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  • 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
  • C12P13/005—Amino acids other than alpha- or beta amino acids, e.g. gamma amino acids
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • 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
  • C12N1/205—Bacterial isolates
• • 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
• • 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
  • C12P13/22—Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine
  • C12P13/227—Tryptophan
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/185—Escherichia
  • C12R2001/19—Escherichia coli

Definitions

• the present invention relates to methods for producing L-amino acids by fermentation.
• L-amino acids are widely used as raw materials of drugs, seasonings, feedstuffs and so forth.
• L-amino acids are industrially produced by fermentation using coryneform bacteria that belong to the genus Brevibacterium or Corynebacterium.
• methods for producing L-amino acids by using bacteria belonging to the genus Escherichia such as Escherichia coli have also been developed.
• various techniques for increasing L-amino acid producing ability by gene recombination techniques are disclosed (Japanese Patent Laid-open Publication (Kokai) No. 57-71397; U.S. Pat. No. 4,371,614).
• An object of the present invention is to provide a technique for improving the L-amino acid productivity of bacteria belonging to the genus Escherichia.
• the inventors of the present invention assiduously studies in order to achieve the aforementioned object. As a result, they found that, if fructose was used as a carbon source of a medium used for culture of a bacterium belonging to the genus Escherichia, L-amino acid producing ability was improved, and accomplished the present invention.
• the present invention provides the followings.
• a method for producing an L-amino acid which comprises culturing a bacterium belonging to the genus Escherichia having an ability to produce the L-amino acid in a medium containing fructose as a main carbon source to produce and accumulate the L-amino acid in the medium and collecting the L-amino acid from the medium.
• L-amino acid producing ability or “ability to produce an L-amino acid” refers to an ability to accumulate a significant amount of an L-amino acid in a medium or to increase L-amino acid content in microbial cells when a bacterium belonging to the genus Escherichia is cultured in the medium.
• L-amino acid there can be mentioned L-tryptophan, L-phenylalanine, L-lysine, L-threonine, L-valine, L-leucine, L-isoleucine, L-homoserine, L-glutamic acid and so forth.
• Bacteria used in the present invention are not particularly limited so long as they are bacteria belonging to the genus Escherichia and have L-amino acid producing ability.
• Specific examples of the bacteria belonging to the genus Escherichia include those mentioned in the work of Neidhardt et al. (Neidhardt, F. C. et al., Escherichia coli and Salmonella Typhimurium, American Society for Microbiology, Washington D.C., 1208, Table 1) and derivatives derived from these bacteria.
• Escherichia coli having L-amino acid producing ability may be a mutant or recombinant strain.
• mutants having a mutation that increases an activity of intracellular enzyme involved in biosynthesis of an L-amino acid specifically, a mutation that increases an expression amount of the enzyme or a mutation that eliminates feedback inhibition.
• recombinant strain there can be mentioned a strain having an increased copy number of a gene coding for an enzyme involved in L-amino acid biosynthesis, a strain of which expression control sequence is modified to increase expression amount of the gene, a strain introduced with a gene coding for an enzyme of which feedback inhibition is eliminated and so forth.
• the mutants can be obtained by treating wild strains of bacteria belinging to the genus Escherichia or derivatives thereof by UV irradiation or with mutagenizing agents used for a usual mutagenesis treatment such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) and nitrous acid.
• NTG N-methyl-N′-nitro-N-nitrosoguanidine
• the target gene and a vector functioning in a bacterium belonging to the genus Escherichia may be ligated to prepare recombinant DNA, and the bacterium belonging to the genus Escherichia may be transformed with the recombinant DNA. Further, the transformation can be performed by the method of D. A. Morrison ( Methods in Enzymology, 68, p.326, 1979), the method of treating recipient cells with calcium chloride so as to increase the permeability of DNA (Mandel, M. and Higa, A., J. Mol. Biol., 53, p. 159, 1970) and so forth.
• phage vectors can also be used.
• the copy number of a gene can be increased also by presence of multiple copies of the target gene on chromosomal DNA of a bacterium belonging to the genus Escherichia.
• homologous recombination is performed by using, as a target, a sequence that is present on the chromosomal DNA in a multiple copy number.
