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WO2010038905A1 - A BACTERIUM BELONGING TO THE GENUS Pantoea PRODUCING AN L-ASPARTIC ACID OR L-ASPARTIC ACID-DERIVED METABOLITES AND A METHOD FOR PRODUCING L-ASPARTIC ACID OR L-ASPARTIC ACID-DERIVED METABOLITES - Google Patents

A BACTERIUM BELONGING TO THE GENUS Pantoea PRODUCING AN L-ASPARTIC ACID OR L-ASPARTIC ACID-DERIVED METABOLITES AND A METHOD FOR PRODUCING L-ASPARTIC ACID OR L-ASPARTIC ACID-DERIVED METABOLITES Download PDF

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WO2010038905A1
WO2010038905A1 PCT/JP2009/067449 JP2009067449W WO2010038905A1 WO 2010038905 A1 WO2010038905 A1 WO 2010038905A1 JP 2009067449 W JP2009067449 W JP 2009067449W WO 2010038905 A1 WO2010038905 A1 WO 2010038905A1
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Prior art keywords
aspartic acid
gene
bacterium
dehydrogenase
activity
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PCT/JP2009/067449
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French (fr)
Inventor
Olga Nikolaevna Mokhova
Tatyana Mikhailovna Kuvaeva
Lyubov Igorevna Golubeva
Aleksandra Viktorovna Kolokolova
Joanna Yosifovna Katashkina
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Ajinomoto Co.,Inc.
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Publication of WO2010038905A1 publication Critical patent/WO2010038905A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/20—Aspartic acid; Asparagine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/12—Methionine; Cysteine; Cystine

Definitions

  • the present invention relates to the microbiological industry, and specifically to a method for producing an L-aspartic acid or L-aspartic acid-derived metabolites using a bacterium belongiung to the genus Pantoea which has been modified to have attenuated expression of genes coding for ⁇ -ketoglutarate dehydrogenase and citrate synthase, as well as enhanced expression of genes coding for phosphoenolpyruvate carboxylase and glutamate dehydrogenase, or glutamate synthase.
  • 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.
  • L-aspartic acid is a useful metabolite having different applications (aspartame sweetener, biodegradable polymers, etc.).
  • aspartic acid is produced enzymatically from fumarate using aspartase.
  • fumarate can be obtained by chemical synthesis, as a product of oil distillation or from hydrocarbons in bacterial fermentation process.
  • the three-step production process includes the steps of 1) production of calcium-fumarate by fermentation of a bacterial strain; 2) conversion of the calcium- fumarate to diammonium fumarate by addition of ammonia, ammonium carbonate, or ammonia in combination with CO 2 ; 3) enzymatic conversion of the obtained diammonium fumarate to ammonium aspartate by aspartase. This process has been previously described (US patent 6,071,728).
  • L-aspartic acid is synthesized in the aspartate aminotransferase reaction. It's keto-precursor is oxaloacetate, the intermediate of TCA (tricarboxylic acid cycle and glyoxylate bypass, reviewed in Neidhardt, F. C. (Ed. in Chief), R. Curtiss III, J.L. Ingraham, E.C.C. Lin, K.B. Low, B. Magasanik, W.S. Reznikoff, M. Riley, M. Schaechter, and H.E. Umbarger (eds). 1996. Escherichia coli and Salmonella: Cellular and Molecular Biology. American Society for Microbiology); L-glutamic acid serves as a donor of amides in this reaction.
  • the carbon flux to oxaloacetate in E. coli grown on glucose can be increased by increasing phosphoenol pyrvate (PEP)-carboxylase activity, in particular, due to expressing feedback resistant PEP-carboxylase enzymes (European Patent EP072301 IBl) or by introducing heterologous pyruvate (Pyr) carboxylases (US Patent 6,455,284).
  • PEP phosphoenol pyrvate
  • inactivation of oxaloacetate or malate consuming enzymes such as PEP carboxykinase encoded by the pckA gene or malic enzymes encoded by the sfcA and maeB genes, is important for the production of aspartic acid-derived metabolites (WO2007/017710).
  • citrate synthase activity was established as a factor for increasing production of L-lysine, an aspartic acid-derived amino acid, by C. glutamicum (Ozaki H., Shiio J. Production of lysine by pyruvate kinase mutants of Brevibacteriumflavum. Agr. and Biol. Chem., Vol.47(7):1569-1576 (1983)).
  • aspects of the present invention include enhancing the productivity of L- aspartic acid-producing strains and providing a method for producing L-aspartic acid using these strains.
  • the present invention provides a bacterium belonging to the genus Pantoea having an increased ability to produce L-aspartic acid or L-aspartic acid-derived metabolite and a method for producing L-aspartic acid or L-aspartic acid-derived metabolite using the bacterium.
  • bacterium as described above, wherein said bacterium has been further modified to have attenuated expression of a gene coding for malate dehydrogenase. It is a further aspect of the present invention to provide the bacterium as described above, wherein said expression of the gene coding for ⁇ -ketoglutarate dehydrogenase, citrate synthase, aspartate ammonia-lyase (aspartase), pyruvate kinase, glucose dehydrogenase or malate dehydrogenase is attenuated by inactivating said gene in the chromosome of the bacterium.
  • L-aspartic acid-derived metabolite is selected from the group consisting of L-threonine, L-lysine, L-methionine, and L-homoserine.
  • L-aspartic acid-derived metabolite is selected from the group consisting of L-threonine, L-lysine, L-methionine, and L-homoserine.
  • Figure 1 shows the construction of the pMW-E ⁇ spC plasmid.
  • Figure 2 shows the construction of the pMIVK620S and pMIV-Ptac-pycA plasmids.
  • Figure 3 is a photograph which shows accumulation of by-products by the 3 ⁇ , 4 ⁇ (ApykA) and 5 ⁇ strains in test-tube cultivation.
  • Figure 4 shows growth curves of ⁇ pgi ⁇ zwf, ⁇ pgi ⁇ gcd, ⁇ zwf ⁇ gcd and ⁇ pgi ⁇ zwf ⁇ gcd strains in glucose minimal medium
  • Figure 5 shows construction of the RSFPlac-pzwf and RSFPlac-pgnd plasmids.
  • Figure 6 shows construction of the RSFPlaclacI plasmid vector.
  • Figure 7 shows construction of the pMW-intxis-cat plasmid vector
  • Figure 8 shows construction of the phMIV-1 helper plasmid.
  • 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 were recently re-classified into Pantoea agglomerans, Pantoea ananatis, Pantoea stewartii or the like, based on a nucleotide sequence analysis of the 16S rRNA, etc. (International Journal of Systematic Bacteriology, July 1989, 39(3).p.337-345).
  • Pantoea ananatis or Pantoea stewartii (International Journal of Systematic Bacteriology, Jan. 1993, 43(1), pp.162-173).
  • Typical strains of the Pantoea bacteria include, but are not limited to, Pantoea ananatis, Pantoea stewartii, Pantoea agglomerans, and Pantoea citrea. Specific examples include the following strains: Pantoea ananatis AJ 13355 (FERM BP-6614, European Patent Publication No. 0952221), Pantoea ananatis AJ13356 (FERM BP-6615, European Patent Publication No.
  • Pantoea ananatis AJ 13601 Pantoea ananatis AJ 13601
  • An exemplary ⁇ -Red resistant strain is Pantoea ananatis SCl 7(0) (VKPM B-9246, RU application 2006134574, WO2008/075483).
  • bacteria producing L-aspartic acid or L-aspartic acid-derived metabolite can refer to the abilities to produce L-aspartic acid and/or one or more L- aspartic acid-derived metabolites, and to cause accumulation of L-aspartic acid or L- aspartic acid-derived metabolite(s) in a medium or cells of the bacterium to such a degree that L-aspartic acid or L-aspartic acid-derived metabolite can be collected from the medium or cells when the bacterium is cultured in the medium.
  • the phrase "bacterium producing L-aspartic acid or L-aspartic acid-derived metabolite” can mean a bacterium which is able to cause accumulation of a target L-amino acid in the culture medium in an amount larger than the wild-type or parental strain, and can also mean that the microorganism is able to cause accumulation in the medium of an amount not less than 0.4 g/L, and in another example not less than 1.0 g/L of the target L-amino acid.
  • the ability to produce L-aspartic acid or L-aspartic acid-derived metabolites in the bacterium can be a native or inherent ability, or can be imparted by modifying the bacterium using mutagenesis or recombinant DNA techniques.
  • L-aspartic acid refers to an L-aspartic acid or to any salt thereof, and can be called aspartic acid.
  • the L-aspartic acid-derived metabolites include L- threonine, L-lysine, L-methionine, and L-homoserine.
  • the expression of the gene coding for aspartate ammonia-lyase can also be attenuated.
  • the activity of ⁇ -ketoglutarate dehydrogenase can mean catalyzing the conversion of 2-ketoglutarate into succinyl-CoA, and production of NADH and CO 2 in an irreversible reaction within the 2-oxoglutarate dehydrogenase complex [(SucA) i 2 ] [(SuCB) 24 ] [(Lpd) 2 ].
  • the nucleotide sequence of the sucA gene and the amino acid sequence of SucA protein from P. ananatis are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
  • nucleotide sequence of the git A gene and the amino acid sequence of GItA protein from P. ananatis are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.
  • the ppc gene from E. coli is known (nucleotides complementary to nucleotides in positions from 4,148,470 to 4,151,121; GenBank accession no. NC_000913.2; gi: 49175990).
  • the ppc gene is located between the yijP and the argE genes on the chromosome of E. coli K- 12.
  • the nucleotide sequence of the ppc gene and the amino acid sequence of Ppc encoded by the ppc gene from E. coli are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.
  • pyruvate carboxylase (synonyms: Pyc, PycA) can mean catalyzing the following reaction:
  • pyruvate carboxylase from Sinorhizobium meliloti is resistant to feedback inhibition by L-aspartic acid (Dunn, M.F. et al, Cloning and characterization of the pyruvate carboxylase from Sinorhizobium meliloti Rm 10211, Arch. Microbiol., 176, 355-363 (2001)). Therefore, the pycA gene from S. meliloti was cloned and integrated into an L-aspartic acid producing P. ananatis strain. The nucleotide sequence of the pycA gene and the amino acid sequence of PycA encoded by the pycA gene from S.
  • GdhA glutamate dehydrogenase
  • the gdhA gene from E. coli is known (nucleotides in positions from 1,840,395 to 1,841,738; GenBank accession no. NC 000913.2; gi: 49175990).
  • the gdhA gene is located between the ynjH and the ynjl ORPs on the chromosome of E. coli K- 12.
  • the nucleotide sequence of the gdhA gene and the amino acid sequence of GdhA encoded by the gdhA gene from E. coli are shown in S ⁇ Q ID NO: 9 and S ⁇ Q ID NO: 10, respectively.
  • the activity of glutamate synthase can mean catalyzing the single-step conversion of L-glutamine and alpha-ketoglutarate into two molecules of L-glutamate:
  • the glutamate synthase from E. coli includes two subunits encoded by the gltB and gltD genes organized into one operon.
  • the gltB and gltD genes from E. coli are known (nucleotides in positions from 3,352,654 to 3,357,207 and 3,357,220 to 3,358,638, respectively; GenBank accession no. NC 000913.2; gi: 49175990).
  • the gltBD operon is located between the yhcC ORP and the gltF gene on the chromosome of E. coli K- 12.
  • the nucleotide sequences of the gltB and gltD genes and the amino acid sequences of GltB and GltD encoded by the gltB and gltD genes from E. coli are shown in S ⁇ Q ID NO: 11, S ⁇ Q ID NO: 12, S ⁇ Q ID NO: 13, and S ⁇ Q ID NO: 14, respectively.
  • aspartate ammonia-lyase can mean catalyzing the reversible conversion of L-aspartic acid to fumaric acid and ammonia:
  • the nucleotide sequence of the asp A gene and the amino acid sequence of AspA protein from P. ananatis are shown in S ⁇ Q ID NO: 15 and S ⁇ Q ID NO: 16, respectively.
  • P. ananatis has two isozymes of pyruvate kinase: PykA and PykF.
  • the nucleotide sequence of the pykA gene and the amino acid sequence of PykA protein from P. ananatis are shown in SEQ ID NO: 114 and SEQ ID NO: 115, respectively.
  • the nucleotide sequence of the pykF gene and the amino acid sequences of PykF protein from P. ananatis are shown in SEQ ID NO: 116 and SEQ ID NO: 117, respectively.
  • nucleotide sequence of the gcd gene and the amino acid sequence of the Gcd protein from P. ananatis are shown in SEQ ID NO: 17 and SEQ ID NO: 18, respectively.
  • nucleotide sequence of the mdh gene and the amino acid sequence of Mdh protein from P. ananatis are shown in SEQ ID NO: 19 and SEQ ID NO: 20, respectively.
  • bacterium has been modified to have attenuated expression of the gene can mean that the bacterium has been modified in such a way that the modified bacterium can contain a reduced amount of the protein encoded by the gene as compared with an unmodified bacterium, or is unable to synthesize the protein encoded by the gene.
  • the phrase "inactivation of the gene” can mean 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..
  • sequences controlling gene expression such as a promoter, enhancer, attenuator, ribosome-binding site, etc.
  • the presence or absence of the sue A, git A, asp A, gcd, or mdh gene in the chromosome of a bacterium can be detected by well-known methods, including PCR, Southern blotting and the like.
  • the expression levels of the genes can be estimated by measuring the amount 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.
  • Expression of the gene can be attenuated by introducing a mutation into the gene on the chromosome so that the intracellular amount of the protein encoded by the gene is decreased as compared to an unmodified strain.
  • a mutation can be the introduction of 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 gene 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)).
  • SD Shine-Dalgarno
  • a mutant gene can be prepared, and the bacterium to be modified can be transformed with a DNA fragment containing the mutant gene. Then, the native gene on the chromosome can be replaced with the mutant gene by homologous recombination, and the resulting strain can be selected.
  • Such gene replacement by homologous recombination can be conducted by employing a linear DNA, which is known as "Red-driven integration" (Datsenko, K.A. and Wanner, B. L., Proc. Natl. Acad. Sci.
  • An exemplary strain is the Pantoea ananatis strain which has been modified to be resistant to the product of the ⁇ -Red genes. Examples include but are not limited to the Pantoea ananatis strain SC 17(0) (VKPM B-9246, RU application 2006134574, WO2008/075483). 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 lacking the ability 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. Patent 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".
  • variant proteins inactivation of genes, and other methods can be applied to other proteins, genes, and in the breeding of bacteria described below.
  • enhancing the expression of the gene means that the expression of the gene is higher than that of a non-modified strain, for example, a wild-type strain.
  • modifications include increasing the copy number of the target gene per cell, increasing the expression level of the gene, and so forth.
  • the quantity of the copy number of the target gene is 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.
  • FISH fluorescence in situ hybridization
  • the level of gene expression can be measured by various known methods including Northern blotting, quantitative RT-PCR, and the like.
  • wild-type strains that can act as a control include, for example, Pantoea ananatis FERM BP-6614.
  • the ppc, pycA, gdhA, or gltBD gene to be modified to enhance its expression is not limited to the genes shown in SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:11, respectively, but can include genes homologous to SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:11, respectively.
  • the protein variant encoded by the ppc, pycA, gdhA ox gltBD gene can have a homology of not less than 80%, in another example not less than 90%, and in another example not less than 95 %, with respect to the entire amino acid sequence shown in SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, or SEQ ID NO:12, respectively, as long as the activity of the corresponding protein is maintained.
  • the phrase "protein variant” means proteins which have changes in the sequences, whether they 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.
  • Homology between two amino acid sequences can be determined using the well-known methods, for example, the computer program BLAST 2.0, which calculates three parameters: score, identity and similarity.
  • substitution, deletion, insertion or addition of one or several amino acid residues can be conservative mutation(s) so that the activity is maintained.
  • the representative conservative mutation can be a conservative substitution.
  • conservative substitutions include substitution of Ser or Thr for Ala, substitution of GIn, His or Lys for Arg, substitution of GIu, GIn, Lys, His or Asp for Asn, substitution of Asn, GIu or GIn for Asp, substitution of Ser or Ala for Cys, substitution of Asn, GIu, Lys, His, Asp or Arg for GIn, substitution of Asn, GIn, Lys or Asp for GIu, substitution of Pro for GIy, substitution of Asn, Lys, GIn, Arg or Tyr for His, substitution of Leu, Met, VaI or Phe for He, substitution of He, Met, VaI or Phe for Leu, substitution of Asn, GIu, GIn, His or Arg for Lys, substitution of He, Leu, VaI or Phe for Met, substitution of Trp,
  • the ppc, pycA, gdhA or git BD gene can be a variant which hybridizes under stringent conditions with the nucleotide sequence complementary to the sequence shown in SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:11, respectively, or a probe which can be prepared from the nucleotide sequence, provided that it encodes a functional protein.
  • Stringent conditions include those under which a specific hybrid, for example, a hybrid having homology of not less than 60%, in another example not less than 70%, in another example not less than 80%, in another example not less than 90%, and and in another example not less than 95%, is formed and a non-specific hybrid, for example, a hybrid having homology lower than the above, is not formed.
  • stringent conditions are exemplified by washing one time or more, and in another example two or three times, at a salt concentration of 1 xSSC, 0.1% SDS, and in another example 0.1 x SSC, 0.1% SDS at 60 0 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.
  • the recommended duration of washing for the HybondTM N+ nylon membrane (Amersham) under stringent conditions is 15 minutes.
  • washing can be performed 2 to 3 times.
  • the length of the probe can be suitably selected depending on the hybridization conditions, and is usually 100 bp to 1 kbp.
