression is attenuated by inactivating the ydiN gene, the ydiB gene, or both genes.
It is a further aspect of the present invention to provide the bacterium as described above, wherein the bacterium belongs to the genus Escherichia. 
It is a further aspect of the present invention to provide the bacterium as described above, wherein the bacterium belongs to the genus Pantoea. 
It is a further aspect of the present invention to provide the bacterium as described above, wherein said aromatic L-amino acid is selected from the group consisting of L-phenylalanine, L-tyrosine, and L-tryptophan.
It is a further aspect of the present invention to provide a method for producing an aromatic L-amino acid comprising:

    
    
        cultivating the bacterium as described above in a medium, and
        collecting said aromatic L-amino acid from the medium.
    
    


It is a further aspect of the present invention to provide the method as described above, wherein said aromatic L-amino acid is selected from the group consisting of L-phenylalanine, L-tyrosine, and L-tryptophan.
It is a further aspect of the present invention to provide a method for producing a lower alkyl ester of α-L-aspartyl-L-phenylalanine, comprising cultivating the bacterium as described above in a culture medium, and synthesizing the lower alkyl ester of α-L-aspartyl-L-phenylalanine from aspartic acid or derivative thereof, and the L-phenylalanine which is produced by the bacterium,
wherein said bacterium is able to produce L-phenylalanine.
It is a further aspect of the present invention to provide the method as described above, further comprising esterifying L-phenylalanine to generate a lower alkyl ester of L-phenylalanine, condensing the lower alkyl ester of L-phenylalanine with an aspartic acid derivative, wherein the derivative is N-acyl-L-aspartic anhydride, separating the lower alkyl ester of N-acyl-α-L-aspartyl-L-phenylalanine from the reaction mixture, and hydrogenating the lower alkyl ester of N-acyl-α-L-aspartyl-L-phenylalanine to generate the lower alkyl ester of α-L-aspartyl-L-phenylalanine.
The present invention is described in detail below.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the relative positions of primers P5/9/5 and P6/10/10 on plasmid pMW118-attL-Cm-attR. This plasmid is used as a template for PCR amplification of the cat gene.
FIG. 2 shows the construction of the chromosomal DNA fragment containing the inactivated target gene(s).



DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
1. Bacterium
The bacterium is an aromatic L-amino acid-producing bacterium of the Enterobacteriaceae family, wherein the bacterium has been modified to attenuate expression of the ydiN gene, the ydiB gene, or both.
The phrase “aromatic L-amino acid-producing bacterium” means a bacterium which has an ability to produce and secrete an aromatic L-amino acid into a medium, when the bacterium is cultured in the medium.
The term “aromatic L-amino acid-producing bacterium” also means a bacterium which is able to produce and cause accumulation of an aromatic L-amino acid in a culture medium in an amount larger than a wild-type, a parental strain, or an unmodified strain of the bacterium, for example, E. coli, such as E. coli K-12, and preferably means that the bacterium is able to cause accumulation in a medium of an amount not less than 0.5 g/L, more preferably not less than 1.0 g/L, of the target L-amino acid. The term “aromatic L-amino acid” includes, at least, L-phenylalanine, L-tyrosine and L-tryptophan.
The Enterobacteriaceae family includes bacteria belonging to the genera Escherichia, Enterobacter, Erwinia, Klebsiella, Pantoea, Photorhabdus, Providencia, Salmonella, Serratia, Shigella, Morganella, Yersinia, etc. Specifically, bacteria classified into the Enterobacteriaceae family according to the taxonomy used by the NCBI (National Center for Biotechnology Information) database (http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=91347) can be used. A bacterium belonging to the genus Escherichia or Pantoea is preferred.
The phrase “a bacterium belonging to the genus Escherichia” means that the bacterium is classified into the genus Escherichia according to the classification known to a person skilled in the art of microbiology. Examples of a bacterium belonging to the genus Escherichia include, but are not limited to, Escherichia coli (E. coli).
The bacterium belonging to the genus Escherichia is not particularly limited, however, for example, bacteria described by Neidhardt, F. C. et al. (Escherichia coli and Salmonella typhimurium, American Society for Microbiology, Washington D.C., 1208, Table 1) are encompassed.
The phrase “a bacterium belonging to the genus Pantoea” means that the bacterium is classified as the genus Pantoea according to the classification known to a person skilled in the art of microbiology. Some species of Enterobacter agglomerans have been recently re-classified into Pantoea agglomerans, Pantoea ananatis, Pantoea stewartii or the like, based on the nucleotide sequence analysis of 16S rRNA, etc. (Int. J. Syst. Bacteriol., 43, 162-173 (1993)).
The phrase “bacterium has been modified to attenuate expression of the ydiN gene, the ydiB gene, or both” means that the bacterium has been modified in such a way that the modified bacterium contains a reduced amount of the YdiN protein, the ydiB protein, or both as compared with an unmodified bacterium, or is unable to synthesize the YdiN protein, the ydiB protein, or both.
The phrase “inactivation of the ydiN gene, the ydiB gene, or both” means that the modified gene encodes a completely non-functional protein. It is also possible that the modified DNA region is unable to naturally express the gene due to a deletion of a part of the gene, shifting of the reading frame of the gene, introduction of missense/nonsense mutation(s), or modification of an adjacent region of the gene, including sequences controlling gene expression, such as a promoter, enhancer, attenuator, ribosome-binding site, etc.
The presence or absence of the ydiN gene and/or the ydiB gene in the chromosome of a bacterium can be detected by well-known methods, including PCR, Southern blotting, and the like. In addition, the expression levels of the genes can be estimated by measuring the amounts of mRNA transcribed from the genes using various known methods including Northern blotting, quantitative RT-PCR, and the like. The amounts or molecular weights of the proteins coded by the genes can be measured by known methods including SDS-PAGE followed by an immunoblotting assay (Western blotting analysis), and the like.
The ydiN gene (synonyms: ECK1689, b1691) encodes the YdiN protein (synonym: B1691). The ydiN gene (nucleotides in positions 1,770,536 to 1,771,801; GenBank accession no. NC—000913.2; gi: 49175990) is located between the ydiM ORF and the ydiB gene on the chromosome of E. coli K-12. The nucleotide sequence of the ydiN gene and the amino acid sequence of YdiN encoded by the ydiN gene are shown in SEQ ID NO: 1 and SEQ ID NO:2, respectively. The ydiB gene (synonyms: ECK1690, b1692) encodes the YdiB protein (synonym: B1692). The ydiB gene (nucleotides in positions 1,771,813 to 1,772,679; GenBank accession no. NC—000913.2; gi: 49175990) is located between the ydiN gene and the aroD gene on the chromosome of E. coli K-12. The nucleotide sequence of the ydiB gene and the amino acid sequence of YdiB encoded by the ydiB gene are shown in SEQ ID NO: 3 and SEQ ID NO:4, respectively.
Since there may be some differences in DNA sequences between the genera or strains of the Enterobacteriaceae family, the gene to be inactivated on the chromosome is not limited to the genes shown in SEQ ID No:1 and SEQ ID No:3, but may include genes homologous to SEQ ID No:1 and SEQ ID No:3 which encode variant proteins of the YdiN and YdiB proteins. The phrase “variant proteins” means proteins which have changes in their sequence, whether the changes are deletions, insertions, additions, or substitutions of amino acids. The number of changes in the variant proteins depends on the position in the three dimensional structure of the protein or the type of amino acid residue to be changed. It may be 1 to 30, preferably 1 to 15, and more preferably 1 to 5 in SEQ ID NO: 2 and SEQ ID NO: 4. These changes are conservative mutations that preserve the function of the protein. In other words, these changes can occur in regions of the protein which are not critical for the three dimensional structure of the protein. This is because some amino acids have high homology to one another so the three dimensional structure is not affected by such a change. A conservative mutation is a mutation wherein substitution takes place mutually among Phe, Trp, Tyr, if the substitution site is an aromatic amino acid; among Leu, Ile, Val, if the substitution site is a hydrophobic amino acid; between Gln, Asn, if it is a polar amino acid; among Lys, Arg, His, if it is a basic amino acid; between Asp, Glu, if it is an acidic amino acid; and between Ser, Thr, if it is an amino acid having a hydroxyl group. Typical conservative mutations are conservative substitutions. 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 for Asp for Asn, substitution of Asn, Glu or Gln for Asp, substitution of Ser or Ala for Cys, substitution of Asn, Glu, Lys, His, Asp or Arg for Gln, substitution of Asn, Gln, Lys or Asp for Glu, substitution of Pro for Gly, substitution of 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 of 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. Substitutions, deletions, insertions, additions, or inversions and the like of the amino acids described above include naturally occurring mutations (mutant or variant) depending on differences in species, or individual differences of microorganisms that retain the ydiN or ydiB gene. Such a gene can be obtained by modifying the nucleotide sequence shown in SEQ ID NO: 1 or 3 using, for example, site-directed mutagenesis, so that the site-specific amino acid residue in the protein encoded includes substitutions, deletions, insertions, or additions.
Moreover, the protein variant encoded by the ydiN gene and ydiB gene may have a homology of not less than 80%, preferably not less than 90%, and most preferably not less than 95%, with respect to the entire amino acid sequence shown in SEQ ID NO. 2 and SEQ ID No:4, respectively. Activity of YdiB protein can be measured by the method described by Benach J., et. al. (J Biol. Chem. 23; 278(21):19176-82 (2003)) or Michel G., et al. (J Biol. Chem. 23; 278(21):19463-72 (2003)).
Moreover, the ydiN gene and ydiB gene may be a variant which hybridizes with the nucleotide sequence shown in SEQ ID NO: 1 and SEQ ID No:3, or a probe which can be prepared from the nucleotide sequence under stringent conditions. “Stringent conditions” include those under which a specific hybrid, for example, a hybrid having homology of not less than 60%, preferably not less than 70%, more preferably not less than 80%, still more preferably not less than 90%, and most preferably not less than 95%, is formed and a non-specific hybrid, for example, a hybrid having homology lower than the above, is not formed. For example, stringent conditions are exemplified by washing one time or more, preferably two or three times at a salt concentration of 1×SSC, 0.1% SDS, preferably 0.1×SSC, 0.1% SDS at 60° C. Duration of washing depends on the type of membrane used for blotting and, as a rule, should be what is recommended by the manufacturer. For example, the recommended duration of washing for the HybondTM N+ nylon membrane (Amersham) under stringent conditions is 15 minutes. Preferably, washing may be performed 2 to 3 times. The length of the probe may be suitably selected, depending on the hybridization conditions, and is usually 100 bp to 1 kbp.
Homology between two amino acid sequences can be determined using the well-known methods, for example, the computer program BLAST 2.0.
Expression of the ydiN gene, the ydiB gene, or both can be attenuated by introducing a mutation into the gene on the chromosome so that the intracellular amount of the protein encoded by the gene is decreased as compared to an unmodified strain. Such a mutation can be the insertion of a drug-resistance gene, or the deletion of a part of the gene or the entire gene (Qiu, Z. and Goodman, M. F., J. Biol. Chem., 272, 8611-8617 (1997); Kwon, D. H. et al, J. Antimicrob. Chemother., 46, 793-796 (2000)). Expression of the ydiN gene, the ydiB gene, or both can also be attenuated by modifying an expression regulating sequence such as the promoter, the Shine-Dalgarno (SD) sequence, etc. (WO95/34672, Carrier, T. A. and Keasling, J. D., Biotechnol Prog 15, 58-64 (1999)).
For example, the following methods may be employed to introduce a mutation by gene recombination. A mutant gene is prepared, and the bacterium to be modified is transformed with a DNA fragment containing the mutant gene. Then, the native gene on the chromosome is replaced with the mutant gene by homologous recombination, and the resulting strain is selected. Such gene replacement by homologous recombination can be conducted by employing a linear DNA, which is known as “Red-driven integration” (Datsenko, K. A. and Wanner, B. L., Proc. Natl. Acad. Sci. USA, 97, 12, p 6640-6645 (2000)), or by methods employing a plasmid containing a temperature-sensitive replication site (U.S. Pat. No. 6,303,383 or JP 05-007491A). Furthermore, the incorporation of a site-specific mutation by gene substitution using homologous recombination such as set forth above can also be conducted with a plasmid which is unable to replicate in the host.