• a sequence present on chromosomal DNA in a multiple copy number repetitive DNA or inverted repeats present at the ends of transposable elements can be used.
• multiple copies of the target gene can be introduced into chromosomal DNA by mounting each of them on a transposon to transfer them.
• Activity of a target enzyme can be increased by replacing an expression control sequence such as a promoter of a gene coding for the target enzyme with a stronger one (Japanese Patent Laid-open Publication (Kokai) No. 1-215280).
• strong promoters there have been known, for example, lac promoter, trp promoter, trc promoter, tac promoter, P R promoter and P L promoter of lamda phage, tet promoter, amyE promoter and so forth.
• Examples of enzymes involved in L-tryptophan biosynthesis include 3-deoxy-D-arabino-heptulonate-7-phosphate synthase of the L-tryptophan biosynthesis system (Japanese Patent Laid-open Publication (Kokai) No. 5-236947), transketolase (U.S. Pat. No. 5,906,925), anthranilate synthase (W094/08031 (International Patent Unexamined Publication in Japanese (Kohyo) No. 7-507693)), phosphoglycerate dehydrogenase (WO94/08031) and so forth.
• anthranilate synthase is known to suffer feedback inhibition by L-tryptophan
• phosphoglycerate dehydrogenase is known to suffer feedback inhibition by L-serine.
• bacteria belonging to the genus Escherichia preferred are bacteria belonging to the genus Escherichia harboring desensitized anthranilate synthase, desensitized phosphoglycerate dehydrogenase, or both of them.
• a bacterium belonging to the genus Escherichia having such a property can be obtained by, for example, mutating the anthranilate synthase gene (trpE) and/or the phosphoglycerate dehydrogenase gene (serA) so as not to suffer feedback inhibition and introducing the obtained mutant gene into the bacterium belonging to the genus Escherichia.
• a transformant strain obtained by introducing a plasmid pGH5 (W094/08031) containing a mutant serA coding for desensitized phosphoglycerate dehydrogenase into Escherichia coli SV164 containing desensitized anthranilate synthase.
• a bacterium belonging to the genus Escherichia introduced with recombinant DNA containing a tryptophan operon is also a preferred L-tryptophan producing bacterium.
• Escherichia coli introduced with a tryptophan operon containing a gene coding for desensitized anthranilate synthase Japanese Patent Laid-open Publication (Kokai) Nos. 57-71397 and 62-244382; U.S. Pat. No. 4,371,614
• L-tryptophan producing bacteria there can be mentioned Escherichia coli AGX17(pGX44) [NRRL B-12263], which is a bacterial strain having a phenotype of L-phenylalanine and L-tyrosine auxotrophy, and the AGX6(pGX50)aroP [NRRL B-12264] strain, which harbors a plasmid pGX50 containing a tryptophan operon (refer to U.S. Pat. No. 4,371,614 for the both).
• the L-tryptophan producing ability can be enhanced by increasing the phosphoenolpyruvate producing ability in a cell of a bacterium belonging to the genus Escherichia having L-tryptophan producing ability (W097/08333).
• genes or operons of enzymes can be obtained by a usual gene isolation method well known to those skilled in the art.
• a target gene can be obtained by synthesizing primers based on a known sequence, and performing PCR by using chromosomal DNA of a bacterium belonging to the genus Escherichia such as the Escherichia coli K-12 strain as a template.
• Escherichla coli AJ12604 FERM BP-3579
• European Patent Laid-open Publication No. 488,424 European Patent Laid-open Publication No. 488,424.
• L-lysine producing bacterium belonging to the genus Escherichia there can be exemplified a mutant having resistance to an L-lysine analogue.
• the L-lysine analogue is such one which inhibits growth of bacteria belonging to the genus Escherichia, but the inhibition is fully or partially eliminated when L-lysine coexists in a medium. Examples thereof include oxalysine, lysine hydroxamate, (S)-2-aminoethyl-L-cysteine (AEC), ⁇ -methyllysine, ⁇ -chlorocaprolactam and so forth.
• Mutants having resistance to these lysine analogues can be obtained by subjecting a microorganism belonging to the genus Escherichia to a usual artificial mutation treatment.