  • Methods of gene expression enhancement include increasing the gene copy number. Introducing a gene into a vector that is able to function in a bacterium belonging to the genus Pantoea increases the copy number of the gene.
  • a plasmid which can be transformed include plasmids which are autonomously replicable in bacteria belonging to the genus Pantoea, for example, pUC19, pUC18, pBR322, RSFlOlO, pHSG299, pHSG298, pHSG399, pHSG398, pSTV28, pSTV29 (pHSG and pSTV can be obtained from Takara Bio), pMWl 19, pMWll[delta], pMW219, pMW218 (plasmids of pMW series can be obtained from Nippon Gene), and so forth.
  • Phage DNA can also be used as a vector, instead of a plasmid.
  • Gene expression can also be enhanced by introducing multiple copies of the gene into the bacterial chromosome by, for example, a method of homologous recombination, Mu integration, or the like.
  • Mu integration allows for the introduction of up to 3 copies of the gene into the bacterial chromosome.
  • Increasing the copy number of a gene can also be achieved by introducing multiple copies of the gene into the chromosomal DNA of the bacterium.
  • homologous recombination is carried out using a sequence having multiple copies of the gene, and these multiple copies function as targets in the chromosomal DNA.
  • Sequences having multiple copies in the chromosomal DNA include, but are not limited to repetitive DNA, or inverted repeats present at the end of a transposable element. Also it is possible to incorporate the gene into a transposon, and allow it to be transferred to introduce multiple copies of the gene into the chromosomal DNA.
  • Gene expression can also be enhanced by placing the DNA under the control of a potent promoter.
  • a potent promoter for example, the lac promoter, the trp promoter, the trc promoter, the P R , or the P L promoters of lambda phage are all known to be potent promoters.
  • the use of a potent promoter can be combined with multiplication of gene copies.
  • the effect of a promoter can be enhanced by, for example, introducing a mutation into the promoter to increase the transcription level of a gene located downstream of the promoter.
  • substitution of several nucleotides in the spacer between the ribosome binding site (RBS) and the start codon, especially the sequences immediately upstream of the start codon profoundly affect the translation of the mRNA. For example, a 20-fold range in the expression levels was found, depending on the nature of the three nucleotides preceding the start codon (Gold et al, Annu. Rev. Microbiol., 35, 365-403, 1981; Hui et al, EMBO J., 3, 623-629, 1984).
  • a nucleotide substitution into a promoter region of a gene on the bacterial chromosome, which results in a stronger promoter function.
  • the alteration of the expression control sequence can be performed, for example, in the same manner as the gene substitution using a temperature-sensitive plasmid, as disclosed in WO 00/18935 and JP 1-215280 A.
  • Bacteria producing L-aspartic acid or L-aspartic acid-derived metabolites include a bacterium which has been modified to have decreased activity of ⁇ -ketoglutarate dehydrogenase; decreased activity of citrate synthase; increased activity of phosphoenolpyruvate carboxylase or pyruvate carboxylase; and increased activity of glutamate dehydrogenase or glutamate synthase.
  • Bacteria can be further modified to have attenuated expression of the gene coding for aspartate ammonia-lyase (aspartase).
  • parent strains which can be used to derive L-threonine-producing bacteria include strains in which expression of one or more genes encoding an L- threonine biosynthetic enzyme are enhanced.
  • the bacterium is modified to enhance expression of one or more of the following genes:
  • mutant thrA gene which codes for aspartokinase homoserine dehydrogenase I resistant to feed back inhibition by threonine;
  • the thrA gene which encodes aspartokinase homoserine dehydrogenase I of Escherichia coli has been elucidated (nucleotide positions 337 to 2799, GenBank accession no.NC_000913.2, gi: 49175990).
  • the thrA gene is located between the thrL and thrB genes on the chromosome of E. coli K- 12.
  • the thrB gene which encodes homoserine kinase of Escherichia coli has been elucidated (nucleotide positions 2801 to 3733, GenBank accession no.NC_000913.2, gi: 49175990).
  • the thrB gene is located between the thrA and thrC genes on the chromosome of E. coli K- 12.
  • the thrC gene which encodes threonine synthase of Escherichia coli has been elucidated (nucleotide positions 3734 to 5020, GenBank accession no.NC_000913.2, gi: 49175990).
  • the thrC gene is located between the thrB gene and the yaaX open reading frame on the chromosome of E. coli K- 12. All three genes function as a single threonine operon.
  • the attenuator region which affects the transcription is desirably removed from the operon (WO2005/049808, WO2003/097839).
  • a mutant thrA gene which codes for aspartokinase homoserine dehydrogenase I and is resistant to feedback inhibition by 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 threonine producing E. coli strain VKPM B-3996. Plasmid pVIC40 is described in detail in U.S. Patent No. 5,705,371.
  • the rhtA gene is present at 18 min on the E. coli chromosome close to the glnHPQ operon, which encodes components of the glutamine transport system.
  • the rhtA gene is identical to ORPl (ybiF gene, nucleotide positions 764 to 1651, GenBank accession number AAA218541, gi:440181) and is located between the pexB and ompX genes.
  • the unit expressing a protein encoded by the ORFl has been designated the rhtA gene (rht: resistance to homoserine and threonine).
  • the asd gene of E. coli has already been elucidated (nucleotide positions 3572511 to 3571408, GenBank accession no. NC 000913.1, gi:16131307), and can be obtained by PCR (polymerase chain reaction; refer to White, TJ. et al., Trends Genet., 5, 185 (1989)) utilizing primers prepared based on the nucleotide sequence of the gene.
  • the asd genes from other microorganisms can be obtained in a similar manner.
  • the aspC gene of E. coli has already been elucidated (nucleotide positions 983742 to 984932, GenBank accession no. NC 000913.1, gi:16128895), and can be obtained by PCR.
  • the aspC genes of other microorganisms can be obtained in a similar manner.
  • L-lysine-producine bacteria examples include 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. Patent No.
  • ppc phosphoenolpyrvate carboxylase
  • aspartate semialdehyde dehydrogenease aspartase
  • aspA aspartase
  • the parent strains can have increased expression of the gene involved in energy efficiency (cyo) (EP 1170376 A), the gene encoding nicotinamide nucleotide transhydrogenase (pntAB) (U.S. Patent No. 5,830,716), the ybjE gene (WO2005/073390), or combinations thereof.
  • parent strains which can be used to derive L-lysine-producing bacteria also include strains having decreased or eliminated activity of an enzyme that catalyzes a reaction for generating a compound other than L-lysine by branching off from the biosynthetic pathway of L-lysine.
  • the enzymes that catalyze a reaction for generating a compound other than L-lysine by branching off from the biosynthetic pathway of L-lysine include homoserine dehydrogenase, lysine decarboxylase (U.S. Patent No. 5,827,698), and the malic enzyme (WO2005010175).
  • Methods are described for producing L-aspartic acid or L-aspartic acid-derived metabolites which include cultivating the bacterium as described herein in a culture medium to produce and excrete the L-aspartic acid or L-aspartic acid-derived metabolite into the medium, and collecting the produced L-aspartic acid or L-aspartic acid-derived metabolite from the medium.
  • the cultivation, collection, and purification of L-aspartic acid or L-aspartic acid-derived metabolites from the medium and the like can be performed in a manner similar to conventional fermentation methods wherein an amino acid is produced using a bacterium.
  • the medium used for culture can be either a synthetic or natural medium, so long as the medium includes a carbon source, a nitrogen source, minerals, and, if necessary, appropriate amounts of nutrients which the bacterium requires for growth.
  • the carbon source can include various carbohydrates such as glucose and sucrose, and various organic acids. Depending on the assimilation mode of the chosen microorganism, alcohol including ethanol and glycerol can 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.
  • potassium monophosphate magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, calcium chloride, and the like can be used.
  • vitamins thiamine, yeast extract, and the like, can be used.
  • the cultivation can be performed under aerobic conditions, such as by shaking and/or stirring with aeration, at a temperature of 30 to 36°C, and in another example 32 to 34°C.
  • the pH of the culture is usually between 5.0 and 7.0, and in another example between 6.0 and 6.5.
  • the pH of the culture can be adjusted with ammonia, calcium carbonate, various acids, various bases, and buffers. Usually, a 1 to 5 -day cultivation leads to accumulation of the target L-amino acid in the liquid medium.
  • solids such as cells can be removed from the liquid medium by centrifugation or membrane filtration, and then the L-amino acid can be collected and purified by ion-exchange, concentration, and/or crystallization methods.
  • the DNA fragment containing a kanamycin resistance gene flanked with attL and attR sites of phage ⁇ with primers presented in Table 1 was amplified with PCR. Primers used in the reaction were homologous with at least 40bp of the target sites of P. ananatis genome on their 5'- ends.
  • pMWl ⁇ 8-( ⁇ attL-Km ⁇ - ⁇ attR) plasmid (RU application 2006134574, WO2008/075483) was used as a template in all the reactions.
  • the obtained DNA fragments were treated for 2 or 3 hours with Dpnl restrictase which recognizes the methylated GATC site to eliminate pMWl 18-( ⁇ Z-Km r - ⁇ //i?).
  • pMW- IntXis-cat plasmid was electroporated to the selected plasmid-less integrant by the same procedure as for the electroporation of the PCR- generated fragments. After electroporation the cells were plated on LB-agar containing 0.5% glucose, 0.5x M9 salt solution and chloramphenicol (50mg/L) and incubated at 37°C overnight to induce synthesis of the Int/Xis proteins. The grown clones were replica-plated on LB-plates with and without kanamycin to select Km s variants. The selected Km s clones were re-checked by PCR with corresponding test primers.
  • Cm R the pMW-IntXis helper plasmid (WO2005010175) was used. Selection of Cm s clones was performed as above. But in this case, cells after electroporation were plated on a medium containing 800 mg/L ampicillin.
  • the desirable plasmids were electroporated to the corresponding strains by the method described above for electroporation of DNA fragments.
  • the 3 ⁇ ::pycA strain was constructed via integration of the mini-Mu cassette carrying the pycA gene from Sinorhizobium meliloti under control of the P, flC promoter and canonical SD sequence AGGAGG.
  • the integrative plasmid pMIV-P toc - pycA was constructed (see Example 2).
  • the pMIV-P tac -#yc ⁇ plasmid was electroporated to 3 ⁇ -S strain (marker- less) (see Table 7) harboring phMIV-1 helper plasmid (see Reference Example 3) providing expression of the Mu integrase according to the procedure described above.
  • the 3 ⁇ -S strain is a marker-less strain of the 3 ⁇ strain (SCIl(O)AaSpAAsUcAAgItA). After electroporation, cells were incubated at 37°C for induction of the integrase synthesis. Cells were plated on L-agar containing 25mg/L chloramphenicol. pMIV -P, ac -pycA carried two antibiotic resistance markers: cat inside the integrative cassette and bla on the vector part of the plasmid. The grown clones were replica-plated and Ap clones were selected. The selected clones did not contain the integrative plasmid. PCR analysis proved the presence of the pycA gene in the genome of the selected clones.
  • the obtained integrants were cured from the helper plasmid. To that, cells were seeded in LB medium and incubated without agitation at 37°C in 3 days. After that, cells were plated on L-agar with addition of chloramphenicol (25mg/L) and incubated at 34°C overnight. The grown clones were replica plated and Tc s Cm R variants were selected and designated 3 ⁇ ::pycA.
  • 5 ⁇ P2-36S strain is a marker-less derivative of the 5 ⁇ -S strain carrying Appc mutation and the E. colippc K620S gene integrated to the genome by the same Mu- dependent procedure as was used for the 3 ⁇ ::pycA strain construction.
  • the k ⁇ n gene flanked by ⁇ ttR/L was amplified by PCR, using primers Dppc-3' (SEQ ID NO: 73) and attR3-XbaI-HindIII (SEQ ID NO: 76).
  • Genomic DNA isolated from P. ⁇ n ⁇ n ⁇ tis SC 17(0) strain Ptac-lacZ (RU application 2006134574, WO2008090770) was used as a template for PCR.
  • the fragment containing the terminator of the leader peptide of the E. coli threonine operon (Tthr) was constructed by PCR.
  • mashl SEQ ID NO: 74
  • mash2 SEQ ID NO: 75
  • Dppc-5' SEQ ID NO: 72
  • Tthr5'-Xbal SEQ ID NO: 77
  • the resulting DNA fragment was used as a template for PCR with Dppc-5'(SEQ ID NO: 72) and Tthr5'-Xbal (SEQ ID NO: 77) primers to create the fragment with a Xb ⁇ l recognition site on its 5 '-end, which is necessary to join the integrative cassette and homologous arm for integration on the 3 '-end.
  • the fragments including Tthr and the removable Km R marker were digested by Xb ⁇ l restrictase and then ligated.
  • the ligated mixture was electroporated to the SC17(0)/RSF-Red-TER (RU application 2006134574) strain for integration according to the procedure for ⁇ Red-dependent integration described above.
  • Integrants were selected on LB-agar plates with kanamycin (40mg/l). The chromosome structure of the integrants was confirmed by PCR using ppc-tl (SEQ ID NO: 78) and ppc-t2 (SEQ ID NO: 79) oligonucleotides as primers. The resulting strain was named SC17(0) ⁇ ppc.
  • the constructed deletion was transferred to 5 ⁇ -S by the chromosome electroporation procedure.
  • 200 ng of genomic DNA isolated from SC17(0) ⁇ ppc using the Genomic DNA Purification Kit provided by "Fermentas” was electro-transformed to 5 ⁇ -S.
  • the cultivation conditions were the same as for the Red- dependent integration procedure except for the addition of IPTG.
  • Preparation of the electrocompetent cells was the same as for Red-dependent integration.
  • the obtained integrants were verified by PCR using ppc-tl (SEQ ID NO: 78) and ppc-t2 (SEQ ID NO: 79) oligonucleotides as primers.
  • the resulting strain was named 5 ⁇ P.
  • the high L-GIu concentration was used to simulate the presence of the glutamate dehydrogenase.
  • the selected strain was cured from the chloramphenicol resistance marker using the ⁇ lnt/Xis-dependent procedure (see above).
  • the resultant strain was named 5 ⁇ P2-36S.
  • 5 ⁇ P2R strain was obtained from 5 ⁇ P2-36S as a spontaneous Asp R mutant selected on the M9 plates containing 10 g/L glucose and 30 g/L L- Asp (L-aspartic acid), pH5.5 having the same producing ability as the parental strain.
  • Section B was adjusted by NaOH to have pH 6.5. Sections B and C were sterilized separately.
  • Seed Plates containing LB-M91/2 medium (LB medium enriched with 0.5x M9 salt solution and 0.5% glucose) with addition of the appropriate antibiotic seeded and incubated at 34°C overnight.
  • the DNA fragment which includes a coding part of the E. coli aspC gene linked with canonical SD-sequence AGGAGG was generated in PCR with the primers E-aspC5'KI (SEQ ID NO: 92) and E-aspC3'BI (SEQ ID NO: 93).
  • Chromosomal DNA isolated from the E. coli strain MGl 655 was used as template in the reaction.
  • the strain MGl 655 can be obtained from American Type Culture Collection. (P.O. Box 1549 Manassas, VA 20108, United States of America).
  • the obtained fragment was digested with Kpnl and BamRl restrictases and ligated with pMWl 18-PlacUV5-lacI vector (Skorokhodova, A. Yu et al, Biotekhnologiya (Rus), 5, 3-21 (2004)) which had been digested with the same endonucleases.
  • the ligated mixture was transformed to E. coli strain TGl (J. Sambrook et al., Molecular Cloning, 1989) and plasmid DNA was isolated from the clones grown on LB plates with ampicillin (100mg/L).
  • the plasmids carrying the desirable insertion were electroporated to the SC17(0) ⁇ aspC ⁇ tyrB strain and several plasmids which resulted in auxotrophy of the strain to L-aspartic acid were selected.
  • One of these plasmids was electro-transformed to 3 ⁇ -S/RSFGP strain (see below) and specific aspartate aminotransferase activity was assayed in crude extracts of the 3 ⁇ - S/RSFGP harboring pMW-EaspC.
  • the plasmid-less strain was used as a control.
  • Table 4 indicate that the pMW-EaspC plasmid provides an increase of aspartate aminotransferase activity in 3 ⁇ -S/RSFGP strain by 1.5 times.
  • the E. colippc ⁇ 2 gene coding for PEP-carboxylase resistant against inhibition by aspartic acid was sub-cloned from the pTK620S plasmid (Masato Yano and Katsura Izui, Eur. J. Biochem. FEBS, 247, 74-81, 1997) to the pMIV-5JS plasmid (RU patent application 2006132818, EP 1942183) in two steps. At first, the Sall-Sphl fragment of pTK620S was sub-cloned into Sall-Sphl recognition sites of pMIV5-JS.
  • the obtained pMIV-ppc-5'plasmid carries a large 5'-terminal portion of the p/7c K620S gene.
  • the 3'-proximal portion of the ppc gene was amplified in PCR with primers ppc-Sphl (SEQ ID NO: 94) and ppc-Hindlll (SEQ ID NO: 95) and the pTK620S plasmid as template.
  • This fragment and pMIV-ppc-5' were digested with Sphl and Hindlll restrictases and ligated.
  • the ligated mixture was electroporated to E.coli strain MG1655 ⁇ ppc (see Reference Example 4).
  • telomere sequence a DNA fragment containing the coding part of the pycA gene from Sinorhizobium meliloti was generated in PCR with primers PycSm5-LNX (SEQ ID NO: 96) and pycSm3-Sal (SEQ ID NO: 97). Chromosomal DNA isolated from the S".
  • meliloti type strain DSM 30135, ATCC 9930, VKPM B- 9293
  • VKPM All-Russian Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1 Dorozhny proezd, 1) was used as a template in this reaction. This strain can also be obtained from ATCC.