Expression of the gene can also be attenuated by insertion of a transposon or an IS factor into the coding region of the gene (U.S. Pat. No. 5,175,107), or by conventional methods, such as mutagenesis with UV irradiation or nitrosoguanidine (N-methyl-N′-nitro-N-nitrosoguanidine).
Inactivation of the gene can also be performed by conventional methods, such as by mutagenesis with UV irradiation or nitrosoguanidine (N-methyl-N′-nitro-N-nitrosoguanidine), site-directed mutagenesis, gene disruption using homologous recombination, or/and insertion-deletion mutagenesis (Yu, D. et al., Proc. Natl. Acad. Sci. USA, 2000, 97:12: 5978-83 and Datsenko, K. A. and Wanner, B. L., Proc. Natl. Acad. Sci. USA, 2000, 97:12: 6640-45) also called “Red-driven integration”.
The above description regarding variant proteins, gene inactivation, and other methods can be applied to other proteins, genes, and the breeding of bacteria described below. Methods for preparation of plasmid DNA, digestion and ligation of DNA, transformation, selection of an oligonucleotide as a primer, and the like may be ordinary methods well-known to one skilled in the art. These methods are described, for instance, in Sambrook, J., Fritsch, E. F., and Maniatis, T., “Molecular Cloning: A Laboratory Manual, Second Edition”, Cold Spring Harbor Laboratory Press (1989).
Aromatic L-Amino Acid-Producing Bacteria
A bacterium which is modified to attenuate expression of the ydiN gene, the ydiB gene, or both is able to produce an aromatic L-amino acids.
The bacterium can be obtained by attenuating expression of the ydiN gene, the ydiB gene, or both in a bacterium which inherently has the ability to produce aromatic L-amino acids. Alternatively, the bacterium can be obtained by imparting the ability to produce aromatic L-amino acids to a bacterium already having the attenuated expression of the ydiN gene, the ydiB gene, or both.
L-tryptophan, L-phenylalanine, and L-tyrosine are all aromatic amino acids and share a common biosynthesis pathway. Examples of the genes encoding biosynthesis enzymes for these aromatic amino acids include deoxyarabino-heptulosonate phosphate synthase (aroG), 3-dehydroquinate synthase (aroB), shikimate dehydratase, shikimate kinase (aroL), 5-enolpyruvylshikimate-3-phosphate synthase (aroA), and chorismate synthase (aroC) (EP763127). Therefore, by placing multiple copies of the genes encoding these enzymes on a plasmid or genome, the aromatic amino acid-producing ability can be improved. It is known that these genes are controlled by the tyrosine repressor (tyrR), so the enzyme activity of an aromatic amino acid biosynthesis may also be increased by deleting the tyrR gene (see EP763127).
In order to enhance an aromatic amino acid productivity of a bacterium, biosynthesis of an amino acid other than the target aromatic amino acid may be attenuated. For example, when the target amino acid is L-tryptophan, biosynthetic pathways of L-phenylalanine and/or L-tyrosine may be attenuated (U.S. Pat. No. 4,371,614). Furthermore, 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase (DS) which endoded by aroF or aroG gene is subject to feedback inhibition by aromatic amino acids. Therefore, the bacterium may be modified so that the bacterium contains mutant DS which is not subject to the feedback inhibition. Such a mutant DS can be obtained, for example, by replacing L-aspartic acid at position 147 or L-serine at position 181 with other amino acids in aroF. In the case of aroG, mutant DS can be obtained, for example, by replacing aspartic acid at position 146, L-methionine at position 147, L-proline at position 150, or L-alanine at position 202 with other amino acids, or replacing L-methionine at position 157 and L-alanine at position 219 with other amino acids. An aromatic L-amino acid producing bacterium can be obtained by introducing a mutant gene which encodes such a mutant DS (EP0488424) to the bacterium. Especially, a mutant aroG gene (aroG4) in which L-proline at position 150 is replaced by L-leucine is preferred. The nucleotide sequence of the wild-type aroG gene and the amino acid sequence of DS encoded by the aroG gene are shown in SEQ ID NO: 19 and SEQ ID NO:20, respectively.
L-Phenylalanine-Producing Bacteria
Examples of parent 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, as a parent strain, E. coli K-12 [W3110 (tyrA)/pPHAB (FERM BP-3566), E. coli K-12 [W3110 (tyrA)/pPHAD] (FERM BP-12659), E. coli K-12 [W3110 (tyrA)/pPHATerm] (FERM BP-12662) and E. coli K-12 [W3110 (tyrA)/pBR-aroG4, pACMAB] named as AJ 12604 (FERM BP-3579) may be used (EP 488-424 B1). Furthermore, L-phenylalanine producing bacteria belonging to the genus Escherichia with an enhanced activity of the protein encoded by the yedA gene or the yddG gene may also be used (U.S. patent applications 2003/0148473 A1 and 2003/0157667 A1).
L-Tryptophan-Producing Bacteria
Examples of parent strains which can be used to derive the L-tryptophan-producing bacteria 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 tryptophanyl-tRNA synthetase encoded by mutant trpS gene (U.S. Pat. No. 5,756,345), E. coli SV164 (pGH5).
E. coli SV164 (pGH5) having a serA allele encoding phosphoglycerate dehydrogenase which is not subject to feedback inhibition by serine and a trpE allele encoding anthranilate synthase which is not subject to feedback inhibition by tryptophan (U.S. Pat. No. 6,180,373). The nucleotide sequence of the wild-type serA gene and the amino acid sequence of phosphoglycerate dehydrogenase encoded by the serA gene are shown in SEQ ID NO: 17 and SEQ ID NO: 18, respectively.
According to the description in WO94/08031 (International Patent Unexamined Publication in Japanese (Kohyo) No. 7-507693), a trpE deficient strain, Escherichia coli KB862 (DSM7196), was introduced with a mutant gene coding for anthranilate synthase to which feedback inhibition was desensitized (also referred to as “desensitized AS” hereafter) to obtain Escherichia coli SV164 (trpE8). This SV164 strain was introduced with a plasmid pGH5 (described in WO94/08031) containing a serA5 gene coding for phosphoglycerate dehydrogenase to which feedback inhibition was desensitized (also referred to as “desensitized PGD” hereafter). The SV164/pGH5 strain has ability to produce tryptophan and serine (U.S. Pat. No. 7,045,320).
Escherichia coli KB862 was designated as AJ13828, and deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology (currently, National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary, Central 6, 1-1 Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan) as an international deposit on Dec. 21, 2000 under the provisions of the Budapest Treaty and received an accession number of FERM BP-7405.
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. L-tryptophan-producing bacteria belonging to the genus Escherichia which have enhanced activity of the protein encoded by the yedA or yddG genes may also be used (U.S. patent applications 2003/0148473 A1 and 2003/0157667 A1).
Examples of parent strains which can be used to derive the L-tryptophan-producing bacteria also include strains in which one or more activities are enhanced of the following enzymes: anthranilate synthase (trpE), phosphoglycerate dehydrogenase (serA), and tryptophan synthase (trpAB). The anthranilate synthase and phosphoglycerate dehydrogenase are both subject to feedback inhibition by L-tryptophan and L-serine, therefore a mutation desensitizing the feedback inhibition may be introduced into these enzymes. Specific examples of strains having such a mutation include E. coli SV164 which harbors desensitized anthranilate synthase and a transformant strain obtained by introducing into the E. coli SV164 the plasmid pGH5 (WO 94/08031), which contains a mutant serA gene encoding feedback-desensitized phosphoglycerate dehydrogenase.
Examples of parent strains which can be used to derive the L-tryptophan-producing bacteria also include strains which have been transformed with the tryptophan operon containing a gene encoding desensitized anthranilate synthase (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). 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).
Examples of parent strains which can be used to derive the L-tryptophan-producing bacteria also include strains which have been transformed with the operon containing a gene encoding feedback-desensitized phosphoglycerate dehydrogenase and a gene encoding feedback-desensitized 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase. Specific examples of such strains include an E. coli SV164(Ptac-ideal→aroG4-serA5), containing operon Ptac-ideal→aroG4-serA5, integrated into chromosome at the position 2.933.542 in expression cassette from the plasmid pMDV3-aroG4-serA5. The integrative plasmid pMDV3-aroG4-serA5 was constructed on the basis of integrative plasmid pMDV3 (Zimenkov D. et al., Biotechnology in Russia, 6, 1-22 (2004)). Two DNA fragments were cloned into pMDV3. The first fragment is the DNA fragment BglII-XbaI of plasmid pMW118-Ptac-ideal→lacZ-ter_rrnB (Mashko S. et. al., Biotechnology in Russia, 5, 3-20 (2001)), containing promoter Ptac-ideal (Olac-ideal-Ptac/Olac). The second one is the DNA fragment XbaI-EcoRI of polylinker from plasmid pMW118 (GenBank/EMBL accession number AB005475). Furthermore, the amplified DNA fragment containing aroG4 was cloned into the plasmid pAROG4 (Kikuchi Y. et. al., Appl. And Env. Microb., 761-2 (1997)), which was used as a template with primers P1 (SEQ ID NO: 5) and P2 (SEQ ID NO: 6) for PCR. These primers contained the recognition sites for XbaI and SmaI endonucleases respectively. The plasmid pAROG4 harbors the aroG4 gene encoding 3-deoxy-D-arabino-heptulosinate-7-phosphate (DAHP) synthase which is not subject to feedback inhibition by phenylalanine. The resulting plasmid pMDV-aroG4 was used as a vector for cloning the structural part of serA5. The amplified DNA fragment containing serA5 was obtained by PCR using the plasmid pGH5 (U.S. Pat. No. 6,180,373) as the template and primers P3 (SEQ ID NO: 7) and P4 (SEQ ID NO: 8). The plasmid pGH5 harbors the serA5 gene encoding phosphoglycerate dehydrogenase which is not subject to feedback inhibition by serine. This amplified fragment has the recognition site for SmaI endonuclease in the 5′-region of serA5 gene and the recognition sites for SalI, SphI, SacI endonucleases in the 3′-region of serA5 gene. This DNA fragment was cloned into plasmid pMDV-aroG4 using the recognition sites for SmaI and SacI endonucleases. The resulting integrative plasmid pMDV3-aroG4-serA5 contained operon Ptac-ideal→aroG4-serA5.