• bacterial strains used for production of L-lysine include Escherichia coli AJ11442 (FERM BP-1543, NRRL B-12185; refer to Japanese Patent Laid-open (Kokai) No. 56-18596 and U.S. Pat. No. 4,346,170) and Escherichia coli VL611.
• the AJ11442 strain was deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (currently, National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary, 1-3 Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan) on May 1, 1981 and received an accession number of FERM P-5084. Then, it was transferred to an international deposit under the provisions of the Budapest Treaty on Oct. 29, 1987 from the above original deposit, and received an accession number of FERM BP-1543.
• the feedback inhibition of aspartokinase by L-lysine is desensitized in the above microorganisms.
• L-threonine producing bacteria there can also be mentioned L-threonine producing bacteria. This is because, in L-threonine producing bacteria, inhibition of aspartokinase by L-lysine is generally eliminated.
• Escherichia coli there can be mentioned Escherichia coli MG442 (refer to Gusyatiner et al., Genetika (in Russian), 14, pp.947-956, 1978).
• a gene encodes an L-lysine biosynthesis enzyme may be enhanced in the aforementioned L-lysine producing bacterium.
• Examples of such a gene include a gene coding for phosphoenolpyruvate carboxylase having a mutation for desensitizing feedback inhibition by aspartic acid (Japanese Patent Publication (Kokoku) No. 7-83714).
• bacteria belonging to the genus Escherichia containing L-valine biosynthesis gene of which control mechanism is substantially eliminated can be obtained by, for example, introducing an ilvGMEDA operon, preferably, an ilvGMEDA operon which does not express threonine deaminase activity and in which attenuation is eliminated, into a bacterium belonging to the genus Escherichia (refer to Japanese Patent Laid-open Publication (Kokai) No. 8-47397).
• the whole nucleotide sequence of the ilvGMEDA operon has been revealed ( Nucleic Acids Res., 5, p.2137, 1987), it can be obtained from chromosomal DNA of Escherichia coli by colony hybridization or PCR using oligonucleotides prepared based on the sequence.
• a DNA fragment containing the ilvGMEDA operon can be introduced into Escherichia coli by the aforementioned method using a plasmid, phage or transposon.
• L-leucine producing bacteria belonging to the genus Escherichia include a strain having ⁇ -2-thienylalanine resistance, a strain having ⁇ -2-thienylalanine resistance and ⁇ -hydroxyleucine resistance (Japanese Patent Publication (Kokoku) No. 62-34397 for the above) and a strain having 4-azaleucine resistance or 5,5,5-trifluoroleucine resistance (Japanese Patent Laid-open Publication (Kokai) No. 8-70879).
• Escherichia coli KX141 VKPM B-4781
• European Patent Publication No. 519,113 European Patent Publication No. 519,113
• Escherichia coli VKPM B-3996 (RIA 1867) (U.S. Pat. No. 5,175,107) and the MG442 strain.
• NZ10 strain which is a Leu + revertant of the C600 strain (Appleyard R. K., Genetics, 39, pp.440-452, 1954).
• L-glutamic acid producing bacteria belonging to the genus Escherichia there can be mentioned L-valine resistant strains such as Escherichia coli B11, Escherichia coli K-12 (ATCC 10798), Escherichia coli B (ATCC 11303) and Escherichla coli W (ATCC 9637).
• a medium containing fructose as a main carbon source is used when a bacterium belonging to the genus Escherichia having L-amino acid producing ability is cultured.
• the yield with respect to sugar and the production rate of L-amino acids are improved by using fructose as a main carbon source.
• the carbon source may substantially consist only of fructose or may also contain carbon sources other than fructose.
• the fructose content is preferably 30 weight % or more, preferably about 30-95 weight %, more preferably about 30-70 weight %, particularly preferably about 50%, with respect to the total carbon source.
• Other carbon sources include glucose, sucrose, maltose and so forth. Among these, glucose is preferred.
• a specific example of the carbon source used in the present invention is a mixture of 30 weight % or more of fructose and 70 weight % or less of glucose.
• Medium components other than the carbon source are usual medium components such as nitrogen source, inorganic ions and organic trace nutrients used as required.