  • the obtained DNA fragment was digested with Sail and Xbal restrictases and ligated with the pMIV5-JS integrative plasmid which had been treated with the same enzymes.
  • the ligated mixture was transformed to E. coli strain TGl and plasmid DNA was isolated from the clones which grew on LB plates with ampicillin (100mg/L) and chloramphenicol (50mg/L).
  • Three plasmids carrying the desirable insertion were selected. These plasmids carrying the promoter-less gene pycA were designated pMIV- pycA (Fig. 2). All three obtained plasmid probes were used for the cloning of P toc promoter in front of the pycA gene.
  • the DNA fragment containing P toc was generated by PCR with primers J56 (SEQ ID NO: 108) and J57(KpnBgl) (SEQ ID NO: 109).
  • the pDR540 plasmid (GenBank accession number U13847, "Pharmacia") was used as a template in this reaction.
  • Primer J56 contains the site for Xbal restrictase at the 5 '-end thereof.
  • Primer J57(KpnBgl) contains sites for Kpnl amd BgHl restrictase at the 5 '-end thereof.
  • the resulting DNA fragment was digested with Kpnl and Klenow fragment of DNA polymerase I to obtain the blunt ends.
  • the fragment was digested with Xbal and ligated with pMIV-pycA plasmid digested with Xbal and Ec/136II (the last enzyme produces the blunt ends).
  • the resulting ligated mixture was transformed to the E. coli strain MG1655 ⁇ ppc. After transformation, cells were plated on M9 glucose minimal medium to select the clones prototrophic to L- glutamic acid. Plasmid DNA from the selected clones was isolated and the structure of the resulting pMIV-Ptac-pycA plasmid was proven by restriction analysis.
  • RSFGP plasmid carrying ppc and gdhA genes from E. coli was constructed on the basis of the RSFCPG plasmid containing the git A, ppc and gdhA genes (US patent application 20070134773).
  • 5'- end phosporylated primers S ⁇ Q ID NO: 118 and 119 were used for PCR to obtain a big part of the RSFCPG plasmid without the git A gene using TaKaRa La TaqTM DNA polymerase (TaKaRa Bio Inc., Japan). The resulting 14 kb PCR fragment was purified and ligated. Thus, the RSFGP plasmid was obtained.
  • pMWgdhA plasmid carrying the gdhA gene from E. coli was obtained by ligation of the DNA fragment carrying the gdhA gene from E. coli into pMWl 19 vector.
  • the DNA fragment carrying the gdhA gene from E. coli was obtained by PCR using primers S ⁇ Q ID NO: 120 and 121.
  • Primer S ⁇ Q ID NO: 120 contains a Sail restriction site at the 5 '-end thereof
  • primer S ⁇ Q ID NO: 121 contains Hindlll restriction site at the 5 '-end thereof.
  • the resulting DNA fragment was treated with Sail and Hindlll restrictases and ligated into a pMWl 19 vector previously treated with the same restrictases.
  • Example 3 Function of the genes involved in L-aspartatic acid/L- glutamic acid metabolism in P. ananatis.
  • E. coli two enzymes possess aspartate aminotransferase activity. They are aspartate aminotransferase encoded by the aspC gene and aromatic amino acids aminotrasferase, the product of the tyrB gene. Only simultaneous inactivation of the aspC and tyrB genes leads to Asp auxotrophy in E. coli (Reitzer, L. J., Ammonia assimilation and the biosynthesis of glutamine, glutamic acid, aspartic acid, asparagine, L-alanine, and D-alanine. In Neidhardt, F. C. (Ed. in Chief), R. Curtiss III, J.L. Ingraham, E.C.C.
  • P. ananatis is a member of Enterobacteriaceae family, and is closely related to E. coli.
  • the putative aspC and tyrB genes were found in the P. ananatis genome using the "Genome” program. These genes were inactivated in P. ananatis SC 17(0) strain using the procedure of ⁇ Red-dependent integration of PCR generated DNA fragments (see Reference Example 1), which were previously adopted for use in this organism (RU application 2006134574).
  • oxaloacetate is a precursor for aspartic acid synthesis and L-glutamic acid is a donor of amides.
  • L-glutamic acid is a donor of amides.
  • an increase of the oxaloacetate intracellular level is necessary.
  • PEP carboxylases and pyruvate carboxylases are inhibited by L-aspartic acid.
  • PEP carboxylase from E. coli is sensitive to this inhibitor (Wohl, R.C. & Markus, G., J. Biol. Chem., Vol. 247(18), pp.
  • the constructed strain could not use aspartic acid as a sole carbon source absolutely (Table 5) and did not display aspartase activity. Therefore, the aspA gene coding for aspartase was identified. It has been shown that, as in E. coli, the aspartase reaction is the major way for aspartic acid utilization in P. ananatis. The aspA gene must be inactivated in an aspartic acid-producing strain to avoid product utilization by the cells.
  • the fumarate which is formed in the aspartase reaction is converted to malate and, after that, to oxaloacetate in TCA.
  • oxaloacetate can be converted to citrate in citrate synthase reaction or decarboxylated to PEP in PEP carboxykinase reaction or to Pyr by oxaloacetate decarboxylase.
  • malic enzyme catalyzes decarboxylation of malate to pyruvate. It was published elsewhere (H Ozaki, J Shiio. Production of lysine by pyruvate kinase mutants of Brevibacterium flavum, Agr. Biol.
  • citrate synthase reaction is the major way of unproductive waste of oxaloacetate.
  • ⁇ -ketoglutarate utilization (due to AsucA mutation) must be blocked, and re-amination of ⁇ -ketoglutarate at a high rate is necessary.
  • Animation of ⁇ -ketoglutarate to L-glutamic acid can be carried out in glutamate dehydrogenase or glutamate synthase reactions. It is well known in Enterobacteriaceae that glutamate dehydrogenase and glutamate synthase/glutamine synthetase form two alternative pathways for L-glutamic acid synthesis. Bacteria use only one of these pathways depending on the availability of energy and nitrogen. In E.
  • the P. ananatis genome does not contain ORFs coding for close homologues of E. coli glutamate dehydrogenase or glutamate synthase. Instead, it contains the gltB gene coding for a close homologue of a large subunit of glutamate synthase from Agrobacterium tumefaciens, and the gdhA gene coding for close homologue of glutamate dehydrogenase from Salmonella typhimurium.
  • We have deleted the gltB gene in P. ananatis see Reference Example 1). The resulting strain has proven to be a strong auxotroph to L-GIu (L-GIu requirement 0.5 g/L).
  • glutamate synthase/glutamine synthetase reactions are the only pathways for the synthesis of L- glutamic acid in P. ananatis.
  • deletion of the putative gdhA gene as well as replacing its regulatory region with a strong V tac promoter linked to a consensus SD sequence did not significantly change the growth of P. ananatis with glucose and L-glutamic acid as the sole carbon sources.
  • glutamate dehydrogenase activity was not detected in P. ananatis crude extracts.
  • the gdhA gene found by computer analysis in this organism is inactive, at least under the chosen cultivation conditions. Therefore, the introduction of a foreign glutamate dehydrogenase or increase of glutamate synthase activity could be necessary for high-performance L- aspartic acid production.
  • enzymes including aspartate ammonia-lyase (aspartase), glucose dehydrogenase and malate dehydrogenase. It is also desirable to increase activity of aspartate aminotransferase.
  • Table 7 Test tube fermentation of the 3 ⁇ -S/RSFGP strain and the strains lacking one of the genetic factors necessary for L- Asp production. 48-hour cultivation; initial glucose concentration 40g/L, initial L-GIu concentration 3g/L. ImM IPTG was added for the strains harboring pMWaspC. Average data for 3 independent cultures are represented.
  • Example 5 Decrease of the unproductive conversion of PEP to Pyr due to inactivation of the pykA and pykF genes.
  • the 3 ⁇ -S (marker-less SC ⁇ 7(0) ⁇ aspA ⁇ sucAAgltA) strain transformed with the RSFGP plasmid carrying the ppc and gdhA genes from E. coli resulted in accumulation of L- Asp up to 0.7g/L in test-tube cultivation. At the same time, accumulation of VaI, Leu and Ala up to 2-3g/L was observed.
  • ⁇ #y£A:: ⁇ attL-Tc R - ⁇ attR and Apyk ⁇ :. ⁇ a.UL- Km R - ⁇ attR deletions were introduced step by step to 3 ⁇ -S, which is the marker-less derivative of 3 ⁇ . The resulting strains were named 4 ⁇ and 5 ⁇ , respectively.
  • the 5 ⁇ strain was cured from the selective markers according to the above- described procedure.
  • the resulting strain 5 ⁇ -S was electro transformed with RSFGP plasmid to increase the PEP-carboxylase and glutamate dehydrogenase activities.
  • 5 ⁇ -S/RSFGP strain showed higher accumulation of aspartic acid than the control strain 3 ⁇ /RSFGP (Table 7).
  • the SC 17(0) ⁇ zwf ⁇ pzwf strain was constructed and the activities of glucose-6P dehydrogenase were determined in this strain, SC 17(0), and in SC17(0) ⁇ zwf. NAD-dependent activity was absent in all strains. NADP-dependent glucose-6P dehydrogenase activity was decreased at least 50 times in the strains carrying deletion of the chromosome zwf gene. Cells for this experiment were grown on glucose- containing medium. We have concluded that the chromosome zwf gene encodes NADP-dependent glucose-6P dehydrogenase whereas the pzwf gene is not expressed under these conditions.
  • the coding part of the /?zw/ ⁇ gene was amplified by PCR using primers pzwf-5 (SEQ ID NO: 98) and pzwf-3 (SEQ ID NO: 99), and genomic DNA isolated from P. ananatis SC 17 strain (US patent 6,596,517) as a template.
  • Primer pzwf-5 (SEQ ID NO: 98) contains the site formal restrictase at the 5 '-end thereof.
  • Primer pzwf-3 (SEQ ID NO: 99) contains the site for BamHl restrictase at the5'-end thereof.
  • the resulting DNA fragment was treated with Xbal and BamRl restrictases and was cloned into the Xbal-BamHl sites of the RSF-based vector RSFPlaclacI (see Reference Example 1) under control of the P/ ⁇ c uvs promoter.
  • the resulting plasmid RSFP /ac -pzwf (Fig 5) was isolated from 4 independent plasmid clones and electro-transformed to the SC17(0) ⁇ zwf ⁇ pgi ⁇ gcd strain, and then plated on glucose minimal medium.
  • the SC17(0) ⁇ zwf ⁇ pgi ⁇ gcd and SC17(0) ⁇ pgi ⁇ gcd strains were used as controls.
  • Glucose-6P dehydrogenase activities were determined in crude extracts of all 4 transformants (assay is described in the Example section below). As it is shown in Table 8, the plasmid isolated from clone 1 resulted in undetectable levels of Zwf (glucose-6P dehydrogenase) activity, while the plasmid isolated from clones 4, 5 and 7 carried a copy of the plasmid zwf gene encoding glucose-6P dehydrogenase with double NAD/NADP co factor specificity. Moreover, the level of NAD-dependent activity was significantly higher than that of NADP-dependent activity in these clones.
  • the pgnd gene was cloned into the RSFPlaclacI plasmid under the control of the P /flC uvs promoter using the primers pgnd-5 (SEQ ID NO: 100) and pgnd-3 (SEQ ID NO: 101) for PCR-amplification of its coding part.
  • Primer pgnd-5 (SEQ ID NO: 100) contains the site formal restrictase at the5'-end thereof.
  • Primer pgnd-3 (SEQ ID NO: 101) contains the site for BamHl restrictase at the5'-end thereof.
  • the DNA fragment obtained by PCR was treated with Xbal and BamHl restrictases and cloned into Xbal-BamHl sites of the RSF-based vector RSFPlaclacI.
  • the resulting plasmid RSFPlac-pgnd is depictured in Fig. 5.
  • Example 7 Gluconic acid accumulation in aspartic acid fermentation process. Further improvement of aspartic acid production due to inactivation of glucose dehydrogenase.
  • gluconic acid is formed from glucose via a glucose dehydrogenase reaction.
  • the gcd gene coding for glucose dehydrogenase was deleted in the 5 ⁇ P2R strain according to the above-described ⁇ Red-dependent procedure using oligonucleotides gcd-attR (SEQ ID NO: 49) and gcd-attL (SEQ ID NO: 50) as primers for generation of the integrative DNA fragment; oligonucleotides gcd-testl (SEQ ID NO: 51) and gcd-test2 (SEQ ID NO: 52) were used for PCR verification of the obtained integrants.
  • the resulting strain 5 ⁇ P2RG was electroporated with pMWgdhA and checked in test tube cultivation. Inactivation of glucose dehydrogenase led to an increase of aspartic acid accumulation by 3.3 times for the plasmid-less strains and by 1.4 times for the plasmid strains (Table 10). Table 10. Effects of the ⁇ gcd mutation on aspartic acid production by P. ananatis. 72- hour cultivation; initial glucose concentration 40g/L, initial L-GIu concentration 3g/L. Average data for 3 independent cultures are represented.
  • Example 8 Further improvement by amplification of the pzwfor psnd gene.
  • the RSFP lac-pzwf and RSFPlac-pgnd plasmids were introduced into the 5 ⁇ P2- 36S strain by the electroporation procedure (see above).
  • the resulting plasmid strains gave significantly increased aspartic acid accumulation in comparison with the plasmid-less parental strain.
  • Amplification of the/?zw/gene led to a 2.6-fold increase of the product accumulation, amplification of the pgm/ gene gave a 1.4-fold increase of aspartic acid accumulation in test tube cultivation (Table 11).
  • Example 9 Further improvement of aspartic acid production by inactivation of malate dehydrogenase.
  • the mdhA gene coding for malate dehydrogenase was deleted in the 5 ⁇ P2R strain according to the above-described ⁇ Red-dependent procedure using oligonucleotides mdhA-attR (SEQ ID NO: 88) and mdhA-attL (SEQ ID NO: 89) as primers for generation of the integrative DNA fragment; oligonucleotides mdhA-testl (SEQ ID NO: 90) and mdhA-test2 (SEQ ID NO: 91) were used for PCR verification of the obtained integrants.
  • the resulting strain 5 ⁇ P2RM was cured from the kanamycin resistance marker using the above-described ⁇ lnt/Xis-dependent procedure.
  • the pMWgdhA plasmid was introduced into the marker-less derivative of 5 ⁇ P2RM and to 5 ⁇ P2R.
  • the results of the experiment on test tube cultivation of the obtained strains represented in Table 12 show a significant positive effect of the AmdhA mutation on aspartic acid accumulation and yield for the both plasmid and plasmid-less variants.
  • RSFPlaclacI (Fig. 6) is a derivative of the RSFPlaclacIsacBcat plasmid previously described in Russian patent application (RU 2006134574).
  • the RSFPlaclacIsacBcat plasmid which is also named pRSFsacB, has been disclosed by Katashkina, J.I. et al. (Use of the ⁇ Red-recombineering method for genetic engineering of Pantoea ananatis. BMC Molecular biology 2009, 10:34).
  • To construct this plasmid Red-dependent integration of the generated in vitro short DNA fragment, which includes a polylinker site into the RSFPlacIsacBcat plasmid, was performed.
  • the oligonucleotides DeI-F (SEQ ID NO: 102) and DeI-R (SEQ ID NO: 103) 200 ng of each were annealed and extended in the polymerase chain reaction mixture prepared from the reagents provided by "Fermentas" accordingly to the manufacturer's instructions (temperature profile was 95°C for 2min, 50°C for 1 min, 72°C for 2 min).
  • the probe was precipitated with ethanol, washed with 70% ethanol twice and dissolved in 10 ⁇ l of fresh de-ionized water.
  • the MG1655/pKD46 strain was used as a recipient strain for Red-dependent integration.
  • Preparation of the electrocompetent cells was as described by Datsenko, K. A. and Wanner, B.L. (Proc. Natl. Acad. Sci. USA, 97, 12, p 6640-6645 (2000)).
  • 200 ng of the prepared DNA fragment and 100 ng of the RSFPlaclacIsacBcat plasmid were used for electroporation.
  • Electroporation was performed using a Gene Pulser apparatus (BioRad, USA, version 2-89). Pulse time was 5 msec, and electric field strength was 12.5 kV/cm. 1 ml of SOC medium was added to the cell suspension immediately after electroporation.
  • the cells were cultivated at 37°C for 2 hours, spread on LB agar containing 50 ⁇ g/ml of chloramphenicol, 5% sucrose, and 1 mM IPTG and cultivated at 37°C overnight.
  • the integrative DNA fragment contained flanks homologous to the upstream and downstream regions of the sacB gene coding for levansucrase. Expression of this gene is highly toxic for many bacterial species and is routinely used as a contra-selective marker (Gay, P. et al., J. Bacterid., 164, 918-921 (1985)).
  • Cm R Suc R clones Fifteen Cm R Suc R clones were selected in this experiment. Plasmid DNA was isolated from these clones, transformed to the E. coli TGl strain and re-isolated from it for the restriction analysis. After digestion of the isolated DNA probes with the Hindlll, Sphl, Pstl, Sail, BamHI, BgIII, Pvul, and Xbal restriction endonucleases, the clone containing the polylinker site of the expected structure was selected.
  • a strain in which the ppc gene is deleted was constructed by the method initially developed by Datsenko, K.A. and Wanner, BX. (Proc. Natl. Acad. Sci. USA, 2000, 97(12), 6640-6645) called "Red-driven integration".
  • the DNA fragment containing the Cm R marker encoded by the cat gene was obtained by PCR, using primers pps-attR (SEQ ID NO: 111) and ppc-attL (SEQ ID NO: 112), and plasmid pMWl 18-attL-Cm-attR as a template (WO2005010175).