2. Method of the Present Invention
The method of the present invention is a method for producing an aromatic L-amino acid by cultivating the bacterium of the present invention in a culture medium to produce and secrete the aromatic L-amino acid into the medium, and collecting the aromatic L-amino acid from the medium.
The cultivation, collection, and purification of an aromatic L-amino acid from the medium and the like may be performed in a manner similar to conventional fermentation methods wherein an amino acid is produced using a bacterium.
The chosen culture medium may be either a synthetic or natural medium, so long as it includes a carbon source and a nitrogen source and minerals and, if necessary, appropriate amounts of nutrients which the bacterium requires for growth. The carbon source may include various carbohydrates such as glucose and sucrose, and various organic acids. Depending on the mode of assimilation of the chosen microorganism, alcohol, including ethanol and glycerol, may be used. As the nitrogen source, various ammonium salts such as ammonia and ammonium sulfate, other nitrogen compounds such as amines, a natural nitrogen source such as peptone, soybean-hydrolysate, and digested fermentative microorganism can be used. As minerals, potassium monophosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, calcium chloride, and the like can be used. As vitamins, thiamine, yeast extract, and the like, can be used.
The cultivation is preferably performed under aerobic conditions, such as a shaking culture, or a stirring culture with aeration, at a temperature of 20 to 40° C., preferably 30 to 38° C. The pH of the culture is usually between 5 and 9, preferably between 6.5 and 7.2. The pH of the culture can be adjusted with ammonia, calcium carbonate, various acids, various bases, and buffers. Usually, a 1 to 5-day cultivation leads to accumulation of the target aromatic L-amino acid in the liquid medium.
After cultivation, solids such as cells can be removed from the liquid medium by centrifugation or membrane filtration, and then the aromatic L-amino acid can be collected and purified by ion-exchange, concentration, and/or crystallization methods. Phenylalanine produced by the method of the present invention may be used for, for example, producing lower alkyl ester of α-L-aspartyl-L-phenylalanine (also referred to as “aspartame”). That is, the method includes a method for producing a lower alkyl ester of α-L-aspartyl-L-phenylalanine by using L-phenylalanine as a raw material. The method includes synthesizing a lower alkyl ester of α-L-aspartyl-L-phenylalanine from L-phenylalanine produced by the method as described above and aspartic acid or its derivative. As a lower alkyl ester, methyl ester, ethyl ester and propyl ester, or the like can be mentioned.
In the method, a process for synthesizing a lower alkyl ester of α-L-aspartyl-L-phenylalanine from L-phenylalanine and aspartic acid or its derivative is not particularly limited and any conventional method can be applied so long as L-phenylalanine or its derivative can be used for the synthesis of the lower alkyl ester of α-L-aspartyl-L-phenylalanine. Concretely, for example, lower alkyl esters of α-L-aspartyl-L-phenylalanine may be produced by the following process (U.S. Pat. No. 3,786,039). L-phenylalanine is esterified to obtain a lower alkyl ester of L-phenylalanine. The L-phenylalanine alkyl ester is reacted with L-aspartic acid derivative of which an amino group and .beta.carboxyl group are protected and a carboxyl group is esterified to activate. The derivative includes N-acyl-L-aspartic anhydrides such as N-formyl-, N-carbobenzoxy-, or N-p-methoxycarbobenzoxy-L-aspartic anhydride. By the condensation reaction, a mixture of N-acyl-α-L-aspartyl-L-phenylalanine and N-acyl-β-L-aspartyl-L-phenylalanine is obtained. If the condensation reaction is performed in the presence of an organic acid of which acid dissociation constant at 37° C. is 10−4 or less, ratio of α form to β form in the mixture is increased (Japanese Patent Laid-Open Publication No. 51-113841). Then, the N-acyl-α-L-aspartyl-L-phenylalanine is separated from the mixture, followed by hydrogenating to obtain α-L-aspartyl-L-phenylalanine.
EXAMPLES
The present invention will be more concretely explained below with reference to the following non-limiting Examples.
Example 1
Construction of a Strain with an Inactivated ydiN Gene
1. Deletion of the ydiN Gene
The ydiN gene was deleted by the method initially developed by Datsenko, K. A. and Wanner, B. L. (Proc. Natl. Acad. Sci. USA, 2000, 97(12), 6640-6645) called “Red-driven integration”. The DNA fragment containing the CmR marker encoded by the cat gene was obtained by PCR, using the primers P5 (SEQ ID NO: 9) and P6 (SEQ ID NO: 10), and the pMW118-attL-Cm-attR plasmid as a template (WO 05/010175). Primer P5 contains both a region complementary to the 36-nt region located at the 5′ end of the ydiN gene, -tt-nucleotides for preventing frame-shift and the 28-nt region complementary to the 3′ end of the attR region. Primer P6 contains both a region complementary to the 36-nt region located at the 3′ end of the ydiN gene, and the 28-nt region complementary to the 5′ end of the attL region. Conditions for PCR were as follows: denaturation step for 3 min at 95° C.; profile for two first cycles: 1 min at 95° C., 30 sec at 50° C., 40 sec at 72° C.; profile for the last 25 cycles: 30 sec at 95° C., 30 sec at 54° C., 40 sec at 72° C.; final step: 5 min at 72° C.
An 1711-bp PCR product (FIG. 1) was obtained and purified in an agarose gel and was used for electroporation of the E. coli strain MG1655 (ATCC 700926), which contains the pKD46 plasmid which has a temperature-sensitive replication origin. The pKD46 plasmid (Datsenko, K. A. and Wanner, B. L., Proc. Natl. Acad. Sci. USA, 2000, 97:12:6640-45) includes a 2,154 nucleotide DNA fragment of phage λ (nucleotide positions 31088 to 33241, GenBank accession no. J02459), and contains genes of the λ Red homologous recombination system (γ, β, exo genes) under the control of the arabinose-inducible ParaB promoter. The plasmid pKD46 is necessary for integration of the PCR product into the chromosome of strain MG1655. The strain MG1655 can be obtained from the American Type Culture Collection. (P.O. Box 1549 Manassas, Va. 20108, U.S.A.).
Electrocompetent cells were prepared as follows: E. coli MG1655/pKD46 was grown overnight at 30° C. in LB medium containing ampicillin (100 mg/l), and the culture was diluted 100 times with 5 ml of SOB medium (Sambrook et al, “Molecular Cloning: A Laboratory Manual, Second Edition”, Cold Spring Harbor Laboratory Press, 1989) containing ampicillin and L-arabinose (1 mM). The cells were grown with aeration at 30° C. to an OD600 of ≈0.6 and then were made electrocompetent by concentrating 100-fold and washing three times with ice-cold deionized H2O. Electroporation was performed using 70 μl of cells and 100 ng of the PCR product. Cells after electroporation were incubated with 1 ml of SOC medium (Sambrook et al, “Molecular Cloning: A Laboratory Manual, Second Edition”, Cold Spring Harbor Laboratory Press, 1989) at 37° C. for 2.5 hours and then were plated onto L-agar containing chloramphenicol (30 μg/ml) and grown at 37° C. to select CmR recombinants. Then, to eliminate the pKD46 plasmid, two passages on L-agar with Cm at 42° C. were performed and the obtained colonies were tested for sensitivity to ampicillin.
2. Verification of the ydiN Gene Deletion by PCR
The mutants having the ydiN gene deleted and marked with the Cm resistance gene were verified by PCR. Locus-specific primers P7 (SEQ ID NO: 11) and P8 (SEQ ID NO: 12) were used in PCR for the verification. Conditions for PCR verification were as follows: denaturation step for 3 min at 94° C.; profile for 30 cycles: 30 sec at 94° C., 30 sec at 54° C., 1 min at 72° C.; final step: 7 min at 72° C. The PCR product obtained using the parental ydiN+ strain MG1655 as a template, was 1461 bp in length. The PCR product obtained using the mutant strain as the template was 1900 bp in length (FIG. 2). The mutant strain was named MG1655 ΔydiN::cat.
Example 2
Production of L-phenylalanine by E. coli Strain AJ12739-ΔydiN
To test the effect of inactivation of the ydiN gene on L-phenylalanine production, DNA fragments from the chromosome of the above-described E. coli MG1655 ΔydiN::cat can be transferred to the phenylalanine-producing E. coli strain AJ12739 by P1 transduction (Miller, J. H. Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, 1972, Plainview, N.Y.) to obtain strain AJ12739-ΔydiN::cat. The strain AJ12739 has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1 Dorozhny proezd, 1) on Nov. 6, 2001 under accession no. VKPM B-8197 and then converted to a deposit under the Budapest Treaty on Aug. 23, 2002.
Furthermore, the Cm resistance gene (cat gene) can be eliminated from the chromosome of the strain AJ12739-ΔydiN::cat using the int-xis system. For that purpose, the AJ12739-ΔydiN::cat strain can be transformed with the pMWts-Int/Xis plasmid (WO 2005 010175). Transformant clones can be selected on the LB-medium containing 100 μg/ml of ampicillin. Plates can be incubated overnight at 30° C. Transformant clones can be cured from the cat gene by spreading the separate colonies at 37° C. (at that temperature repressor CIts is partially inactivated and transcription of the int/xis genes is derepressed) followed by selection of CmSApR variants. Elimination of the cat gene from the chromosome of the strain can be verified by PCR. Locus-specific primers P7 (SEQ ID NO: 11) and P8 (SEQ ID NO: 12) can be used in PCR for verification. Conditions for PCR verification can be as described above. The PCR product obtained in the reaction with cells not having the cat gene, should be 294 bp in length. Thus, the AJ12739-ΔydiN strain with the inactivated ydiN gene and eliminated cat gene can be obtained.
Both strains, AJ12739-ΔydiN and AJ12739, can each be cultivated at 37° C. for 18 hours in a nutrient broth, and 0.3 ml of the obtained culture can each be inoculated into 3 ml of a fermentation medium in a 20×200-mm test tube and cultivated at 37° C. for 48 hours with shaking on a rotary shaker. After cultivation, the amount of phenylalanine which accumulates in the medium can be determined by TLC. The 10×15-cm TLC plates coated with 0.11-mm layers of Sorbfil silica gel containing no fluorescent indicator (Stock Company Sorbpolymer, Krasnodar, Russia) can be used. The Sorbfil plates can be developed with a mobile phase consisting of propan-2-ol: ethylacetate: 25% aqueous ammonia: water=40:40:7:16 (v/v). A solution of ninhydrin (2%) in acetone can be used as a visualizing reagent.
The composition of the fermentation medium (g/l) is as follows:
