• the nitrogen source there can be used inorganic ammonium salts such as ammonium sulfate, ammonium chloride and ammonium phosphate, organic nitrogen such as soybean hydrolysate, ammonia gas, aqueous ammonia and so forth.
• inorganic ammonium salts such as ammonium sulfate, ammonium chloride and ammonium phosphate
• organic nitrogen such as soybean hydrolysate, ammonia gas, aqueous ammonia and so forth.
• inorganic ions or source thereof small amounts of potassium phosphate, magnesium sulfate, iron ion, manganese ion and so forth are added.
• organic trace nutrients required substances such as vitamin B 1 , yeast extract and so forth are desirably contained In appropriate amounts as required.
• the culture may be performed under a condition selected depending on the used bacterial strain, but, specifically, it is preferably performed under an aerobic condition for 16-72 hours.
• the culture temperature is regulated to be 30-45° C. and pH is regulated to be 5-7 during the culture.
• An inorganic or organic, acidic or alkaline substance and further an ammonia gas or the like can be used for pH adjustment.
• Collection of L-amino acids from fermented liquor can be attained by appropriately combining known methods such as those utilizing ion exchange resin, precipitation and others.
• the yield with respect to sugar and/or the production rate can be improved in methods for producing L-amino acids such as L-tryptophan by using bacteria belonging to the genus Escherichia.
• This SV164 strain was introduced with a plasmid pGH5 (described in W094/08031) containing a gene coding for phosphoglycerate dehydrogenase of which feedback inhibition was desensitized (also referred to as “desensitized PGD” hereafter).
• the SV164/pGH5 strain had ability to produce tryptophan and serine.
• Escherichia coli KB862 was designated as AJ13828, and deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology (currently, National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary, 1-3 Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan) as an international deposit on Dec. 21, 2000 and received an accession number of FERM BP-7405.
• a mutant strain containing inhibition desensitized AS was screened by using 5-methyltryptophan, which is a tryptophan analogue.
• E. coli K12 YMC9 (ATCC 33927) was subjected to a mutagenesis treatment with N-methyl-N′-nitro-N-nitrosoguanidine (NG) according to the method of Miller (Miller J. H., Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., pp.125-129, 1972). That is, about 2 ⁇ 10 9 of YMC9 cells were incubated in 4 ml of 0.1 M sodium citrate buffer (pH 5.5) containing 50 ⁇ g/ml of NG at 37° C. for 30 minutes.
• NG N-methyl-N′-nitro-N-nitrosoguanidine
• the cells were washed twice with 0.1 M sodium phosphate buffer (pH 7.0), and the cells in an amount of 0.1 ml were cultured overnight at 37° C. in LB medium with shaking. Subsequently, the culture broth was diluted with 0.9% NaCl to dilutions of 10 ⁇ 3 , 10 ⁇ 4 and 10 ⁇ 5, and 0.1 ml of each diluted solution was applied on a minimal medium plate containing 100 ⁇ g/ml of 5-methyltryptophan.
• the composition of the minimal medium included 5 g/l of glucose, 5 mg/l of vitamin B 1 , 3 g/l of KH 2 PO 4 , 12 g/l of K 2 HPO 4 , 0.3 g/l of MgSO 4 ⁇ 7H 2 O, 0.1 g/l of NaCl, 5 g/l of (NH 4 ) 2 SO 4 , 14.7 mg/l of CaCl 2 ⁇ 2H 2 O, 2 mg/l of FeSO 4 ⁇ 7H 2 O, 1 g/l of trisodium citrate and 15 g/l of agar.
• Chromosomal DNA of each mutant strain was isolated and digested with restriction enzymes NheI and ClaI to isolate a fragment of about 5 kb, and this fragment was ligated with the NheI/ClaI fragment (4158 bp) of pBR322.
• E. coli KB 862 (trpE) (DSM7196) was transformed with the ligation reaction product. A clone that could grow on a minimal medium not containing L-tryptophan was selected. All the plasmids complementing the trpE mutation contained the NheI/ClaI fragment of 5 kb. Further, this 5 kb NheI/ClaI fragment contained trpE, trpD, a sequence about 0.8 kb upstream from trpE, and a sequence about 1 kb downstream from trpD.