  • Primer pps-attR contains both a region complementary to the region located at the 5' end of the ppc gene and a region complementary to the attR region. Primer pps-attR contains both a region complementary to the region located at the 3' end of the ppc gene and a region complementary to the attL region.
  • a 1,7 kbp PCR product was obtained and purified in agarose gel, and was used for electroporation of the E. coli strain MG 1655 (ATCC 700926), which contains the plasmid pKD46 having a temperature-sensitive replication.
  • the plasmid pKD46 (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 ⁇ (nucleotide positions 31088 to 33241, GenBank accession no.
  • J02459 contains genes of the ⁇ Red homologous recombination system ( ⁇ , ⁇ , exo genes) under the control of the arabinose-inducible P araB promoter.
  • the plasmid pKD46 is necessary for integration of the PCR product into the chromosome of strain MG 1655.
  • Electrocompetent cells were prepared as follows: E. coli MG1655/pKD46 was grown overnight at 30 °C in LB medium containing ampicillin (100 mg/1), 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 OD OOO of «0.6, and then were made electrocompetent by concentrating 100-fold and washing three times with ice-cold deionized H 2 O. Electroporation was performed using 70 ⁇ l of cells and «100 ng of the PCR product.
  • the mutants having the ppc gene deleted and marked with the Cm resistance gene were verified by PCR.
  • Locus-specific checking primers ppc-testl (SEQ ID NO: 112) and ppc-test2 (SEQ ID NO: 113) were used in PCR for the verification.
  • the PCR product obtained in the reaction with the cells of parental ppc + strain MG1655 as a template was 2,8 kbp in length.
  • the PCR product obtained in the reaction with the cells of mutant strain as the template was 1.7 kbp in length.
  • the mutant strain was named MG1655 ⁇ ppc::cat.
  • Cm marker was excised from the chromosome using standard techniques (int-xis system) described in WO2005010175. Length of the DNA obtained in the PCR using primers ppc-testl and ppc-test2 in the case when the Cm marker is excised was 280 bp.
  • the reaction mixture contained 10OmM TRIS HCl (pH7.5), 1OmM L- Asp (pH7.0), 5mM ⁇ -ketoglutarate (pH7.0), 0,ImM pyridoxal phosphate. Oxaloacetate formation was detected by measuring the absorption at 265 nm.
  • Cells were collected by centrifugation, washed by 5OmM potassium phosphate buffer pH7.5, and disrupted by sonication. The activity determination was conducted in the coupled reaction with malate dehydrogenase.
  • the reaction mixture included: 10OmM TRIS HCl (pH7.5), 5mM PEP, 4mM DTTE, 5mM MnSO 4 , 0.15mM NADH, 10OmM NaHCO 3 , O.lmM Acetyl-CoA, 2u/ml malate dehydrogenase. The change in NADH extinction at 340 nm was measured. The reactions without PEP or Acetyl-CoA were used as controls.
  • the reaction mixture contained 5OmM TRIS-HCl (pH 8.0), 4mM NAD + /NADP + , 2mM MgCl 2 and 4mM 6P-gluconate. The change in NADH extinction at 340 nm was measured. The average data for two independent cultures are shown in each case.

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Abstract

The present invention provides a bacterium belonging to the genus Pantoea which has been modified to have decreased activity of α-ketoglutarate dehydrogenase; decreased activity of citrate synthase; increased activity of phosphoenolpyruvate carboxylase or pyruvate carboxylase; and increased activity of glutamate dehydrogenase or glutamate synthase. This bacterium has the ability to produce L-aspartic acid or L-aspartic acid-derived metabolites, such as L-threonine, L-lysine, L-methionine and L-homoserine.The present invention also provides a method for producing L-aspartic acid or L-aspartic acid-derived metabolites using the bacterium.

Description

DESCRIPTION
A BACTERIUM BELONGING TO THE GENUS Pantoea PRODUCING AN L- ASPARTIC ACID OR L-ASPARTIC ACID-DERIVED METABOLITES AND A METHOD FOR PRODUCING L-ASPARTIC ACID OR L-ASPARTIC ACID- DERIVED METABOLITES
Technical field
The present invention relates to the microbiological industry, and specifically to a method for producing an L-aspartic acid or L-aspartic acid-derived metabolites using a bacterium belongiung to the genus Pantoea which has been modified to have attenuated expression of genes coding for α-ketoglutarate dehydrogenase and citrate synthase, as well as enhanced expression of genes coding for phosphoenolpyruvate carboxylase and glutamate dehydrogenase, or glutamate synthase.
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 (U.S. Patent 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 to feedback inhibition by the resulting L-amino acid (U.S. Patent Nos. 4,346,170, 5,661,012 and 6,040,160).
L-aspartic acid is a useful metabolite having different applications (aspartame sweetener, biodegradable polymers, etc.). In the currently used industrial process, aspartic acid is produced enzymatically from fumarate using aspartase. In turn, fumarate can be obtained by chemical synthesis, as a product of oil distillation or from hydrocarbons in bacterial fermentation process. The three-step production process includes the steps of 1) production of calcium-fumarate by fermentation of a bacterial strain; 2) conversion of the calcium- fumarate to diammonium fumarate by addition of ammonia, ammonium carbonate, or ammonia in combination with CO2; 3) enzymatic conversion of the obtained diammonium fumarate to ammonium aspartate by aspartase. This process has been previously described (US patent 6,071,728).
Direct methods for producing L-aspartic acid from hydrocarbons in bacterial fermentation have been previously developed on the basis of bacteria belonging to the genus Corynebacterium or Brevibacterium (US Patent 4,000,040). Some wild-type strains of these genera are the natural producers of L-aspartic acid. These inventions relate to the process of selecting the induced mutants with increased producing ability.
As for the genetic factors that could influence L-aspartic acid production, the metabolic pathways providing its synthesis and degradation in Escherichia coli and some other Enterobacteriaceae are well-studied. It is known that L-aspartic acid is synthesized in the aspartate aminotransferase reaction. It's keto-precursor is oxaloacetate, the intermediate of TCA (tricarboxylic acid cycle and glyoxylate bypass, reviewed in Neidhardt, F. C. (Ed. in Chief), R. Curtiss III, J.L. Ingraham, E.C.C. Lin, K.B. Low, B. Magasanik, W.S. Reznikoff, M. Riley, M. Schaechter, and H.E. Umbarger (eds). 1996. Escherichia coli and Salmonella: Cellular and Molecular Biology. American Society for Microbiology); L-glutamic acid serves as a donor of amides in this reaction.
The carbon flux to oxaloacetate in E. coli grown on glucose can be increased by increasing phosphoenol pyrvate (PEP)-carboxylase activity, in particular, due to expressing feedback resistant PEP-carboxylase enzymes (European Patent EP072301 IBl) or by introducing heterologous pyruvate (Pyr) carboxylases (US Patent 6,455,284). In addition, inactivation of oxaloacetate or malate consuming enzymes, such as PEP carboxykinase encoded by the pckA gene or malic enzymes encoded by the sfcA and maeB genes, is important for the production of aspartic acid-derived metabolites (WO2007/017710).
Some proposals concerning the possible genetic factors that could influence production of L-aspartic acid by Actinobacillus succinogenes were made in the patent application WO2007019301; in particular, an increase of aspartate aminotransferase activity was suggested as an important factor of aspartic acid production. In addition, inactivation of PEP-utilizing enzymes, such as Pyr kinases, Pyr dehydrogenase or Pyr- formate lyase was described as the possible factors for increasing aspartic acid production.
Moreover, some increase in production of several aspartic acid-derived products, but not aspartic acid, by E. coli strains with increased activities of the glyoxylate shunt, which is known to enhance aspartic acid production, has been disclosed (WO 2007/017710 Al).
Inactivation of α-ketoglutarate dehydrogenase, increase of PEP-carboxylase, citrate synthase, and glutamate dehydrogenase activities, and selection of the mutations providing high resistance against glutamate as essential factors for L-glutamic acid production by Pantoea ananatis have been disclosed (US patent application 2002004231).
The positive effect of the inactivation of aspartase on the production of threonine, the aspartic acid-derived amino acid, has been shown (US patent application 2004115780).
The decrease of citrate synthase activity was established as a factor for increasing production of L-lysine, an aspartic acid-derived amino acid, by C. glutamicum (Ozaki H., Shiio J. Production of lysine by pyruvate kinase mutants of Brevibacteriumflavum. Agr. and Biol. Chem., Vol.47(7):1569-1576 (1983)).
But currently, there have been no reports of using a bacterium belongiung to the genus Pantoea which has been modified to have attenuated expression of genes coding for 2-oxoglutarate dehydrogenase and citrate synthase; enhanced expression of genes coding for phosphoenolpyruvate carboxylase and glutamate dehydrogenase, or pyruvate kinase.
Summary of the Invention
Aspects of the present invention include enhancing the productivity of L- aspartic acid-producing strains and providing a method for producing L-aspartic acid using these strains.
The above aspects were achieved by finding that construction of the bacterium belonging to the genus Pantoea modified to have decreased activity of α-ketoglutarate dehydrogenase; decreased activity of citrate synthase; increased activity of phosphoenolpyruvate carboxylase or pyruvate carboxylase; and increased activity of glutamate dehydrogenase or glutamate synthase can enhance production of L-aspartic acid.
The present invention provides a bacterium belonging to the genus Pantoea having an increased ability to produce L-aspartic acid or L-aspartic acid-derived metabolite and a method for producing L-aspartic acid or L-aspartic acid-derived metabolite using the bacterium.
It is an aspect of the present invention to provide a bacterium belonging to the genus Pantoea which is able to produce L-aspartic acid or an L-aspartic acid derived- metabolite, wherein said bacterium has been modified to have:
- decreased activity of α-ketoglutarate dehydrogenase;
- decreased activity of citrate synthase;
- increased activity of phosphoenolpyruvate carboxylase or pyruvate carboxylase; and
- increased activity of glutamate dehydrogenase or glutamate synthase.
It is a further aspect of the present invention to provide the bacterium as described above, wherein the activities of α-ketoglutarate dehydrogenase and citrate synthase are decreased by attenuation of expression of the genes coding for α- ketoglutarate dehydrogenase and citrate synthase.
It is a further aspect of the present invention to provide the bacterium as described above, wherein said bacterium has been further modified to have attenuated expression of a gene coding for aspartate ammonia-lyase (aspartase).
It is a further aspect of the present invention to provide the bacterium as described above, wherein said bacterium has been further modified to have attenuated expression of a gene coding for pyruvate kinase.
It is a further aspect of the present invention to provide the bacterium as described above, wherein said bacterium has been further modified to have attenuated expression of a gene coding for glucose dehydrogenase.
It is a further aspect of the present invention to provide the bacterium as described above, wherein said bacterium has been further modified to have attenuated expression of a gene coding for malate dehydrogenase. It is a further aspect of the present invention to provide the bacterium as described above, wherein said expression of the gene coding for α-ketoglutarate dehydrogenase, citrate synthase, aspartate ammonia-lyase (aspartase), pyruvate kinase, glucose dehydrogenase or malate dehydrogenase is attenuated by inactivating said gene in the chromosome of the bacterium.
It is a further aspect of the present invention to provide the bacterium as described above, wherein said activity of phosphoenolpyruvate carboxylase or pyruvate carboxylase is increased by enhancing the expression of the gene coding for phosphoenolpyruvate carboxylase or pyruvate carboxylase; and activity of glutamate dehydrogenase or glutamate synthase is increased by enhancing the expression of the gene coding for glutamate dehydrogenase or glutamate synthase .
It is a further aspect of the present invention to provide the bacterium as described above, wherein said expression is enhanced by modifying an expression control sequence of said gene or by increasing the copy number of said gene.
It is a further aspect of the present invention to provide the bacterium as described above, wherein said genes coding for phosphoenolpyruvate carboxylase and glutamate dehydrogenase are derived from Escherichia coli.
It is a further aspect of the present invention to provide the bacterium as described above, wherein the gene coding for glutamate dehydrogenase codes for NAD-dependent glutamate dehydrogenase derived from Pantoea ananatis.
It is a further aspect of the present invention to provide the bacterium as described above, wherein the gene coding pyruvate carboxylase is derived from Sinorhizobium meliloti..
It is a further aspect of the present invention to provide the bacterium as described above, wherein said bacterium is Pantoea ananatis.
It is a further aspect of the present invention to provide the bacterium as described above, wherein the L-aspartic acid-derived metabolite is selected from the group consisting of L-threonine, L-lysine, L-methionine, and L-homoserine.
It is a further aspect of the present invention to provide a method for producing an L-aspartic acid or an L-aspartic acid-derived metabolite comprising:
- cultivating the bacterium as described above in a culture medium, and - collecting the L-aspartic acid or L-aspartic acid-derived metablite from the medium.
It is a further aspect of the present invention to provide the method as described above, wherein the L-aspartic acid-derived metabolite is selected from the group consisting of L-threonine, L-lysine, L-methionine, and L-homoserine.
Brief description of the drawings
Figure 1 shows the construction of the pMW-EαspC plasmid.
Figure 2 shows the construction of the pMIVK620S and pMIV-Ptac-pycA plasmids.
Figure 3 is a photograph which shows accumulation of by-products by the 3Δ, 4Δ (ApykA) and 5Δ strains in test-tube cultivation.
Figure 4 shows growth curves of ΔpgiΔzwf, ΔpgiΔgcd, ΔzwfΔgcd and ΔpgiΔzwfΔgcd strains in glucose minimal medium
Figure 5 shows construction of the RSFPlac-pzwf and RSFPlac-pgnd plasmids.
Figure 6 shows construction of the RSFPlaclacI plasmid vector.
Figure 7 shows construction of the pMW-intxis-cat plasmid vector
Figure 8 shows construction of the phMIV-1 helper plasmid.
Detailed description of the preferred embodiments 1. Bacterium
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 were recently re-classified into Pantoea agglomerans, Pantoea ananatis, Pantoea stewartii or the like, based on a nucleotide sequence analysis of the 16S rRNA, etc. (International Journal of Systematic Bacteriology, July 1989, 39(3).p.337-345). Furthermore, some bacteria belonging to the genus Erwinia were re-classified as Pantoea ananatis or Pantoea stewartii (International Journal of Systematic Bacteriology, Jan. 1993, 43(1), pp.162-173). Typical strains of the Pantoea bacteria include, but are not limited to, Pantoea ananatis, Pantoea stewartii, Pantoea agglomerans, and Pantoea citrea. Specific examples include the following strains: Pantoea ananatis AJ 13355 (FERM BP-6614, European Patent Publication No. 0952221), Pantoea ananatis AJ13356 (FERM BP-6615, European Patent Publication No. 0952221), Pantoea ananatis AJ 13601 (FERM BP-7207, European Patent Publication No. 0952221). An exemplary λ-Red resistant strain is Pantoea ananatis SCl 7(0) (VKPM B-9246, RU application 2006134574, WO2008/075483).
The phrase "bacterium producing L-aspartic acid or L-aspartic acid-derived metabolite" can refer to the abilities to produce L-aspartic acid and/or one or more L- aspartic acid-derived metabolites, and to cause accumulation of L-aspartic acid or L- aspartic acid-derived metabolite(s) in a medium or cells of the bacterium to such a degree that L-aspartic acid or L-aspartic acid-derived metabolite can be collected from the medium or cells when the bacterium is cultured in the medium. Alternatively, the phrase "bacterium producing L-aspartic acid or L-aspartic acid-derived metabolite" can mean a bacterium which is able to cause accumulation of a target L-amino acid in the culture medium in an amount larger than the wild-type or parental strain, and can also mean that the microorganism is able to cause accumulation in the medium of an amount not less than 0.4 g/L, and in another example not less than 1.0 g/L of the target L-amino acid. The ability to produce L-aspartic acid or L-aspartic acid-derived metabolites in the bacterium can be a native or inherent ability, or can be imparted by modifying the bacterium using mutagenesis or recombinant DNA techniques.
The term "L-aspartic acid" refers to an L-aspartic acid or to any salt thereof, and can be called aspartic acid. The L-aspartic acid-derived metabolites include L- threonine, L-lysine, L-methionine, and L-homoserine.
A minimal combination of the factors which cause L-aspartic acid production in a bacterium belonging to the genus Pantoea, for example, P. ananatis, is described. The combination includes the following modifications:
- decreasing activity of α-ketoglutarate dehydrogenase;
- decreasing activity of citrate synthase;
- increasing activity of phosphoenolpyruvate carboxylase or pyruvate carboxylase; and
- increasing activity of glutamate dehydrogenase or glutamate synthase.
The expression of the gene coding for aspartate ammonia-lyase (aspartase) can also be attenuated. The activity of α-ketoglutarate dehydrogenase (synonyms include SucA, 2- oxoglutarate dehydrogenase) can mean catalyzing the conversion of 2-ketoglutarate into succinyl-CoA, and production of NADH and CO2 in an irreversible reaction within the 2-oxoglutarate dehydrogenase complex [(SucA) i2] [(SuCB)24] [(Lpd)2]. The nucleotide sequence of the sucA gene and the amino acid sequence of SucA protein from P. ananatis are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
The activity of citrate synthase (synonyms: GItA, citrogenase, oxalaoacetate transacetylase) can mean catalyzing the following reaction: oxaloacetate + acetyl-CoA + H2O <=> citrate + coenzyme A.
The nucleotide sequence of the git A gene and the amino acid sequence of GItA protein from P. ananatis are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.
The activity of phosphoenolpyruvate carboxylase (synonyms include Ppc, orthophosphate:oxaloacetate carboxy-lyase (phosphorylating)) can mean catalyzing the following reaction: oxaloacetate + phosphate <=> phosphoenolpyruvate + H2O + CO2.