Glucose
40.0



(NH4)2SO4
16.0



K2HPO4
0.1



MgSO4•7H2O
1.0



FeSO4•7H2O
0.01



MnSO4•5H2O
0.01



Thiamine HCl
0.0002



Yeast extract
2.0



Tyrosine
0.125



CaCO3
20.0










Glucose and magnesium sulfate are sterilized separately. CaCO3 is dry-heat sterilized at 180° C. for 2 hours. The pH is adjusted to 7.0.
Example 3
Production of L-tryptophan by E. coli Strain SV164 (Ptac-ideal→aroG4-serA5)-ΔydiN
To test the effect of inactivation of the ydiN gene on L-tryptophan production, DNA fragments from the chromosome of the above-described E. coli strain MG1655 ΔydiN::cat can be transferred to the tryptophan-producing E. coli strain SV164 (Ptac-ideal→aroG4-serA5) by P1 transduction to obtain the strain SV164(Ptac-ideal→aroG4-serA5)-ΔydiN::cat. The SV164 strain has the trpE allele encoding anthranilate synthase which is not subjected to feedback inhibition by tryptophan. The SV164 strain (Ptac-ideal→-aroG4-serA5) contains the Ptac-ideal→aroG4-serA5 operon, which is integrated into the chromosome at the 2.933.542 position in the expression cassette from the pMDV3-aroG4-serA5 plasmid. The nucleotide sequence of the Ptac-ideal→aroG4-serA5 operon is shown in SEQ ID NO: 21. The position of the genes in the operon is as follows: Ptac-ideal (1 to 116), aroG4 (133 to 1185) and serA5 (1209 to 2438). In aroG4, L-proline at position 150 in the wild-type deoxyarabino-heptulosonate phosphate synthase (SEQ ID NO: 20) is replaced by L-leucine. In serA5, the tyrosine residue at position 410 in the wild-type phosphoglycerate dehydrogenase (SEQ ID NO: 18) is deleted.
Furthermore, the Cm resistance gene (cat gene) can be eliminated from the chromosome of the SV164 strain (Ptac-ideal→-aroG4-serA5)-ΔydiN::cat using the int-xis system. For that purpose, the SV164 strain (Ptac-ideal→aroG4-serA5)-ΔydiN::cat can be transformed with the pMWts-Int/Xis plasmid. Transformant clones can be selected on the LB-medium containing 100 μg/ml of ampicillin. Plates can be incubated overnight at 30° C. Transformant clones can be cured from the cat gene by spreading the separate colonies at 37° C. (at that temperature repressor CIts is partially inactivated and transcription of the int/xis genes is derepressed) followed by selection of CmSApR variants. Elimination of the cat gene from the chromosome of the strain can be verified by PCR. Locus-specific primers P7 (SEQ ID NO: 11) and P8 (SEQ ID NO: 12) can be used in PCR for verification. Conditions for PCR verification can be as described above. The PCR product obtained using the cells not having the eliminated cat gene, should be 294 bp in length. Thus, the SV164 strain (Ptac-ideal→aroG4-serA5)-ΔydiN with the inactivated ydiN gene and eliminated cat gene can be obtained.
Both of the SV164(Ptac-ideal→aroG4-serA5)-ΔydiN and SV164(Ptac-ideal→-aroG4-serA5) strains can each be cultivated with shaking at 32° C. for 18 hours in 3 ml of nutrient broth. The obtained cultures (0.3 ml each) can be inoculated into 3 ml of a fermentation medium in 20×200-mm test tubes, and cultivated at 32° C. for 72 hours with a rotary shaker at 250 rpm. After cultivation, the amount of tryptophan which accumulates in the medium can be determined by TLC as described in Example 2.
The fermentation medium components are listed in Table 1, but should be sterilized in separate groups (A, B, C, D, E, F, G and H), as shown, to avoid adverse interactions during sterilization.