• the two mutant classes were combined to highly express the gene coding for the desensitized AS.
• the ⁇ trpL1 mutation was used for the class 2 strain.
• a 1.6 kb NruI fragment having the ⁇ trpL1 mutation was isolated from the plasmid p ⁇ trpL and replaced with a corresponding NruI fragment in the plasmid pE0, pE5, pE6 or pE8.
• the obtained plasmids were designated as pIE0, pIE5, pIE6 and pIE8 and used for incorporation into chromosome by homologous recombination.
• the mutant trpE gene coding for the desensitized AS and having the ⁇ trpL1 mutation was transferred from each bacterial strain to the KB862 strain by P1 transduction (Miller J. H., Experiments in Molecular Genetics, Cold Spring Harbor, N.Y., pp.201-205, 1972) and selected in a minimal medium not containing tryptophan.
• the obtained bacterial strains were designated as PD103 (trpE0), KB862 (trpE5), SV164 (trpE8) and SV163 (trpE6).
• a serA gene coding for PGD was cloned from the E. coli B strain (ATCC 23226) into the plasmid vector pUC18.
• the B strain was cultured overnight at 37° C. in LB.
• the cells were collected by centrifugation (4000 ⁇ g), and chromosomal DNA was prepared by the method described in Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates 2.4.1-2.4.2, 1987.
• chromosomal DNA was digested with SphI.
• About 3 ⁇ g of the digested product was ligated with 0.2 ⁇ g of plasmid pUC18 similarly digested with SphI.
• the serA mutant PC1523 (CGSC No. 5411) (CGSC: E. coli Genetic Stock Center, Department of Biology 255 OML, Yale University, Postbox 6666, New Haven, Conn., USA) was transformed with the ligation reaction mixture by the aforementioned method of Cohen et al.
• the transformant strains were applied on a minimal medium not containing L-serine.
• the grown clones contained the E. coli serA gene in a 3.5 kb SphI fragment.
• the sequence of the wild type serA gene is described in W094/08031.
• the recombinant vector having the serA gene was designated as pGC3.
• a serA gene coding for desensitized PGD of which C-terminus amino acid was deleted was prepared.
• pGC3 was digested with SalI and KpnI, and the obtained fragment was separated by agarose gel electrophoresis.
• a 2.0 kb SalI-KpnI fragment including the full length of the serA gene was purified from the gel.
• the aforementioned tryptophan producing bacterium Escherichia coli SV164/pGH5
• LB medium 1% trypton, 0.5% yeast extract, 0.5% sodium chloride
• [Main culture medium composition] Glucose 17.5 g/L KH 2 PO 4 1.5 g/L (NH 4 ) 2 SO 4 5 g/L NaCl 0.5 g/L MgSO 4 • 7H 2 O 0.3 g/L CaCl 2 • 2H 2 O 15 mg/L FeSO 4 • 7H 2 O 75 mg/L Na 2 Citrate•2H 2 O 1 g/L Trace element solution 1 mg/L L-Phenylalanine 750 mg/L L-Tyrosine 750 mg/L Vitamin B 1 5 mg/L Yeast extract 2.5 g/L Tryptone 2.5 g/L Tetracycline 20 mg/L (Trace element solution) Na 3 MoO 4 0.15 g/L H 3 BO 3 2.5 g/L CoCl 2 • 6H 2 O 0.7 g/L CuSO 4 • 5H 2 O 0.25 g/L MnCl 2 • 4H 2 O 1.5 g/L ZnSO 4 • 7H 2 O 0.3 g/L
• the sugar concentration in the small-size fermenter was adjusted to 5-20 g/L by pumping 700 g/L (W/V) of sugar solution having one of the compositions shown in Table 2 (sterilized by autoclaving). After 48 hours of culture, the L-tryptophan concentration in the medium was measured. The yield with respect to sugar and the production rate are shown in Table 3. Table 3 shows ratios when the value of G100 is taken as 1. TABLE 2 Composition of carbon source G100 G30F70 G50F50 G70F30 F100 Glucose (%) 100 30 50 70 0 Fructose (%) 0 70 50 30 100

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  • 2001-01-19 JP JP2001011847A patent/JP2002209596A/ja active Pending
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