The ppc gene from E. coli is known (nucleotides complementary to nucleotides in positions from 4,148,470 to 4,151,121; GenBank accession no. NC_000913.2; gi: 49175990). The ppc gene is located between the yijP and the argE genes on the chromosome of E. coli K- 12. The nucleotide sequence of the ppc gene and the amino acid sequence of Ppc encoded by the ppc gene from E. coli are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.
The activity of pyruvate carboxylase (synonyms: Pyc, PycA) can mean catalyzing the following reaction:
HCO3 " + pyruvate + ATP <=> phosphate + oxaloacetate + ADP.
It is known that pyruvate carboxylase from Sinorhizobium meliloti is resistant to feedback inhibition by L-aspartic acid (Dunn, M.F. et al, Cloning and characterization of the pyruvate carboxylase from Sinorhizobium meliloti Rm 10211, Arch. Microbiol., 176, 355-363 (2001)). Therefore, the pycA gene from S. meliloti was cloned and integrated into an L-aspartic acid producing P. ananatis strain. The nucleotide sequence of the pycA gene and the amino acid sequence of PycA encoded by the pycA gene from S. meliloti are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively. The activity of glutamate dehydrogenase (synonym: GdhA) can mean catalyzing the NADPH-dependent animation of alpha-ketoglutarate to yield L- glutamate:
L-glutamate + H2O + NADP+ <=> 2-ketoglutarate + ammonia + NADPH.
The gdhA gene from E. coli is known (nucleotides in positions from 1,840,395 to 1,841,738; GenBank accession no. NC 000913.2; gi: 49175990). The gdhA gene is located between the ynjH and the ynjl ORPs on the chromosome of E. coli K- 12. The nucleotide sequence of the gdhA gene and the amino acid sequence of GdhA encoded by the gdhA gene from E. coli are shown in SΕQ ID NO: 9 and SΕQ ID NO: 10, respectively.
The activity of glutamate synthase can mean catalyzing the single-step conversion of L-glutamine and alpha-ketoglutarate into two molecules of L-glutamate:
L-glutamine + 2-ketoglutarate + NADPH <=> 2 L-glutamate + NADP+.
The glutamate synthase from E. coli includes two subunits encoded by the gltB and gltD genes organized into one operon. The gltB and gltD genes from E. coli are known (nucleotides in positions from 3,352,654 to 3,357,207 and 3,357,220 to 3,358,638, respectively; GenBank accession no. NC 000913.2; gi: 49175990). The gltBD operon is located between the yhcC ORP and the gltF gene on the chromosome of E. coli K- 12. The nucleotide sequences of the gltB and gltD genes and the amino acid sequences of GltB and GltD encoded by the gltB and gltD genes from E. coli are shown in SΕQ ID NO: 11, SΕQ ID NO: 12, SΕQ ID NO: 13, and SΕQ ID NO: 14, respectively.
The activity of aspartate ammonia-lyase (synonyms: Asp A, aspartase) can mean catalyzing the reversible conversion of L-aspartic acid to fumaric acid and ammonia:
L-aspartic acid <=> fumaric acid + ammonia.
The nucleotide sequence of the asp A gene and the amino acid sequence of AspA protein from P. ananatis are shown in SΕQ ID NO: 15 and SΕQ ID NO: 16, respectively.
The activity of pyruvate kinase can mean catalyzing the following reversible reaction: pyruvate + ATP <=> ADP + phosphoenolpyruvate. P. ananatis has two isozymes of pyruvate kinase: PykA and PykF. The nucleotide sequence of the pykA gene and the amino acid sequence of PykA protein from P. ananatis are shown in SEQ ID NO: 114 and SEQ ID NO: 115, respectively. The nucleotide sequence of the pykF gene and the amino acid sequences of PykF protein from P. ananatis are shown in SEQ ID NO: 116 and SEQ ID NO: 117, respectively.
The activity of glucose dehydrogenase (synonyms: Gcd, D- glucose:(pyrroloquinoline-quinone) 1-oxidoreductase, PQQGDH) can mean catalyzing the following reaction: β-D-glucose + ubiquinone (UQ) <=> glucono-δ-lactone + UQH2.
The nucleotide sequence of the gcd gene and the amino acid sequence of the Gcd protein from P. ananatis are shown in SEQ ID NO: 17 and SEQ ID NO: 18, respectively.
The activity of malate dehydrogenase (synonyms: Mdh, malate oxidoreductase , L-malate oxidoreductase , L-malate:NAD(+) oxidoreductase) can mean catalyzing the following reaction: malate + NAD+ <=> oxaloacetate + NADH.
The nucleotide sequence of the mdh gene and the amino acid sequence of Mdh protein from P. ananatis are shown in SEQ ID NO: 19 and SEQ ID NO: 20, respectively.
The phrase "bacterium has been modified to have attenuated expression of the gene" can mean that the bacterium has been modified in such a way that the modified bacterium can contain a reduced amount of the protein encoded by the gene as compared with an unmodified bacterium, or is unable to synthesize the protein encoded by the gene.
The phrase "inactivation of the gene" can mean 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 sue A, git A, asp A, gcd, or mdh 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 amount 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.
Expression of the gene can be attenuated by introducing a mutation into the gene on the chromosome so that the intracellular amount of the protein encoded by the gene is decreased as compared to an unmodified strain. Such a mutation can be the introduction of 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 gene 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 can be employed to introduce a mutation by gene recombination. A mutant gene can be prepared, and the bacterium to be modified can be transformed with a DNA fragment containing the mutant gene. Then, the native gene on the chromosome can be replaced with the mutant gene by homologous recombination, and the resulting strain can be selected. Such gene replacement by homologous recombination can be conducted by employing a linear DNA, which is known as "Red-driven integration" (Datsenko, K.A. and Wanner, B. L., Proc. Natl. Acad. Sci. USA, 97, 12, p 6640-6645 (2000)), or by methods employing a plasmid containing a temperature-sensitive replication (U.S. Patent 6,303,383 or JP 05- 007491 A). An exemplary strain is the Pantoea ananatis strain which has been modified to be resistant to the product of the λ-Red genes. Examples include but are not limited to the Pantoea ananatis strain SC 17(0) (VKPM B-9246, RU application 2006134574, WO2008/075483). 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 lacking the ability 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. Patent 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 descriptions regarding variant proteins, inactivation of genes, and other methods can be applied to other proteins, genes, and in the breeding of bacteria described below.
The phrase "enhancing the expression of the gene" means that the expression of the gene is higher than that of a non-modified strain, for example, a wild-type strain. Examples of such modification include increasing the copy number of the target gene per cell, increasing the expression level of the gene, and so forth. The quantity of the copy number of the target gene is 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 measured by various known methods including Northern blotting, quantitative RT-PCR, and the like. Furthermore, wild-type strains that can act as a control include, for example, Pantoea ananatis FERM BP-6614.
Since there may be some differences in DNA sequences between the genera or strains, the ppc, pycA, gdhA, or gltBD gene to be modified to enhance its expression is not limited to the genes shown in SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:11, respectively, but can include genes homologous to SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:11, respectively. Therefore, the protein variant encoded by the ppc, pycA, gdhA ox gltBD gene can have a homology of not less than 80%, in another example not less than 90%, and in another example not less than 95 %, with respect to the entire amino acid sequence shown in SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, or SEQ ID NO:12, respectively, as long as the activity of the corresponding protein is maintained. The phrase "protein variant" means proteins which have changes in the sequences, whether they 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. It can be 1 to 30, in another example 1 to 15, and in another example 1 to 5 in SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, or SEQ ID NO: 12, respectively. These changes in the variants 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.
Homology between two amino acid sequences can be determined using the well-known methods, for example, the computer program BLAST 2.0, which calculates three parameters: score, identity and similarity.
The substitution, deletion, insertion or addition of one or several amino acid residues can be conservative mutation(s) so that the activity is maintained. The representative conservative mutation can be a conservative substitution. Examples of conservative substitutions include substitution of Ser or Thr for Ala, substitution of GIn, His or Lys for Arg, substitution of GIu, GIn, Lys, His or Asp for Asn, substitution of Asn, GIu or GIn for Asp, substitution of Ser or Ala for Cys, substitution of Asn, GIu, Lys, His, Asp or Arg for GIn, substitution of Asn, GIn, Lys or Asp for GIu, substitution of Pro for GIy, substitution of Asn, Lys, GIn, Arg or Tyr for His, substitution of Leu, Met, VaI or Phe for He, substitution of He, Met, VaI or Phe for Leu, substitution of Asn, GIu, GIn, His or Arg for Lys, substitution of He, Leu, VaI or Phe for Met, substitution of Trp, Tyr, Met, He 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, He or Leu for VaI.
Therefore, the ppc, pycA, gdhA or git BD gene can be a variant which hybridizes under stringent conditions with the nucleotide sequence complementary to the sequence shown in SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:11, respectively, or a probe which can be prepared from the nucleotide sequence, provided that it encodes a functional protein. "Stringent conditions" include those under which a specific hybrid, for example, a hybrid having homology of not less than 60%, in another example not less than 70%, in another example not less than 80%, in another example not less than 90%, and and in another example 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, and in another example two or three times, at a salt concentration of 1 xSSC, 0.1% SDS, and in another example 0.1 x SSC, 0.1% SDS at 600C. 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 Hybond™ N+ nylon membrane (Amersham) under stringent conditions is 15 minutes. Preferably, washing can be performed 2 to 3 times. The length of the probe can be suitably selected depending on the hybridization conditions, and is usually 100 bp to 1 kbp.
Methods of gene expression enhancement include increasing the gene copy number. Introducing a gene into a vector that is able to function in a bacterium belonging to the genus Pantoea increases the copy number of the gene. Examples of a plasmid which can be transformed include plasmids which are autonomously replicable in bacteria belonging to the genus Pantoea, for example, pUC19, pUC18, pBR322, RSFlOlO, pHSG299, pHSG298, pHSG399, pHSG398, pSTV28, pSTV29 (pHSG and pSTV can be obtained from Takara Bio), pMWl 19, pMWll[delta], pMW219, pMW218 (plasmids of pMW series can be obtained from Nippon Gene), and so forth. Phage DNA can also be used as a vector, instead of a plasmid.
Gene expression can also be enhanced by introducing multiple copies of the gene into the bacterial chromosome by, for example, a method of homologous recombination, Mu integration, or the like. For example, one act of Mu integration allows for the introduction of up to 3 copies of the gene into the bacterial chromosome.
Increasing the copy number of a gene can also be achieved by introducing multiple copies of the gene into the chromosomal DNA of the bacterium. In order to introduce multiple copies of the gene into a bacterial chromosome, homologous recombination is carried out using a sequence having multiple copies of the gene, and these multiple copies function as targets in the chromosomal DNA. Sequences having multiple copies in the chromosomal DNA include, but are not limited to repetitive DNA, or inverted repeats present at the end of a transposable element. Also it is possible to incorporate the gene into a transposon, and allow it to be transferred to introduce multiple copies of the gene into the chromosomal DNA.
Gene expression can also be enhanced by placing the DNA under the control of a potent promoter. For example, the lac promoter, the trp promoter, the trc promoter, the PR, or the PL promoters of lambda phage are all known to be potent promoters. The use of a potent promoter can be combined with multiplication of gene copies.
Alternatively, the effect of a promoter can be enhanced by, for example, introducing a mutation into the promoter to increase the transcription level of a gene located downstream of the promoter. Furthermore, it is known that substitution of several nucleotides in the spacer between the ribosome binding site (RBS) and the start codon, especially the sequences immediately upstream of the start codon, profoundly affect the translation of the mRNA. For example, a 20-fold range in the expression levels was found, depending on the nature of the three nucleotides preceding the start codon (Gold et al, Annu. Rev. Microbiol., 35, 365-403, 1981; Hui et al, EMBO J., 3, 623-629, 1984).
Moreover, it is also possible to introduce a nucleotide substitution into a promoter region of a gene on the bacterial chromosome, which results in a stronger promoter function. The alteration of the expression control sequence can be performed, for example, in the same manner as the gene substitution using a temperature-sensitive plasmid, as disclosed in WO 00/18935 and JP 1-215280 A.
Methods for preparation of plasmid DNA, digestion and ligation of DNA, transformation, selection of an oligonucleotide as a primer and the like are 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).
Bacteria producing L-aspartic acid or L-aspartic acid-derived metabolites Bacteria which are able to produce L-aspartic acid or L-aspartic acid-derived metabolites include a bacterium which has been modified to have decreased activity of α-ketoglutarate dehydrogenase; decreased activity of citrate synthase; increased activity of phosphoenolpyruvate carboxylase or pyruvate carboxylase; and increased activity of glutamate dehydrogenase or glutamate synthase. Bacteria can be further modified to have attenuated expression of the gene coding for aspartate ammonia-lyase (aspartase).
L-threonine-producing bacteria
Examples of parent strains which can be used to derive L-threonine-producing bacteria include strains in which expression of one or more genes encoding an L- threonine biosynthetic enzyme are enhanced. In one example, the bacterium is modified to enhance expression of one or more of the following genes:
- the mutant thrA gene which codes for aspartokinase homoserine dehydrogenase I resistant to feed back inhibition by threonine;
- the thrB gene which codes for homoserine kinase;
- the thrC gene which codes for threonine synthase;
- the rhtA gene which codes for a putative transmembrane protein;
- the asd gene which codes for aspartate-β-semialdehyde dehydrogenase; and
- the aspC gene which codes for aspartate aminotransferase (aspartate transaminase).
Genes from Escherichia coli bacteria can be used. The thrA gene which encodes aspartokinase homoserine dehydrogenase I of Escherichia coli has been elucidated (nucleotide positions 337 to 2799, GenBank accession no.NC_000913.2, gi: 49175990). The thrA gene is located between the thrL and thrB genes on the chromosome of E. coli K- 12. The thrB gene which encodes homoserine kinase of Escherichia coli has been elucidated (nucleotide positions 2801 to 3733, GenBank accession no.NC_000913.2, gi: 49175990). The thrB gene is located between the thrA and thrC genes on the chromosome of E. coli K- 12. The thrC gene which encodes threonine synthase of Escherichia coli has been elucidated (nucleotide positions 3734 to 5020, GenBank accession no.NC_000913.2, gi: 49175990). The thrC gene is located between the thrB gene and the yaaX open reading frame on the chromosome of E. coli K- 12. All three genes function as a single threonine operon. To enhance expression of the threonine operon, the attenuator region which affects the transcription is desirably removed from the operon (WO2005/049808, WO2003/097839).
A mutant thrA gene which codes for aspartokinase homoserine dehydrogenase I and is resistant to feedback inhibition by 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 threonine producing E. coli strain VKPM B-3996. Plasmid pVIC40 is described in detail in U.S. Patent No. 5,705,371.
The rhtA gene is present at 18 min on the E. coli chromosome close to the glnHPQ operon, which encodes components of the glutamine transport system. The rhtA gene is identical to ORPl (ybiF gene, nucleotide positions 764 to 1651, GenBank accession number AAA218541, gi:440181) and is located between the pexB and ompX genes. The unit expressing a protein encoded by the ORFl has been designated the rhtA gene (rht: resistance to homoserine and threonine). Also, it was revealed that the rhtA23 mutation is an A-for-G substitution at position -1 with respect to the ATG start codon (ABSTRACTS of the 17th International Congress of Biochemistry and Molecular Biology in conjugation with Annual Meeting of the American Society for Biochemistry and Molecular Biology, San Francisco, California August 24-29, 1997, abstract No. 457, EP 1013765 A).
The asd gene of E. coli has already been elucidated (nucleotide positions 3572511 to 3571408, GenBank accession no. NC 000913.1, gi:16131307), and can be obtained by PCR (polymerase chain reaction; refer to White, TJ. et al., Trends Genet., 5, 185 (1989)) utilizing primers prepared based on the nucleotide sequence of the gene. The asd genes from other microorganisms can be obtained in a similar manner.
Also, the aspC gene of E. coli has already been elucidated (nucleotide positions 983742 to 984932, GenBank accession no. NC 000913.1, gi:16128895), and can be obtained by PCR. The aspC genes of other microorganisms can be obtained in a similar manner.
L-lysine-producine bacteria Examples of parent strains which can be used to derive L-lysine-producing bacteria include 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. Patent No. 6,040,160), phosphoenolpyrvate carboxylase (ppc), aspartate semialdehyde dehydrogenease (asd), and aspartase (aspA) (EP 1253195 A). In addition, the parent strains can have increased expression of the gene involved in energy efficiency (cyo) (EP 1170376 A), the gene encoding nicotinamide nucleotide transhydrogenase (pntAB) (U.S. Patent No. 5,830,716), the ybjE gene (WO2005/073390), or combinations thereof.
Examples of parent strains which can be used to derive L-lysine-producing bacteria also include strains having decreased or eliminated activity of an enzyme that catalyzes a reaction for generating a compound other than L-lysine by branching off from the biosynthetic pathway of L-lysine. Examples of the enzymes that catalyze a reaction for generating a compound other than L-lysine by branching off from the biosynthetic pathway of L-lysine include homoserine dehydrogenase, lysine decarboxylase (U.S. Patent No. 5,827,698), and the malic enzyme (WO2005010175).
2. Methods
Methods are described for producing L-aspartic acid or L-aspartic acid-derived metabolites which include cultivating the bacterium as described herein in a culture medium to produce and excrete the L-aspartic acid or L-aspartic acid-derived metabolite into the medium, and collecting the produced L-aspartic acid or L-aspartic acid-derived metabolite from the medium.
The cultivation, collection, and purification of L-aspartic acid or L-aspartic acid-derived metabolites from the medium and the like can be performed in a manner similar to conventional fermentation methods wherein an amino acid is produced using a bacterium.