TABLE 1





Solutions
Component
Final concentration, g/l

















A
KH2PO4
1.5



NaCl
0.5



(NH4)2SO4
15.0



L-Methionine
0.05



L-Phenylalanine
0.1



L-Tyrosine
0.1



Mameno (total N)
0.35


B
Glucose
40.0



MgSO4 7H2O
0.3


C
CaCl2 2H2O
14.7


D
FeSO4 7H2O
0.075


E
Na2MoO4 2H2O
0.00015



H3BO3
0.0025



CoCl2 6H2O
0.0007



CuSO4 5H2O
0.00025



MnCl2 4H2O
0.0016



ZnSO4 7H2O
0.0003


F
Thiamine HCl
0.005


G
CaCO3
30.0


H
Pyridoxine
0.03









The pH of solution A is adjusted to 7.1 with NH4OH. Each group is sterilized separately, chilled, and then mixed together.
Example 4
Construction of a Strain with an Inactivated ydiB Gene
1. Deletion of the ydiB Gene
The ydiB gene was deleted by “Red-driven integration”. The DNA fragment containing the CmR marker encoded by the cat gene was obtained by PCR, using primers P9 (SEQ ID NO: 13) and P10 (SEQ ID NO: 14), and the pMW118-attL-Cm-attR plasmid as a template. Primer P9 contains both a region complementary to the 36-nt region located at the 5′ end of the ydiB gene, -tt-nucleotides for preventing frame-shift and the 28-nt region complementary to the 3′ end of the attR region. Primer P10 contains both a region complementary to the 36-nt region located at the 3′ end of the ydiB gene and the 28-nt region complementary to the 5′ end of the attL region. Conditions for PCR were as follows: denaturation step for 3 min at 95° C.; profile for two first cycles: 1 min at 95° C., 30 sec at 50° C., 40 sec at 72° C.; profile for the last 25 cycles: 30 sec at 95° C., 30 sec at 54° C., 40 sec at 72° C.; final step: 5 min at 72° C.
An 1711-bp PCR product (FIG. 1) was obtained and purified in agarose gel and was used for electroporation of the E. coli strain MG1655, which contains the plasmid pKD46 having a temperature-sensitive replication. The plasmid pKD46 is necessary for integration of the PCR product into the chromosome of strain MG1655.
Electrocompetent cells were prepared as follows: E. coli MG1655/pKD46 was grown overnight at 30° C. in LB medium containing ampicillin (100 mg/l), and the culture was diluted times with 5 ml of SOB medium containing ampicillin and L-arabinose (1 mM). The cells were grown with aeration at 30° C. to an OD600 of ≈0.6 and then were made electrocompetent by concentrating 100-fold and washing three times with ice-cold deionized H2O. Electroporation was performed using 70 μl of cells and ≈100 ng of the PCR product. Cells after electroporation were incubated with 1 ml of SOC medium at 37° C. for 2.5 hours and then were plated onto L-agar containing chloramphenicol (30 μg/ml) and grown at 37° C. to select CmR recombinants. Then, to eliminate the pKD46 plasmid, two passages on L-agar with Cm at 42° C. were performed and the obtained colonies were tested for sensitivity to ampicillin.
2. Verification of the ydiB Gene Deletion by PCR
The mutants having the ydiB gene deleted and marked with the Cm resistance gene were verified by PCR. Locus-specific primers P11 (SEQ ID NO: 15) and P12 (SEQ ID NO: 16) were used in PCR for verification. Conditions for PCR verification were as follows: denaturation step for 3 min at 94° C.; profile for 30 cycles: 30 sec at 94° C., 30 sec at 54° C., 1 min at 72° C.; final step: 7 min at 72° C. The PCR product obtained using the parental ydiB+ strain MG1655 as a template, was 1083 bp in length. The PCR product obtained using the mutant strain as the template was 1927 bp in length (FIG. 2). The mutant strain was named MG1655 ΔydiB::cat.
Example 5
Production of L-phenylalanine by E. coli Strain AJ12739-ΔydiB
To test the effect of inactivation of the ydiB gene on L-phenylalanine production, DNA fragments from the chromosome of the above-described E. coli MG1655 ΔydiB::cat can be transferred to the phenylalanine-producing E. coli strain AJ12739 by P1 transduction to obtain strain AJ12739-ΔydiB::cat.
Furthermore, the Cm resistance gene (cat gene) can be eliminated from the chromosome of the AJ12739-ΔydiB::cat strain using the int-xis system. For that purpose, the AJ12739-ΔydiB::cat strain can be transformed with the pMWts-Int/Xis plasmid. Transformant clones can be selected on LB-medium containing 100 μg/ml of ampicillin. Plates can be incubated overnight at 30° C. Transformant clones can be cured from the cat gene by spreading the separate colonies at 37° C. (at that temperature repressor CIts is partially inactivated and transcription of the int/xis genes is derepressed) followed by selection of CmSApR variants. Elimination of the cat gene from the chromosome of the strain can be verified by PCR. Locus-specific primers P11 (SEQ ID NO: 15) and P12 (SEQ ID NO: 16) can be used in PCR for verification. Conditions for PCR verification can be as described above. The PCR product obtained in reaction with cells not having the cat gene as a template, should be 321 bp in length. Thus, the AJ12739-ΔydiB strain with the inactivated ydiB gene and eliminated cat gene can be obtained.
Both of the AJ12739-ΔydiB and AJ12739 strains can each be cultivated at 37° C. for 18 hours in a nutrient broth, and 0.3 ml of the obtained culture can each be inoculated into 3 ml of a fermentation medium in a 20×200-mm test tube and cultivated at 37° C. for 48 hours with shaking on a rotary shaker. After cultivation, the amount of phenylalanine which accumulates in the medium can be determined by TLC as described in Example 2.
The composition of the fermentation medium (g/l) is as follows:
