The medium used for culture can be either a synthetic or natural medium, so long as the medium includes a carbon source, a nitrogen source, minerals, and, if necessary, appropriate amounts of nutrients which the bacterium requires for growth. The carbon source can include various carbohydrates such as glucose and sucrose, and various organic acids. Depending on the assimilation mode of the chosen microorganism, alcohol including ethanol and glycerol can 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 can be performed under aerobic conditions, such as by shaking and/or stirring with aeration, at a temperature of 30 to 36°C, and in another example 32 to 34°C. The pH of the culture is usually between 5.0 and 7.0, and in another example between 6.0 and 6.5. The pH of the culture can be adjusted with ammonia, calcium carbonate, various acids, various bases, and buffers. Usually, a 1 to 5 -day cultivation leads to accumulation of the target L-amino acid in the liquid medium.
After cultivation, solids such as cells can be removed from the liquid medium by centrifugation or membrane filtration, and then the L-amino acid can be collected and purified by ion-exchange, concentration, and/or crystallization methods.
Examples
The present invention will be more concretely explained below with reference to the following non-limiting examples.
Example 1. Construction of strains.
The method of λ Red-dependent integration of PCR-generated fragments followed by λlnt/Xis-dependent removing of the antibiotic resistance marker used for selection of the integrants previously adjusted for use in P. ananatis (RU application 2006134574, WO2008/075483) was applied for construction of all the strains which were constructed using PCR primers listed in Table 1.
To prepare the integrative DNA fragments, the DNA fragment containing a kanamycin resistance gene flanked with attL and attR sites of phage λ with primers presented in Table 1 was amplified with PCR. Primers used in the reaction were homologous with at least 40bp of the target sites of P. ananatis genome on their 5'- ends. pMWl \8-(λattL-Kmτ-λattR) plasmid (RU application 2006134574, WO2008/075483) was used as a template in all the reactions. The obtained DNA fragments were treated for 2 or 3 hours with Dpnl restrictase which recognizes the methylated GATC site to eliminate pMWl 18-(λα^Z-Kmr-λα//i?).
P. ananatis strain SC17(0) (VKPM B-9246, RU application 2006134574, WO2008/075483) which is resistant to the expression of all three Red-genes from phage λ {gam, bet and exo) harbors the RSF-Red-TER plasmid, and was used as a recipient in integration experiments. The SC 17(0) strain was deposited at the Russian National Collection of Industrial Microorganisms (VKPM, GNII Genetika, address: Russia, 117545 Moscow, 1 Dorozhny proezd. 1) on September 21, 2005, and assigned an accession number of VKPM B-9246.
In order to obtain electrocompetent P. ananatis cells, the SC 17(0) strain transformed with the RSF-Red-TER plasmid was grown overnight at 34°C on LB medium with 50 μg/ml chloramphenicol. Then, the culture was diluted 100 x with fresh LB medium containing 50 μg/ml of chloramphenicol and grown up to OD600 of 0.3 at 34°C under aeration. After that, IPTG was added to ImM and cultivation was continued up to OD600=0.7. 10 ml samples were washed three times with an equal volume of deionized ice water and resuspended in 40 μl of 10% cold glycerol. Just before electroporation, 100-200 ng of the in vitro amplified DNA preparation dissolved in 5 μl of deionized water was added to the cell suspension. The procedure was performed using the device for bacterial electrotransformation ("BioRad", USA, catalog number 165-2089, version 2-89). The pulse parameters applied were the following: electric field intensity of 20 kV/cm, pulse time of 5 msec. After electroporation, 1 ml of LB medium enriched with glucose (0.5%) was immediately added to the cell suspension. Then the cells were grown under aeration at 34°C for 2 h and plated on solid LB medium containing 40 μg/ml of kanamycin followed by an overnight incubation at 34°C. To select the integrants among grown KmR clones, their chromosome structure was verified by PCR with the test primers presented in Table 1. To cure the selected KmR-integrants from the RSF-Red-TER helper plasmid, they were streaked on the plates containing LB medium with the addition of IPTG (ImM) and sucrose (5g/L) at 340C and grown to form single colonies. KmR, Cms plasmid-less clones were isolated.
Table 1. Primers used to construct ene deletions.
Figure imgf000022_0001
To eliminate kanamycin or tetracycline antibiotic resistance marker, pMW- IntXis-cat plasmid (see Reference Example 2) was electroporated to the selected plasmid-less integrant by the same procedure as for the electroporation of the PCR- generated fragments. After electroporation the cells were plated on LB-agar containing 0.5% glucose, 0.5x M9 salt solution and chloramphenicol (50mg/L) and incubated at 37°C overnight to induce synthesis of the Int/Xis proteins. The grown clones were replica-plated on LB-plates with and without kanamycin to select Kms variants. The selected Kms clones were re-checked by PCR with corresponding test primers.
To eliminate the chloramphenicol resistance marker, CmR, the pMW-IntXis helper plasmid (WO2005010175) was used. Selection of Cms clones was performed as above. But in this case, cells after electroporation were plated on a medium containing 800 mg/L ampicillin.
To construct the strains carrying multiple mutations, all the procedures were repeated with the corresponding pairs of primers.
To construct the plasmid strains listed in Table 7, the desirable plasmids were electroporated to the corresponding strains by the method described above for electroporation of DNA fragments.
The 3Δ::pycA strain was constructed via integration of the mini-Mu cassette carrying the pycA gene from Sinorhizobium meliloti under control of the P,flC promoter and canonical SD sequence AGGAGG. To that, the integrative plasmid pMIV-Ptoc- pycA was constructed (see Example 2). The pMIV-Ptac-#ycΛ plasmid was electroporated to 3Δ-S strain (marker- less) (see Table 7) harboring phMIV-1 helper plasmid (see Reference Example 3) providing expression of the Mu integrase according to the procedure described above. The 3Δ-S strain is a marker-less strain of the 3Δ strain (SCIl(O)AaSpAAsUcAAgItA). After electroporation, cells were incubated at 37°C for induction of the integrase synthesis. Cells were plated on L-agar containing 25mg/L chloramphenicol. pMIV -P, ac-pycA carried two antibiotic resistance markers: cat inside the integrative cassette and bla on the vector part of the plasmid. The grown clones were replica-plated and Ap clones were selected. The selected clones did not contain the integrative plasmid. PCR analysis proved the presence of the pycA gene in the genome of the selected clones. The obtained integrants were cured from the helper plasmid. To that, cells were seeded in LB medium and incubated without agitation at 37°C in 3 days. After that, cells were plated on L-agar with addition of chloramphenicol (25mg/L) and incubated at 34°C overnight. The grown clones were replica plated and TcsCmR variants were selected and designated 3Δ::pycA.
5ΔP2-36S strain is a marker-less derivative of the 5Δ-S strain carrying Appc mutation and the E. colippcK620S gene integrated to the genome by the same Mu- dependent procedure as was used for the 3Δ::pycA strain construction.
To construct appc deletion, the kαn gene flanked by λαttR/L was amplified by PCR, using primers Dppc-3' (SEQ ID NO: 73) and attR3-XbaI-HindIII (SEQ ID NO: 76). Genomic DNA isolated from P. αnαnαtis SC 17(0) strain Ptac-lacZ (RU application 2006134574, WO2008090770) was used as a template for PCR. In parallel, the fragment containing the terminator of the leader peptide of the E. coli threonine operon (Tthr) was constructed by PCR. To that, two pairs of oligonucleotides were used: mashl (SEQ ID NO: 74), mash2 (SEQ ID NO: 75), and Dppc-5' (SEQ ID NO: 72), Tthr5'-Xbal (SEQ ID NO: 77). At first, mashl (SEQ ID NO: 74) and mash2 (SEQ ID NO: 75) were annealed to each other. As a result, the terminator was generated. The resulting DNA fragment was used as a template for PCR with Dppc-5'(SEQ ID NO: 72) and Tthr5'-Xbal (SEQ ID NO: 77) primers to create the fragment with a Xbαl recognition site on its 5 '-end, which is necessary to join the integrative cassette and homologous arm for integration on the 3 '-end. The fragments including Tthr and the removable KmR marker were digested by Xbαl restrictase and then ligated. The ligated mixture was electroporated to the SC17(0)/RSF-Red-TER (RU application 2006134574) strain for integration according to the procedure for λRed-dependent integration described above. Integrants were selected on LB-agar plates with kanamycin (40mg/l). The chromosome structure of the integrants was confirmed by PCR using ppc-tl (SEQ ID NO: 78) and ppc-t2 (SEQ ID NO: 79) oligonucleotides as primers. The resulting strain was named SC17(0)Δppc.
The constructed deletion was transferred to 5Δ-S by the chromosome electroporation procedure. To that, 200 ng of genomic DNA isolated from SC17(0)Δppc using the Genomic DNA Purification Kit provided by "Fermentas" was electro-transformed to 5Δ-S. The cultivation conditions were the same as for the Red- dependent integration procedure except for the addition of IPTG. Preparation of the electrocompetent cells was the same as for Red-dependent integration. Pulse parameters were E=12.5kV/cm, pulse time 10msec. The obtained integrants were verified by PCR using ppc-tl (SEQ ID NO: 78) and ppc-t2 (SEQ ID NO: 79) oligonucleotides as primers. The resulting strain was named 5ΔP.
After that, Mu-dependent integration of the E. coli ppcK620S gene encoding feedback-resistant PEP-carboxylase to 5ΔP was performed using the integrative plasmid pMIVK620S (see Example 2). The integration procedure was the same as for thepycA gene (see above). The obtained integrants (59 independent clones) were named 5ΔP2. The clone No.36 gave the highest aspartic acid and biomass accumulation in a 48-hour test tube cultivation with increased L-GIu (L-glutamic acid) concentration (6.0 g/L) (Table 3, data for 15 best clones are represented), and was chosen for further improvement. Test tube cultivation was performed using a medium shown in Table 2. The high L-GIu concentration was used to simulate the presence of the glutamate dehydrogenase. The selected strain was cured from the chloramphenicol resistance marker using the λlnt/Xis-dependent procedure (see above). The resultant strain was named 5ΔP2-36S.
5ΔP2R strain was obtained from 5ΔP2-36S as a spontaneous AspR mutant selected on the M9 plates containing 10 g/L glucose and 30 g/L L- Asp (L-aspartic acid), pH5.5 having the same producing ability as the parental strain.
Table 2. Medium for test-tube cultivation.
Figure imgf000026_0001
Section B was adjusted by NaOH to have pH 6.5. Sections B and C were sterilized separately.
Seed: Plates containing LB-M91/2 medium (LB medium enriched with 0.5x M9 salt solution and 0.5% glucose) with addition of the appropriate antibiotic seeded and incubated at 34°C overnight.
Cultivation conditions: 34°C, CT=48h.
Table 3. Test tube cultivation of the strains with the integrated E. coli ppc(K620S) gene. Initial L-GIu concentration 6.0 g/L, CT=48h.
Figure imgf000026_0002
Example 2. Construction of the plasmids.
Construction of the pMW-EaspC plasmid. To construct the pMW-EaspC plasmid, the DNA fragment which includes a coding part of the E. coli aspC gene linked with canonical SD-sequence AGGAGG was generated in PCR with the primers E-aspC5'KI (SEQ ID NO: 92) and E-aspC3'BI (SEQ ID NO: 93). Chromosomal DNA isolated from the E. coli strain MGl 655 (ATCC 700926) was used as template in the reaction. The strain MGl 655 can be obtained from American Type Culture Collection. (P.O. Box 1549 Manassas, VA 20108, United States of America). The obtained fragment was digested with Kpnl and BamRl restrictases and ligated with pMWl 18-PlacUV5-lacI vector (Skorokhodova, A. Yu et al, Biotekhnologiya (Rus), 5, 3-21 (2004)) which had been digested with the same endonucleases. The ligated mixture was transformed to E. coli strain TGl (J. Sambrook et al., Molecular Cloning, 1989) and plasmid DNA was isolated from the clones grown on LB plates with ampicillin (100mg/L). The plasmids carrying the desirable insertion were electroporated to the SC17(0)ΔaspCΔtyrB strain and several plasmids which resulted in auxotrophy of the strain to L-aspartic acid were selected. One of these plasmids was electro-transformed to 3Δ-S/RSFGP strain (see below) and specific aspartate aminotransferase activity was assayed in crude extracts of the 3Δ- S/RSFGP harboring pMW-EaspC. The plasmid-less strain was used as a control. The data shown in Table 4 indicate that the pMW-EaspC plasmid provides an increase of aspartate aminotransferase activity in 3Δ-S/RSFGP strain by 1.5 times.
Table 4. Specific activity of aspartate aminotransferase.
Figure imgf000027_0001
Construction of the pMIVK620S plasmid.
The E. colippcκ 2 gene coding for PEP-carboxylase resistant against inhibition by aspartic acid was sub-cloned from the pTK620S plasmid (Masato Yano and Katsura Izui, Eur. J. Biochem. FEBS, 247, 74-81, 1997) to the pMIV-5JS plasmid (RU patent application 2006132818, EP 1942183) in two steps. At first, the Sall-Sphl fragment of pTK620S was sub-cloned into Sall-Sphl recognition sites of pMIV5-JS. The obtained pMIV-ppc-5'plasmid carries a large 5'-terminal portion of the p/7cK620S gene. The 3'-proximal portion of the ppc gene was amplified in PCR with primers ppc-Sphl (SEQ ID NO: 94) and ppc-Hindlll (SEQ ID NO: 95) and the pTK620S plasmid as template. This fragment and pMIV-ppc-5' were digested with Sphl and Hindlll restrictases and ligated. The ligated mixture was electroporated to E.coli strain MG1655Δppc (see Reference Example 4). Cells after transformation were plated on M9 glucose (5g/l) minimal media with chloramphenicol (50mg/l) to select the colonies harboring plasmids providing PEP-carboxylase activity. After isolation of the plasmid DNA from the grown colonies and restriction analysis, the plasmids of the expected structure were selected.
Construction of the pMIV-P/gr-pycA plasmid.
To construct pMIV-Prαc-pycA plasmid, a DNA fragment containing the coding part of the pycA gene from Sinorhizobium meliloti was generated in PCR with primers PycSm5-LNX (SEQ ID NO: 96) and pycSm3-Sal (SEQ ID NO: 97). Chromosomal DNA isolated from the S". meliloti type strain (DSM 30135, ATCC 9930, VKPM B- 9293) provided by All-Russian Collection of Industrial Microorganisms (VKPM) (Russia, 117545 Moscow, 1 Dorozhny proezd, 1) was used as a template in this reaction. This strain can also be obtained from ATCC.
The obtained DNA fragment was digested with Sail and Xbal restrictases and ligated with the pMIV5-JS integrative plasmid which had been treated with the same enzymes. The ligated mixture was transformed to E. coli strain TGl and plasmid DNA was isolated from the clones which grew on LB plates with ampicillin (100mg/L) and chloramphenicol (50mg/L). Three plasmids carrying the desirable insertion were selected. These plasmids carrying the promoter-less gene pycA were designated pMIV- pycA (Fig. 2). All three obtained plasmid probes were used for the cloning of Ptoc promoter in front of the pycA gene. To that, the DNA fragment containing Ptoc was generated by PCR with primers J56 (SEQ ID NO: 108) and J57(KpnBgl) (SEQ ID NO: 109). The pDR540 plasmid (GenBank accession number U13847, "Pharmacia") was used as a template in this reaction. Primer J56 contains the site for Xbal restrictase at the 5 '-end thereof. Primer J57(KpnBgl) contains sites for Kpnl amd BgHl restrictase at the 5 '-end thereof. The resulting DNA fragment was digested with Kpnl and Klenow fragment of DNA polymerase I to obtain the blunt ends. After that, the fragment was digested with Xbal and ligated with pMIV-pycA plasmid digested with Xbal and Ec/136II (the last enzyme produces the blunt ends). The resulting ligated mixture was transformed to the E. coli strain MG1655Δppc. After transformation, cells were plated on M9 glucose minimal medium to select the clones prototrophic to L- glutamic acid. Plasmid DNA from the selected clones was isolated and the structure of the resulting pMIV-Ptac-pycA plasmid was proven by restriction analysis.
RSFGP plasmid carrying ppc and gdhA genes from E. coli was constructed on the basis of the RSFCPG plasmid containing the git A, ppc and gdhA genes (US patent application 20070134773). 5'- end phosporylated primers SΕQ ID NO: 118 and 119 were used for PCR to obtain a big part of the RSFCPG plasmid without the git A gene using TaKaRa La Taq™ DNA polymerase (TaKaRa Bio Inc., Japan). The resulting 14 kb PCR fragment was purified and ligated. Thus, the RSFGP plasmid was obtained. pMWgdhA plasmid carrying the gdhA gene from E. coli was obtained by ligation of the DNA fragment carrying the gdhA gene from E. coli into pMWl 19 vector. The DNA fragment carrying the gdhA gene from E. coli was obtained by PCR using primers SΕQ ID NO: 120 and 121. Primer SΕQ ID NO: 120 contains a Sail restriction site at the 5 '-end thereof, primer SΕQ ID NO: 121 contains Hindlll restriction site at the 5 '-end thereof. The resulting DNA fragment was treated with Sail and Hindlll restrictases and ligated into a pMWl 19 vector previously treated with the same restrictases.
Example 3. Function of the genes involved in L-aspartatic acid/L- glutamic acid metabolism in P. ananatis.
Three different biochemical reactions which produce L-aspartic acid, at least in vitro, have been found to date. They are the aspartate aminotransferase reaction, reverse aspartase reaction, and aspartate dehydrogenase reaction. The aspartate aminotransferase reaction is used by all organisms studied to date for L-aspartic acid biosynthesis.