Glucose
40.0



(NH4)2SO4
16.0



K2HPO4
0.1



MgSO4 7H2O
1.0



FeSO4•7H2O
0.01



MnSO4•5H2O
0.01



Thiamine HCl
0.0002



Yeast extract
2.0



Tyrosine
0.125



CaCO3
20.0










Glucose and magnesium sulfate are sterilized separately. CaCO3 is dry-heat sterilized at 180° C. for 2 hours. The pH is adjusted to 7.0.
Example 6
Production of L-tryptophan by E. coli Strain SV164 (Ptac-ideal→aroG4-serA5)-ΔydiB
To test the effect of inactivation of the ydiB gene on L-tryptophan production, DNA fragments from the chromosome of the above-described E. coli strain MG1655 ΔydiB::cat was transferred to the tryptophan-producing E. coli strain SV164 (Ptac-ideal→aroG4-serA5) by P1 transduction to obtain the strain SV164(Ptac-ideal→aroG4-serA5)-ΔydiB::cat.
Furthermore, the Cm resistance gene (cat gene) was eliminated from the chromosome of the SV164(Ptac-ideal→-aroG4-serA5)-ΔydiB::cat strain using the int-xis system. For that purpose, the SV164(Ptac-ideal→-aroG4-serA5)-ΔydiB::cat strain was transformed with the pMWts-Int/Xis plasmid. Transformant clones were selected on the LB-medium containing 100 μg/ml of ampicillin. Plates were incubated overnight at 30° C. Transformant clones were cured from the cat gene by spreading the separate colonies at 37° C. (at that temperature repressor CIts is partially inactivated and transcription of the int/xis genes is derepressed) followed by selection of CmSApR variants. Elimination of the cat gene from the chromosome of the strain was verified by PCR. Locus-specific primers P11 (SEQ ID NO: 15) and P12 (SEQ ID NO: 16) were used in PCR for the verification. Conditions for PCR verification were as described above. The PCR product obtained using the cells not having the cat gene, should be 321 bp in length. Thus, the SV164(Ptac-ideal→aroG4-serA5)-ΔydiB strain with the inactivated ydiB gene and eliminated cat gene was obtained.
Both of the SV164(Ptac-ideal→aroG4-serA5)-ΔydiB and SV164(Ptac-ideal→aroG4-serA5) strains were each cultivated with shaking at 32° C. for 18 hours in 3 ml of nutrient broth. The obtained cultures (0.3 ml each) were inoculated into 3 ml of a fermentation medium in 20×200-mm test tubes, and cultivated at 32° C. for 50 hours with a rotary shaker at 250 rpm. It was determined that all glucose was utilized at that moment. After cultivation, the amount of tryptophan which accumulates in the medium was determined by TLC as described in Example 2. Results of at least three independent fermentations are presented in the Table 2. As it is seen, inactivation of ydiB gene increases tryptophan production.
The fermentation medium components are listed in Table 1, but should be sterilized in separate groups (A, B, C, D, E, F, G and H), as shown, to avoid adverse interactions during sterilization.









TABLE 2







Amount of


Strain
OD540
tryptophan, g/l







SV164(Ptac-idealΔaroG4-serA5)
16.9 ± 0.5
3.8 ± 0.1


SV164(Ptac-ideal→aroG4-serA5)-ΔydiB
17.2 ± 0.3
4.1 ± 0.1









Example 7
Construction of a Strain with Inactivated ydiN and ydiB Genes
1. Deletion of the ydiN and the ydiB Genes
The ydiN and the ydiB genes were deleted by the “Red-driven integration”. The DNA fragment containing the CmR marker encoded by the cat gene was obtained by PCR, using primers P5 (SEQ ID NO: 9) and P10 (SEQ ID NO: 14), and the pMW118-attL-Cm-attR plasmid as a template. 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.
An 1711-bp PCR product (FIG. 1) was obtained and purified in agarose gel and was used for electroporation of the E. coli strain MG1655, which contains the pKD46 plasmid having a temperature-sensitive replication origin. The pKD46 plasmid is necessary for integration of the PCR product into the chromosome of the MG1655 strain.
Electrocompetent cells were prepared as follows: E. coli MG1655/pKD46 was grown overnight at 30° C. in LB medium containing ampicillin (100 mg/l), and the culture was diluted times with 5 ml of SOB medium containing ampicillin and L-arabinose (1 mM). The cells were grown with aeration at 30° C. to an OD600 of ≈0.6 and then were made electrocompetent by concentrating 100-fold and washing three times with ice-cold deionized H2O. Electroporation was performed using 70 μl of cells and 100 ng of the PCR product. Cells after electroporation were incubated with 1 ml of SOC medium at 37° C. for 2.5 hours and then were plated onto L-agar containing chloramphenicol (30 μg/ml) and grown at 37° C. to select CmR recombinants. Then, to eliminate the pKD46 plasmid, two passages on L-agar with Cm at 42° C. were performed and the obtained colonies were tested for sensitivity to ampicillin.
2. Verification of the ydiN and the ydiB Genes Deletion by PCR
The mutants having the ydiN and the ydiB genes deleted and marked with the Cm resistance gene were verified by PCR. Locus-specific primers P7 (SEQ ID NO: 11) and P12 (SEQ ID NO: 16) were used in PCR for verification. Conditions for PCR verification were as follows: denaturation step for 3 min at 94° C.; profile for 30 cycles: 30 sec at 94° C., 30 sec at 54° C., 1 min at 72° C.; final step: 7 min at 72° C. The PCR product using the parental ydiB+ strain MG1655 as a template, was 2339 bp in length. The PCR product obtained using the mutant strain as the template was 1900 bp in length (FIG. 2). The mutant strain was named MG1655 ΔydiNB::cat.
Example 8
Production of L-phenylalanine by E. coli Strain AJ12739-ΔydiNB
To test the effect of inactivation of the ydiN and the ydiB genes on L-phenylalanine production, DNA fragments from the chromosome of the above-described E. coli MG1655 ΔydiNB::cat can be transferred to the phenylalanine-producing E. coli strain AJ12739 by P1 transduction to obtain strain AJ12739-ΔydiNB::cat.
Furthermore, Cm resistance gene (cat gene) can be eliminated from the chromosome of the strain AJ12739-ΔydiNB::cat using the int-xis system. For that purpose the strain AJ12739-ΔydiNB::cat can be transformed with the pMWts-Int/Xis plasmid. Transformant clones can be selected on the LB-medium containing 100 μg/ml of ampicillin. Plates can be incubated overnight at 30° C. Transformant clones can be cured from the cat gene by spreading the separate colonies at 37° C. (at that temperature repressor CIts is partially inactivated and transcription of the int/xis genes is derepressed) followed by selection of the CmSApR variants. Elimination of the cat gene from the chromosome of the strain can be verified by PCR. Locus-specific primers P7 (SEQ ID NO: 11) and P12 (SEQ ID NO: 16) can be used in PCR for verification. Conditions for PCR verification can be as described above. The PCR product obtained in reaction with cells not having the cat gene should be 294 bp in length. Thus, the strain AJ12739-ΔydiNB with the inactivated ydiN and ydiB genes and the eliminated cat gene can be obtained.
Both strains, AJ12739-ΔydiNB and AJ12739, can each be cultivated at 37° C. for 18 hours in a nutrient broth, and 0.3 ml of the obtained culture can each be inoculated into 3 ml of a fermentation medium in a 20×200-mm test tube and cultivated at 37° C. for 48 hours with shaking on a rotary shaker. After cultivation, the amount of phenylalanine which accumulates in the medium can be determined by TLC as described in Example 2.
The composition of the fermentation medium (g/l) is as follows:
















Glucose
40.0



(NH4)2SO4
16.0



K2HPO4
0.1



MgSO4•7H2O
1.0



FeSO4•7H2O
0.01



MnSO4•5H2O
0.01



Thiamine HCl
0.0002



Yeast extract
2.0



Tyrosine
0.125



CaCO3
20.0










Glucose and magnesium sulfate are sterilized separately. CaCO3 is dry-heat sterilized at 180° C. for 2 hours. The pH is adjusted to 7.0.
Example 9
Production of L-tryptophan by E. coli strain SV164 (Ptac-ideal→-aroG4-serA5)-ΔydiNB
To test the effect of inactivation of the ydiN and the ydiB genes on L-tryptophan production, DNA fragments from the chromosome of the above-described E. coli strain MG1655 ΔydiNB::cat can be transferred to the tryptophan-producing E. coli strain SV164 (Ptac-ideal→aroG4-serA5) by P1 transduction to obtain the strain SV164(Ptac-ideal→aroG4-serA5)-ΔydiNB::cat.
Furthermore, the Cm resistance gene (cat gene) can be eliminated from the chromosome of the SV164(Ptac-ideal→aroG4-serA5)-ΔydiNB::cat strain using the int-xis system. For that purpose, the SV164(Ptac-ideal→aroG4-serA5)-ΔydiNB::cat strain can be transformed with the pMWts-Int/Xis plasmid. Transformant clones can be selected on the LB-medium containing 100 μg/ml of ampicillin. Plates can be incubated overnight at 30° C. Transformant clones can be cured from the cat gene by spreading the separate colonies at 37° C. (at that temperature, the repressor CIts is partially inactivated and transcription of the int/xis genes is derepressed) followed by selection of CmSApR variants. Elimination of the cat gene from the chromosome of the strain can be verified by PCR. Locus-specific primers P7 (SEQ ID NO: 11) and P12 (SEQ ID NO: 16) can be used in PCR for verification. Conditions for PCR verification can be as described above. The PCR product using the cells not having the cat gene, should be 294 bp in length. Thus, the SV164(Ptac-ideal→aroG4-serA5)-ΔydiNB strain with the inactivated ydiN and the ydiB genes and the eliminated cat gene can be obtained.
Both of the SV164(Ptac-ideal→aroG4-serA5)-ΔydiNB and SV164(Ptac-ideal→-aroG4-serA5) strains can each be cultivated with shaking at 32° C. for 18 hours in 3 ml of nutrient broth. The obtained cultures (0.3 ml each) can be inoculated into 3 ml of a fermentation medium in 20×200-mm test tubes, and cultivated at 32° C. for 72 hours with a rotary shaker at 250 rpm. After cultivation, the amount of tryptophan which accumulates in the medium can be determined by TLC as described in Example 2.
The fermentation medium components are listed in Table 1, but should be sterilized in separate groups (A, B, C, D, E, F, G and H), as shown, to avoid adverse interactions during sterilization.
While the invention has been described in detail with reference to preferred embodiments thereof, it will be apparent to one skilled in the art that various changes can be made, and equivalents employed, without departing from the scope of the invention. All the cited references herein are incorporated as a part of this application by reference.
INDUSTRIAL APPLICABILITY
According to the present invention, production of an aromatic L-amino acid by a bacterium of the Enterobacteriaceae family can be enhanced.

## Claims

1. An aromatic L-amino acid-producing Escherichia coli bacterium, wherein said bacterium has been modified to attenuate expression of a gene selected from the group consisting of Escherichia coli ydiN, Escherichia coli ydiB, and combinations thereof.

2. The bacterium according to claim 1, wherein said expression is attenuated by inactivating said gene.

3. The bacterium according to claim 1, wherein said aromatic L-amino acid is selected from the group consisting of L-phenylalanine, L-tyrosine, and L-tryptophan.

4. A method for producing an aromatic L-amino acid comprising:
cultivating the bacterium according to claim 1 in a medium, and
collecting said aromatic L-amino acid from the medium.

5. The method according to claim 4, wherein said aromatic L-amino acid is selected from the group consisting of L-phenylalanine, L-tyrosine, and L-tryptophan.

6. A method for producing a lower alkyl ester of α-L-aspartyl-L-phenylalanine comprising:
A) cultivating the bacterium according to claim 1 in a culture medium to produce and accumulate L-phenylalanine in the medium, and
B) synthesizing the lower alkyl ester of α-L-aspartyl-L-phenylalanine from aspartic acid or its derivative and the accumulated L-phenylalanine,
wherein said bacterium is able to produce L-phenylalanine.

7. The method according to claim 6, further comprising:
A) esterifying the accumulated L-phenylalanine to generate a lower alkyl ester of L-phenylalanine,
B) condensing the lower alkyl ester of L-phenylalanine with N-acyl-L-aspartic anhydride to generate a lower alkyl ester of N-acyl-α-L-aspartyl-L-phenylalanine,
C) separating the lower alkyl ester of N-acyl-α-L-aspartyl-L-phenylalanine from the culture medium, and
D) hydrogenating the lower alkyl ester of N-acyl-α-L-aspartyl-L-phenylalanine to generate the lower alkyl ester of α-L-aspartyl-L-phenylalanine.

8. A method for producing an aromatic L-amino acid comprising:
cultivating the bacterium according to claim 2 in a medium, and
collecting said aromatic L-amino acid from the medium.

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

10. A method for producing a lower alkyl ester of α-L-aspartyl-L-phenylalanine comprising:
A) cultivating the bacterium according to claim 2 in a culture medium to produce and accumulate L-phenylalanine in the medium, and
B) synthesizing a lower alkyl ester of α-L-aspartyl-L-phenylalanine from aspartic acid or its derivative and the accumulated L-phenylalanine,
wherein said bacterium is able to produce L-phenylalanine.

11. The method according to claim 10, further comprising:
A) esterifying the accumulated L-phenylalanine to generate a lower alkyl ester of L-phenylalanine,
B) condensing the lower alkyl ester of L-phenylalanine with N-acyl-L-aspartic anhydride to generate a lower alkyl ester of N-acyl-α-L-aspartyl-L-phenylalanine,
C) separating the lower alkyl ester of N-acyl-α-L-aspartyl-L-phenylalanine from the culture medium, and
D) hydrogenating the lower alkyl ester of N-acyl-α-L-aspartyl-L-phenylalanine to generate the lower alkyl ester of α-L-aspartyl-L-phenylalanine.