It was known that in E. coli two enzymes possess aspartate aminotransferase activity. They are aspartate aminotransferase encoded by the aspC gene and aromatic amino acids aminotrasferase, the product of the tyrB gene. Only simultaneous inactivation of the aspC and tyrB genes leads to Asp auxotrophy in E. coli (Reitzer, L. J., Ammonia assimilation and the biosynthesis of glutamine, glutamic acid, aspartic acid, asparagine, L-alanine, and D-alanine. In Neidhardt, F. C. (Ed. in Chief), R. Curtiss III, J.L. Ingraham, E.C.C. Lin, K.B. Low, B. Magasanik, W.S. Reznikoff, M. Riley, M. Schaechter, and H.E. Umbarger (eds). 1996. Escherichia coli and Salmonella: Cellular and Molecular Biology. American Society for Microbiology).
P. ananatis is a member of Enterobacteriaceae family, and is closely related to E. coli. The putative aspC and tyrB genes were found in the P. ananatis genome using the "Genome" program. These genes were inactivated in P. ananatis SC 17(0) strain using the procedure of λRed-dependent integration of PCR generated DNA fragments (see Reference Example 1), which were previously adopted for use in this organism (RU application 2006134574).
As for E. coli, inactivation of the aspC gene in the P. ananatis SC 17(0) strain led to only slight growth retardation in M9 glucose minimal medium without aspartic acid or tyrosine supplementation. SC17(0)ΔtyrB strain required tyrosine, whereas the SC17(0)ΔaspCΔtyrB double mutant strain proved to be a strong auxotroph to L- aspartic acid and L-tyrosine (Table 5). Therefore, as expected, the aspartate aminotransferase reaction is the only way for aspartic acid biosynthesis to function in wild-type P. ananatis strain.
Table 5. Growth of the constructed mutants on M9-plates, containing glucose, aspartic acid or glutamic acid (10g/L for each) as a sole carbon source. For aspartic acid and glutamic acid minimal media initial H5.5 was used.
Figure imgf000030_0001
In the aspartate aminotransferase reaction, oxaloacetate is a precursor for aspartic acid synthesis and L-glutamic acid is a donor of amides. To produce aspartic acid, an increase of the oxaloacetate intracellular level is necessary. The problem is that most of the known anaplerotic enzymes (PEP carboxylases and pyruvate carboxylases) are inhibited by L-aspartic acid. In particular, PEP carboxylase from E. coli is sensitive to this inhibitor (Wohl, R.C. & Markus, G., J. Biol. Chem., Vol. 247(18), pp. 5785-5792 (1972); Yano, M, & Izui, K., Eur. J. Biochem., 247: 74-81 (1997)). The only anaplerotic gene previously identified in the P. ananatis genome encodes a close homologue of PEP carboxylase from E. coli (data not published). An assay of PEP carboxylase activity in crude extracts off. ananatis showed strong inhibition of this enzyme by aspartic acid (Table 6). Therefore, enhancement of anaplerotic activity is a key factor of aspartic acid production by P. ananatis.
Table 6. Specific activity of PEP-carboxylase in the derivative of P. ananatis SC 17(0) strain in which the wild-type ppc gene is replaced by the E. colippcK620S gene encoding the enzyme resistant to inhibition by aspartic acid.
Figure imgf000031_0001
Mutations in PEP carboxylase gene from E. coli which result in resistance of the enzyme to feedback inhibition by L-aspartic acid have been previously selected (Yano, M, & Izui, K., Eur. J. Biochem., 247: 74-81 (1997)). Introduction of this mutant allele to an aspartic acid-producing strain can provide high PEP-carboxylase activity even in the presence of 2OmM aspartic acid (Table 6, construction of SC17(0)::ppcK620S is described below). Moreover, it was published elsewhere that pyruvate carboxylase from Synorhizobium meliloti is not inhibited by aspartic acid (Dunn, M.F. et al, Cloning and characterization of the pyruvate carboxylase from Sinorhizobium meliloti RmlO211, Arch. Microbiol., 176, 355-363 (2001)). These enzymes can be used to enhance oxaloacetate formation in an aspartic acid-producing strain. The wild-type strain of P. ananatis can efficiently utilize L-aspartic acid as the sole carbon source. It can grow on M9 medium supplemented with aspartic acid instead of glucose at a high rate. In E. coli, the major way to utilize L- Asp is an aspartase reaction (McFaIl, E., Newman E.B., Amino acids as carbon sources. In Neidhardt, F.C. (Ed. in Chief), R. Curtiss III, J.L. Ingraham, E.C.C. Lin, K.B. Low, B. Magasanik, W.S. Reznikoff, M. Riley, M. Schaechter, and H.E. Umbarger (eds). 1996. Escherichia coli and Salmonella: Cellular and Molecular Biology. American Society for Microbiology). The ORF coding for a homologue of aspartase has been found from E. coli in the P. ananatis genome and the putative aspA gene was deleted (see Reference Example 1). The constructed strain could not use aspartic acid as a sole carbon source absolutely (Table 5) and did not display aspartase activity. Therefore, the aspA gene coding for aspartase was identified. It has been shown that, as in E. coli, the aspartase reaction is the major way for aspartic acid utilization in P. ananatis. The aspA gene must be inactivated in an aspartic acid-producing strain to avoid product utilization by the cells.
The fumarate which is formed in the aspartase reaction is converted to malate and, after that, to oxaloacetate in TCA. In turn, oxaloacetate can be converted to citrate in citrate synthase reaction or decarboxylated to PEP in PEP carboxykinase reaction or to Pyr by oxaloacetate decarboxylase. Moreover, malic enzyme catalyzes decarboxylation of malate to pyruvate. It was published elsewhere (H Ozaki, J Shiio. Production of lysine by pyruvate kinase mutants of Brevibacterium flavum, Agr. Biol. Chem., 47(7): 1569-1576 (1983)) that a significant decrease in citrate synthase activity was necessary for high-performance production of lysine (the amino acid synthesized from L-aspartic acid) by Brevibacterium flavum. The citrate synthase (git A) gene from P. ananatis was previously identified by the inventors of the present invention (data not shown), and was deleted in the P. ananatis strain SC 17(0) (see Reference Example 1). The resulting strain could not use aspartic acid as a sole carbon source absolutely (Table 5). Therefore, we proposed that the citrate synthase reaction is the major way of unproductive waste of oxaloacetate. Inactivation of these genes or a significant decrease in citrate synthase activity in an aspartic acid-producing strain is necessary to provide a high pool of oxaloacetate in the cells. As for E. coli, inactivation of citrate synthase in P. ananatis led to a L-GIu requirement, although the P. ananatis AgItA strain is a leaky auxotroph to L-GIu. Therefore, addition of L-GIu to the medium for cultivation of aspartic acid producing strain is necessary to provide the donor of an amino group. In the aspartate aminotransferase reaction, L-GIu is converted to α-ketoglutarate. To synthesize a large amount of the product from a small amount of L-GIu, α-ketoglutarate utilization (due to AsucA mutation) must be blocked, and re-amination of α-ketoglutarate at a high rate is necessary. Animation of α-ketoglutarate to L-glutamic acid can be carried out in glutamate dehydrogenase or glutamate synthase reactions. It is well known in Enterobacteriaceae that glutamate dehydrogenase and glutamate synthase/glutamine synthetase form two alternative pathways for L-glutamic acid synthesis. Bacteria use only one of these pathways depending on the availability of energy and nitrogen. In E. coli, inactivation of one of the pathways does not lead to an L-GIu requirement (Reitzer, LJ. Ammonia assimilation and the biosynthesis of glutamine, glutamic acid, aspartic acid, asparagine, L-alanine, and D-alanine. In Neidhardt, F.C. (Ed. in Chief), R. Cuitiss III, J.L. Ingraham, E.C.C. Lin, K.B. Low, B. Magasanik, W.S. Reznikoff, M. Riley, M. Schaechter, and H.E. Umbarger (eds). 1996. Escherichia coli and Salmonella: Cellular and Molecular Biology. American Society for Microbiology).
The P. ananatis genome does not contain ORFs coding for close homologues of E. coli glutamate dehydrogenase or glutamate synthase. Instead, it contains the gltB gene coding for a close homologue of a large subunit of glutamate synthase from Agrobacterium tumefaciens, and the gdhA gene coding for close homologue of glutamate dehydrogenase from Salmonella typhimurium. We have deleted the gltB gene in P. ananatis (see Reference Example 1). The resulting strain has proven to be a strong auxotroph to L-GIu (L-GIu requirement 0.5 g/L). Therefore, glutamate synthase/glutamine synthetase reactions are the only pathways for the synthesis of L- glutamic acid in P. ananatis. On the other hand, deletion of the putative gdhA gene as well as replacing its regulatory region with a strong Vtac promoter linked to a consensus SD sequence, did not significantly change the growth of P. ananatis with glucose and L-glutamic acid as the sole carbon sources. Moreover, glutamate dehydrogenase activity was not detected in P. ananatis crude extracts. Probably, the gdhA gene found by computer analysis in this organism is inactive, at least under the chosen cultivation conditions. Therefore, the introduction of a foreign glutamate dehydrogenase or increase of glutamate synthase activity could be necessary for high-performance L- aspartic acid production.
Example 4. Minimal combination of the genetic factors providing L- Asp production in P. ananatis.
Taking into account data described in Example 3 and summarized in Table 7, the combination of four factors was considered as an essential and sufficient combination for aspartic acid production by P. ananatis, namely:
1. Decrease of α-ketoglutarate dehydrogenase activity.
2. Decrease of citrate synthase activity.
3. Increase of anaplerotic (PEP carboxylase or pyruvate carboxylase) activity.
4. Increase of α-ketoglutarate amination (glutamate dehydrogenase or glutamate synthase) activity.
The absence of any of the above factors led to a decrease of L-aspartic acid accumulation or to the predominant L-glutamate accumulation.
It is further desirable to decrease activity of one or several of enzymes including aspartate ammonia-lyase (aspartase), glucose dehydrogenase and malate dehydrogenase. It is also desirable to increase activity of aspartate aminotransferase.
Table 7. Test tube fermentation of the 3Δ-S/RSFGP strain and the strains lacking one of the genetic factors necessary for L- Asp production. 48-hour cultivation; initial glucose concentration 40g/L, initial L-GIu concentration 3g/L. ImM IPTG was added for the strains harboring pMWaspC. Average data for 3 independent cultures are represented.
Figure imgf000035_0001
Example 5. Decrease of the unproductive conversion of PEP to Pyr due to inactivation of the pykA and pykF genes.
As it is shown in Table 7, the 3Δ-S (marker-less SC\7(0)ΔaspAΔsucAAgltA) strain transformed with the RSFGP plasmid carrying the ppc and gdhA genes from E. coli resulted in accumulation of L- Asp up to 0.7g/L in test-tube cultivation. At the same time, accumulation of VaI, Leu and Ala up to 2-3g/L was observed. To prevent unproductive conversion of PEP to Pyr, Δ#y£A::λattL-TcR-λattR and Apyk¥:.λa.UL- KmR-λattR deletions were introduced step by step to 3Δ-S, which is the marker-less derivative of 3Δ. The resulting strains were named 4Δ and 5Δ, respectively.
A check of these strains in test-tube cultivation showed practically the same accumulation of VaI, Leu, and Ala by the 4Δ strain (containing only the pykA deletion) as for 3Δ. On the other hand, the levels of the mentioned by-products in the culture liquids of the 5Δ strain carrying deletions of the both the pykA and pykF pyruvate kinase genes were significantly decreased (Fig. 3). As expected, only trace accumulation of the aspartic acid by the 4Δ and 5Δ strains was observed because of low PEP-carboxylase activity.
The 5Δ strain was cured from the selective markers according to the above- described procedure. The resulting strain 5Δ-S was electro transformed with RSFGP plasmid to increase the PEP-carboxylase and glutamate dehydrogenase activities. In test-tube fermentation, 5Δ-S/RSFGP strain showed higher accumulation of aspartic acid than the control strain 3Δ/RSFGP (Table 7).
Example 6. Glucose utilization by P. ananatis.
A search for the orthologues of E. coli genes in the P. ananatis genome showed that at least three pathways for glucose utilization could be proposed in this organism: glycolysis, the pentose-phosphate pathway, and the PQQ-dependent glucose dehydrogenase pathway. To check real functioning of all these pathways, we have constructed the double mutant strains on the basis of SC 17(0) carrying the following pairs of deletions of the first gene of each pathway: ΔpgiΔzwf, ΔzwfΔgcd and ΔpgiΔgcd. As it is shown in Fig. 4, all of the constructed strains kept the ability to grow on glucose. Therefore, all the predicted pathways actually function in P. ananatis.
To test the existence of some additional pathways, a triple mutant strain carrying Δpgi, Δzwf, and Δgcd deletions was constructed. Unexpectedly, this strain could grow on glucose, although at very low rate.
Further computer analysis of the P. ananatis genome revealed an operon including 4 genes, and is located on a 300-kbp plasmid present in the P. ananatis genome. The second and the third genes of the operon encode close homologues of NAD+-dependent 6P-gluconate dehydrogenase and NAD+-dependent glucose-6P dehydrogenase from Mesorhizobium loti, respectively. These genes were designated as pzwf and pgnd.
Cloning of P. αnαnαtis gene encoding NAD+ZNADP+ glucose-6P dehydrogenase.
The SC 17(0)ΔzwfΔpzwf strain was constructed and the activities of glucose-6P dehydrogenase were determined in this strain, SC 17(0), and in SC17(0)Δzwf. NAD- dependent activity was absent in all strains. NADP-dependent glucose-6P dehydrogenase activity was decreased at least 50 times in the strains carrying deletion of the chromosome zwf gene. Cells for this experiment were grown on glucose- containing medium. We have concluded that the chromosome zwf gene encodes NADP-dependent glucose-6P dehydrogenase whereas the pzwf gene is not expressed under these conditions.
The coding part of the /?zw/~ gene was amplified by PCR using primers pzwf-5 (SEQ ID NO: 98) and pzwf-3 (SEQ ID NO: 99), and genomic DNA isolated from P. ananatis SC 17 strain (US patent 6,596,517) as a template. Primer pzwf-5 (SEQ ID NO: 98) contains the site formal restrictase at the 5 '-end thereof. Primer pzwf-3 (SEQ ID NO: 99) contains the site for BamHl restrictase at the5'-end thereof. The resulting DNA fragment was treated with Xbal and BamRl restrictases and was cloned into the Xbal-BamHl sites of the RSF-based vector RSFPlaclacI (see Reference Example 1) under control of the P/αcuvs promoter. The resulting plasmid RSFP/ac-pzwf (Fig 5) was isolated from 4 independent plasmid clones and electro-transformed to the SC17(0)ΔzwfΔpgiΔgcd strain, and then plated on glucose minimal medium. The SC17(0)ΔzwfΔpgiΔgcd and SC17(0)ΔpgiΔgcd strains were used as controls. After a 48h cultivation, clones of the plasmid-less SC17(0)ΔzwfΔpgiΔgcd strain were very small, while the clones carrying RSFP/αc-pzwf plasmid were of normal size (the same as of the clones of SC17(0)ΔpgiΔgcd strain). So, complementation of Δzw/mutation was observed for all plasmid preparations.
Glucose-6P dehydrogenase activities were determined in crude extracts of all 4 transformants (assay is described in the Example section below). As it is shown in Table 8, the plasmid isolated from clone 1 resulted in undetectable levels of Zwf (glucose-6P dehydrogenase) activity, while the plasmid isolated from clones 4, 5 and 7 carried a copy of the plasmid zwf gene encoding glucose-6P dehydrogenase with double NAD/NADP co factor specificity. Moreover, the level of NAD-dependent activity was significantly higher than that of NADP-dependent activity in these clones. Determination of the DNA sequence of the cloned fragment by the Sanger method showed 100% identity of the sequence of the cloned DNA fragment to the previously determined sequence of pzwf gene for the plasmid isolated from clone No.4. Table 8. Activity of glucose-6P dehydrogenase in crude extracts of the
SC 17(0)ΔzwfΔpzwf strain harboring RSFP/αc-pzwf plasmid. Average data for two independently prepared robes are re resented for each culture.
Figure imgf000038_0001
Cloning of P. ananatis gene encoding NAD+TNADP+ 6P-gluconate dehydrogenase.
In a similar manner, the pgnd gene was cloned into the RSFPlaclacI plasmid under the control of the P/flCuvs promoter using the primers pgnd-5 (SEQ ID NO: 100) and pgnd-3 (SEQ ID NO: 101) for PCR-amplification of its coding part. Primer pgnd-5 (SEQ ID NO: 100) contains the site formal restrictase at the5'-end thereof. Primer pgnd-3 (SEQ ID NO: 101) contains the site for BamHl restrictase at the5'-end thereof. The DNA fragment obtained by PCR was treated with Xbal and BamHl restrictases and cloned into Xbal-BamHl sites of the RSF-based vector RSFPlaclacI. The resulting plasmid RSFPlac-pgnd is depictured in Fig. 5. An assay OfNAD+ and NADP+- dependent 6P-gluconate dehydrogenase activity in crude extracts (assay is described in the Example section below) of SC 17(0) strain with inactivated gnd and pgnd genes and transformed with the constructed plasmid have shown that NAD+-dependent 6P- gluconate dehydrogenase (Gnd) specific activity of this enzyme is approximately 30- fold higher than NADP+-dependent Gnd activity (Table 9). Therefore, as expected, the cloned gene encodes NAD+-dependent 6P-gluconate dehydrogenase. Determination of the DNA sequence of the cloned fragment by the Sanger method showed 100% identity of the sequence of the cloned DNA fragment to the previously determined sequence of pgnd gene for the plasmid isolated from clone No.21. Table 9. Activity of 6P-gluconate dehydrogenase in crude extracts of the
SC17(0)ΔgndΔpgnd strain harboring RSFPlac-pgnd. Average data for two independent cultures are represented in each case.
Figure imgf000039_0001
Example 7. Gluconic acid accumulation in aspartic acid fermentation process. Further improvement of aspartic acid production due to inactivation of glucose dehydrogenase.
Accumulation of up to 50 g/L gluconic acid was observed in the experiments using S-Jar batch fermentation of the aspartic acid-producing strains. Gluconic acid is formed from glucose via a glucose dehydrogenase reaction. To prevent unproductive glucose conversion and toxic effects caused by gluconic acid accumulation, the gcd gene coding for glucose dehydrogenase was deleted in the 5ΔP2R strain according to the above-described λRed-dependent procedure using oligonucleotides gcd-attR (SEQ ID NO: 49) and gcd-attL (SEQ ID NO: 50) as primers for generation of the integrative DNA fragment; oligonucleotides gcd-testl (SEQ ID NO: 51) and gcd-test2 (SEQ ID NO: 52) were used for PCR verification of the obtained integrants. The resulting strain 5ΔP2RG was electroporated with pMWgdhA and checked in test tube cultivation. Inactivation of glucose dehydrogenase led to an increase of aspartic acid accumulation by 3.3 times for the plasmid-less strains and by 1.4 times for the plasmid strains (Table 10). Table 10. Effects of the Δgcd mutation on aspartic acid production by P. ananatis. 72- hour cultivation; initial glucose concentration 40g/L, initial L-GIu concentration 3g/L. Average data for 3 independent cultures are represented.
Figure imgf000040_0001
Example 8. Further improvement by amplification of the pzwfor psnd gene.
The RSFP lac-pzwf and RSFPlac-pgnd plasmids were introduced into the 5ΔP2- 36S strain by the electroporation procedure (see above). The resulting plasmid strains gave significantly increased aspartic acid accumulation in comparison with the plasmid-less parental strain. Amplification of the/?zw/gene led to a 2.6-fold increase of the product accumulation, amplification of the pgm/ gene gave a 1.4-fold increase of aspartic acid accumulation in test tube cultivation (Table 11).
Table 11. Amplification of pzwfor pgnd gene in the Asp-producing strain. 48-hour cultivation; initial glucose concentration 40g/L, initial L-GIu concentration 3 g/L. Average data for 3 independent cultures are represented.
Figure imgf000040_0002
Example 9. Further improvement of aspartic acid production by inactivation of malate dehydrogenase.
To prevent unproductive conversion of oxaloacetate to malate, fumarate and succinate, the mdhA gene coding for malate dehydrogenase was deleted in the 5ΔP2R strain according to the above-described λRed-dependent procedure using oligonucleotides mdhA-attR (SEQ ID NO: 88) and mdhA-attL (SEQ ID NO: 89) as primers for generation of the integrative DNA fragment; oligonucleotides mdhA-testl (SEQ ID NO: 90) and mdhA-test2 (SEQ ID NO: 91) were used for PCR verification of the obtained integrants. The resulting strain 5ΔP2RM was cured from the kanamycin resistance marker using the above-described λlnt/Xis-dependent procedure. The pMWgdhA plasmid was introduced into the marker-less derivative of 5ΔP2RM and to 5ΔP2R. The results of the experiment on test tube cultivation of the obtained strains represented in Table 12 show a significant positive effect of the AmdhA mutation on aspartic acid accumulation and yield for the both plasmid and plasmid-less variants.
Table 12. Effects of the Δmdh mutation on aspartic acid production by P. ananatis. 72 -hour cultivation; initial glucose concentration 40g/L, initial L-GIu concentration 3g/L. Average data for 3 independent cultures are represented.
Figure imgf000041_0001
Reference Example 1. Construction of RSFPlaclacI vector.
RSFPlaclacI (Fig. 6) is a derivative of the RSFPlaclacIsacBcat plasmid previously described in Russian patent application (RU 2006134574). The RSFPlaclacIsacBcat plasmid, which is also named pRSFsacB, has been disclosed by Katashkina, J.I. et al. (Use of the λRed-recombineering method for genetic engineering of Pantoea ananatis. BMC Molecular biology 2009, 10:34). To construct this plasmid, Red-dependent integration of the generated in vitro short DNA fragment, which includes a polylinker site into the RSFPlaclacIsacBcat plasmid, was performed. To prepare this fragment, the oligonucleotides DeI-F (SEQ ID NO: 102) and DeI-R (SEQ ID NO: 103) (200 ng of each) were annealed and extended in the polymerase chain reaction mixture prepared from the reagents provided by "Fermentas" accordingly to the manufacturer's instructions (temperature profile was 95°C for 2min, 50°C for 1 min, 72°C for 2 min). The probe was precipitated with ethanol, washed with 70% ethanol twice and dissolved in 10 μl of fresh de-ionized water.
The MG1655/pKD46 strain was used as a recipient strain for Red-dependent integration. Preparation of the electrocompetent cells was as described by Datsenko, K. A. and Wanner, B.L. (Proc. Natl. Acad. Sci. USA, 97, 12, p 6640-6645 (2000)). 200 ng of the prepared DNA fragment and 100 ng of the RSFPlaclacIsacBcat plasmid were used for electroporation. Electroporation was performed using a Gene Pulser apparatus (BioRad, USA, version 2-89). Pulse time was 5 msec, and electric field strength was 12.5 kV/cm. 1 ml of SOC medium was added to the cell suspension immediately after electroporation. The cells were cultivated at 37°C for 2 hours, spread on LB agar containing 50 μg/ml of chloramphenicol, 5% sucrose, and 1 mM IPTG and cultivated at 37°C overnight. The integrative DNA fragment contained flanks homologous to the upstream and downstream regions of the sacB gene coding for levansucrase. Expression of this gene is highly toxic for many bacterial species and is routinely used as a contra-selective marker (Gay, P. et al., J. Bacterid., 164, 918-921 (1985)). Expression of the sacB gene located in RSFPlaclacIsacBcat plasmid is provided by P/αcuv5 promoter and induced by IPTG. The integration had to result in elimination of sacB from the plasmid. That is why, to select the integrants, we used medium containing sucrose and IPTG.
Fifteen CmR SucR clones were selected in this experiment. Plasmid DNA was isolated from these clones, transformed to the E. coli TGl strain and re-isolated from it for the restriction analysis. After digestion of the isolated DNA probes with the Hindlll, Sphl, Pstl, Sail, BamHI, BgIII, Pvul, and Xbal restriction endonucleases, the clone containing the polylinker site of the expected structure was selected.
Reference Example 2. Construction of pMW-intxis-cat vector.
To construct pMW-intxis-cat (Figure 7), the DNA fragment containing the cat gene was amplified by PCR with primers cat5' BgIII (SEQ ID NO: 104) and cat3'SacI (SEQ ID NO: 105), and pACYC184 plasmid as a template. Pfu polymerase ("Fermentas") was used in this reaction to generate blunt ends. The obtained DNA fragment was cloned in the unique Seal recognition site (Seal restriction endonuclease generates blunt ends) of the pMW-intxis plasmid (WO2005010175). A plasmid with the orientation of the cat gene shown in Figure 7 was confirmed by restriction analysis using Ncol restrictase (lengths of necessary DNA fragments are 3758 and 3395 bp).
Reference Example 3. Construction of phMIV-1 helper plasmid. The DNA fragment including the MuC (cts62), ner, MuA, and MuB genes coding for the integrase and thermosensitive repressor was amplified by PCR using primers MuC5 (SEQ ID NO: 106) and MuB3 (SEQ ID NO: 107), and the pMHIO plasmid (US patent 6,960,455) as a template. Primer MuC5 contains the site for EcoRl restrictase at the 5 '-end thereof. Primer MuB3 contains the site for Ncol restrictase at the 5 '-end thereof. The resulting fragment was cloned into the EcoRl-Ncol recognition sites of the pACYC184 plasmid. Thus, the phMIV-1 helper plasmid was obtained (Figure 8).
Reference Example 4. Construction of E.coli strain MG1655Δppc
A strain in which the ppc gene is deleted was constructed by the method initially developed by Datsenko, K.A. and Wanner, BX. (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 primers pps-attR (SEQ ID NO: 111) and ppc-attL (SEQ ID NO: 112), and plasmid pMWl 18-attL-Cm-attR as a template (WO2005010175). Primer pps-attR contains both a region complementary to the region located at the 5' end of the ppc gene and a region complementary to the attR region. Primer pps-attR contains both a region complementary to the region located at the 3' end of the ppc gene and a region complementary to the attL region.
A 1,7 kbp PCR product was obtained and purified in agarose gel, and was used for electroporation of the E. coli strain MG 1655 (ATCC 700926), which contains the plasmid pKD46 having a temperature-sensitive replication. The plasmid pKD46 (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 MG 1655.
Electrocompetent cells were prepared as follows: E. coli MG1655/pKD46 was grown overnight at 30 °C in LB medium containing ampicillin (100 mg/1), 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 ODOOO 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 370C 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 ppc gene deletion by PCR
The mutants having the ppc gene deleted and marked with the Cm resistance gene were verified by PCR. Locus-specific checking primers ppc-testl (SEQ ID NO: 112) and ppc-test2 (SEQ ID NO: 113) were used in PCR for the verification. The PCR product obtained in the reaction with the cells of parental ppc+ strain MG1655 as a template, was 2,8 kbp in length. The PCR product obtained in the reaction with the cells of mutant strain as the template was 1.7 kbp in length. The mutant strain was named MG1655Δppc::cat. Cm marker was excised from the chromosome using standard techniques (int-xis system) described in WO2005010175. Length of the DNA obtained in the PCR using primers ppc-testl and ppc-test2 in the case when the Cm marker is excised was 280 bp.
Reference Example 5. Assays
Assay for aspartate aminotransferase specific activity.
Overnight cultures grown in LB were diluted 1:50 with a medium containing 1.2 g/L (NH4)2SO4, 6g/L Na2HPO4- 12H2O, 3g/L KH2PO4, O.5g/L NaCl with addition of ImM MgSO4, O.lmM CaCl2, lOg/L glucose and ImM IPTG, and incubated in tubes (10ml) up to OD595=0.9-1.0. Cells were harvested by centrifugation, washed by 5OmM KP buffer and frozen. Cells were re-suspended in 5OmM KP buffer pH7.2 and disrupted by sonication. The reaction mixture contained 10OmM TRIS HCl (pH7.5), 1OmM L- Asp (pH7.0), 5mM α-ketoglutarate (pH7.0), 0,ImM pyridoxal phosphate. Oxaloacetate formation was detected by measuring the absorption at 265 nm.
Assay of PEP-carboxylase specific activity provided by pMIVK620S.
Cells were grown in M9 medium (MG1655Δppc strain was grown in LB medium) overnight. Cells were collected by centrifugation and disrupted by ultra- sonication in 10OmM TRIS-HCl pH7.5 with the addition of 2mM DTT. The activity was assayed in the coupled reaction with malate dehydrogenase. Reaction mixture contained 10OmM TRIS-HCl pH7.5, 5mM PEP, 2mM DTT, 5mM MnSO4, 0.15mM NADH, 1OmM NaHCC-3, 0.ImM acetyl-CoA, 2u/ml malate dehydrogenase. Change of NADH extinction at 340 nm was measured. The results are shown in Table 13.
Table 13. Specific activity of PEP-carboxylase
Figure imgf000045_0001
Assay for the PEP-carboxylase specific activity in the derivative of P. ananatis SC 17(0) strain in which the wild-type ppc gene is replaced by the E. coli ppcK620S gene encoding the enzyme resistant to inhibition by aspartic acid.
Overnight cultures grown in LB medium (or in LB-M91/2 medium for 5ΔP2R) were diluted by 1 :50 with fresh M9 medium containing 1% glucose, L-Lys (L-lysine), L-Met (L-methionine), L5D-DAP (diaminopimeric acid) (100 mg/L of each) and 1 g/L L-GIu, and cultivated at 34°C with aeration up to OD595=LO. Cells were collected by centrifugation, washed by 5OmM potassium phosphate buffer pH7.5, and disrupted by sonication. The activity determination was conducted in the coupled reaction with malate dehydrogenase. The reaction mixture included: 10OmM TRIS HCl (pH7.5), 5mM PEP, 4mM DTTE, 5mM MnSO4, 0.15mM NADH, 10OmM NaHCO3, O.lmM Acetyl-CoA, 2u/ml malate dehydrogenase. The change in NADH extinction at 340 nm was measured. The reactions without PEP or Acetyl-CoA were used as controls.
Assay for glucose-6P dehydrogenase activity in crude extracts of the
Figure imgf000046_0001
Cells were grown overnight at 340C in LBG-1/2M9 medium with the addition of chloramphenicol (50μg/ml) and ImM IPTG, diluted 1 :100 with fresh medium and cultivated at 34°C up to OD595=LO. Cells were harvested by centrifugation and disrupted by sonication. The method described by Deutsch, J. (Glucose-6-phosphate dehydrogenase, p.190- 197. In J.Bergmeyer and M.Grassl (ed.), Methods of enzymatic analysis. 3rd ed., vol.3; VCH, Weinheim, Germany, 1983) was applied for glucose-6P dehydrogenase specific activity determination. The average data for two independently prepared probes are shown for each culture.
Assay for 6P-gluconate dehydrogenase activity in crude extracts of the SC17(O)ΔgndΔpgnd strain harboring RSFPlac-pgnd.
Fresh transformants were used. Overnight cultures grown in LB medium containing 50 mg/1 chloramphenicol (for plasmid strains) were diluted by 50 times with M9 medium (pH6.0) supplemented with 0.5% glucose and ImM IPTG and chloramphenicol 50μg/L (for plasmid strains) and cultivated at 34°C in 10ml volume up to OD595=0.6 — 1.1. Cells were collected by centrifugation, washed by 5OmM TRIS- HCl pH7.5 and sonicated in the buffer containing 50 mM TRIS-HCl (pH7.5), 2mM MgCl2 and 2mM DTT. The reaction mixture contained 5OmM TRIS-HCl (pH 8.0), 4mM NAD+/NADP+, 2mM MgCl2 and 4mM 6P-gluconate. The change in NADH extinction at 340 nm was measured. The average data for two independent cultures are shown in each case.
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.

Claims

1. A bacterium belonging to the genus Pantoea which is able to produce L- aspartic acid or an L-aspartic acid-derived metabolite, wherein said bacterium has been modified to have:
- decreased activity of α-ketoglutarate dehydrogenase;
- decreased activity of citrate synthase;
- increased activity of phosphoenolpyruvate carboxylase or pyruvate carboxylase; and
- increased activity of glutamate dehydrogenase or glutamate synthase.
2. The bacterium according to claim 1, wherein the activities of α-ketoglutarate dehydrogenase and citrate synthase are decreased by attenuation of expression of the genes coding for α-ketoglutarate dehydrogenase and citrate synthase.
3. The bacterium according to claim 1, wherein said bacterium has been further modified to have attenuated expression of a gene coding for aspartate ammonia-lyase (aspartase).
4. The bacterium according to claim 1, wherein said bacterium has been further modified to have attenuated expression of a gene coding for pyruvate kinase.
5. The bacterium according to claim 1, wherein said bacterium has been further modified to have attenuated expression of a gene coding for glucose dehydrogenase.
6. The bacterium according to claim 1, wherein said bacterium has been further modified to have attenuated expression of a gene coding for malate dehydrogenase.
7. The bacterium according to any of claims 2 to 6, wherein said expression of the gene coding for α-ketoglutarate dehydrogenase, citrate synthase, aspartate ammonia-lyase (aspartase), pyruvate kinase, glucose dehydrogenase or malate dehydrogenase is attenuated by inactivating said gene in the chromosome of the bacterium.
8. The bacterium according to claim 1, wherein activity of phosphoenolpyruvate carboxylase or pyruvate carboxylase is increased by enhancing the expression of the gene coding for phosphoenolpyruvate carboxylase or pyruvate carboxylase; and activity of glutamate dehydrogenase or glutamate synthase is increased by enhancing the expression of the gene coding for glutamate dehydrogenase or glutamate synthase.
9. The bacterium according to claim 8, wherein said expression is enhanced by modifying an expression control sequence of said gene or by increasing the copy number of said gene.
10. The bacterium according to claim 8, wherein the genes coding for phosphoenolpyruvate carboxylase and glutamate dehydrogenase are derived from Escherichia coli.
11. The bacterium according to claim 8, wherein the gene coding for glutamate dehydrogenase codes for NAD-dependent glutamate dehydrogenase derived from Pantoea ananatis.
12. The bacterium according to claim 8, wherein the gene coding pyruvate carboxylase is derived from Sinorhizobium meliloti.
13 The bacterium according to claim 1, wherein said bacterium is Pantoea ananatis.
14. The bacterium according to claim 1, wherein the L-aspartic acid-derived metabolite is selected from the group consisting of L-threonine, L-lysine, L- methionine, and L-homoserine.
15. A method for producing an L-aspartic acid or L-aspartic acid-derived metabolite comprising:
- cultivating the bacterium according to any one of claims 1 to 14 in a culture medium, and
- collecting L-aspartic acid or L-aspartic acid-derived metabolite from the medium.
16. The method according to claim 15, wherein L-aspartic acid-derived metabolite is selected from the group consisting of L-threonine, L-lysine, L- methionine, and L-homoserine.
PCT/JP2009/067449 2008-09-30 2009-09-30 A BACTERIUM BELONGING TO THE GENUS Pantoea PRODUCING AN L-ASPARTIC ACID OR L-ASPARTIC ACID-DERIVED METABOLITES AND A METHOD FOR PRODUCING L-ASPARTIC ACID OR L-ASPARTIC ACID-DERIVED METABOLITES WO2010038905A1 (en)

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