The present invention relates to biotechnology, specifically to a method for producing L-amino acids by fermentation and more specifically to genes derived from bacteria Escherichia coli. The genes are useful for improvement of L-amino acid productivity, for example, L-threonine, L-valine, L-proline, L-leucine, L-methionine and L-arginine.
Conventionally the L-amino acids have been industrially produced by method of fermentation utilizing strains of microorganisms obtained from natural sources or mutant of the same especially modified to enhance L-amino acid productivity.
There have been disclosed many techniques to enhance L-amino acid productivity, for example, by transformation of microorganism by recombinant DNA (see, for example,
On the other hand, increased L-amino acid excretion can enhance the productivity of strain producing L-amino acid. Strain of bacterium belonging to the genus Corynebacterium having increased expression of L-lysine excretion gene (lysE gene) is disclosed (
An object of present invention is to enhance the productivity of L-amino acid producing strains and to provide a method for producing L-amino acid, for example, L-threonine, L-valine, L-proline, L-leucine or L-methionine or L-arginine, using the strains.
This aim was achieved by identifying genes coding for proteins, which are not involved into biosynthetic pathway of target L-amino acid but enhance its production. An example of such protein could be a membrane protein having L-amino acid excretion activity. Based on the analysis of complete genome sequence of Escherichia coli, proteins with 4 or more putative transmembrane segments (TMS) were selected. As a result of diligent screening, the present inventors have identified several genes among them, that is, b2682, b2683, b1242 and b3434, and thoroughly studied it. The genes b2682 and b2683 have been known as putative CDS which may encode functionally unknown proteins (nucleotide numbers 92 to 829 and 819 to 1154 in the sequence of GenBank accession AE000353 U00096, respectively). The gene b2683 is also known as ygaH. The gene b1242 has been known as putative CDS which may encode functionally unknown protein (numbers 8432 to 9079 in the sequence of GenBank accession AE000222 U00096). The gene b1242 is also known as ychE. The gene b3434 also has been known as putative CDS which may encode functionally unknown protein (numbers 1463 to 2056 in the sequence of GenBank accession AE000420 U00096). The gene b3434 is also known as yhgN.
The present inventors have found that by enhancing the activity of the protein encoded by b1242 gene the productivity of L-amino acid producing strain is enhanced. Thus the present invention has been completed.
The present inventions are as follows:
- 1) An L-amino acid producing bacterium belonging to the genus Escherichia, wherein the bacterium has been modified so that the L-amino acid production by the bacterium should be enhanced by enhancing activities of proteins as defined in the following (E) or (F) in a cell of the bacterium:
- (E) a protein which comprises the amino acid sequence shown in SEQ ID NO:11 in Sequence listing;
- (F) a protein which comprises an amino acid sequence including deletion, substitution, insertion or addition of 1 to 22 amino acids in the amino acid sequence shown in SEQ ID NO:11 in Sequence listing, and which has an activity of making bacterium having enhanced resistance to the L-amino acids and/or its analogs;
(hereinafter, the proteins as defined in the above (E) or (F) are sometimes referred to as "proteins of the present invention" and the bacterium belonging to the genus Escherichia which is enhanced the activities of the proteins (E) or (F) is sometimes referred to as "a bacterium of the present invention");
- 2) The bacterium according to the above bacterium, wherein the transformation is performed with a multicopy vector.
- 3) A method for producing L-amino acid, which comprises cultivating the bacterium according to the above bacterium in a culture medium and collecting from the culture medium L-amino acid to be produced and accumulated.
- 4) The method according to the above method, wherein L-amino acid is L-threonine.
- 5) The method according to the above method, wherein the bacterium has been modified so that the bacterium should have enhanced expression of threonine operon.
- 6) The method according to the above method, wherein L-amino acid is L-valine.
- 7) The method according to the above method,
The method for producing L-amino acid includes production of L-threonine using L-threonine producing bacterium wherein activities of the proteins of the present invention such as that comprising amino acid sequence shown in SEQ ID NO:11 are enhanced. Also a method for producing L-amino acid includes production of
L-valine using L-valine producing bacterium wherein activities of the proteins of the present invention such as that comprising amino acid sequence shown in SEQ ID NO:11 are enhanced.
The present invention will be explained in detail below.
The bacterium of the present invention is an L-amino acid producing bacterium belonging to the genus Escherichia, wherein the bacterium has been modified so that the L-amino acid production by the bacterium should be enhanced by enhancing activities of the proteins of the present invention in a cell of the bacterium.
In the present invention, "L-amino acid producing bacterium" means a bacterium which has an ability to accumulate L-amino acid in a medium, when the bacterium is cultured in the medium. The L-amino acid producing ability may be possessed by the bacterium as a property of a wild strain of the bacterium or may be imparted or enhanced by breeding.
The bacterium of the present invention is L-amino acid producing bacterium belonging to the genus Escherichia having enhanced activities of proteins, which enhance the productivity of the target L-amino acid. Concretely the bacterium of present invention is L-amino acid producing bacterium belonging to the genus Escherichia which has enhanced activity of at least one or two of the proteins of the present invention.
The term "enhancing an activity of a protein " means that the activity per cell has become higher than that of a non-modified strain, for example, a wild-type bacterium belonging to the genus Esherichia. For example, there can be mentioned a case where number of the protein molecules per cell increases, a case where specific activity per the protein molecule increases and so forth. Further, as a wild-type bacterium belonging to the genus Eshcerichia that serves as an object for comparison, for example, the wild type strain of Escherichia coli can be mentioned.
The proteins of the present invention include ones as defined in the following (E) or (F):
- (E) a protein which comprises the amino acid sequence shown in SEQ ID NO:11 in Sequence listing;
- (F) a protein which comprises an amino acid sequence including deletion, substitution, insertion or addition of 1 to 22 amino acids in the amino acid sequence shown in SEQ ID NO:11 in Sequence listing, and which has an activity of making bacterium having enhanced resistance to the L-amino acids and/or its analogs;
Enhanced resistance to L-amino acids and/or its analogs means ability for bacterium to grow on a minimal medium containing L-amino acid or its analog in concentration under which the unmodified strain or the wild type strain, or the parental strain of the bacterium cannot grow, or ability for bacterium to grow faster on a medium containing L-amino acid or its analog than the unmodified strain or the wild type strain, or the parental strain of the bacterium.
More concretely, it can be said that E. coli strain has enhanced resistance to the L-amino acid or its analog if the strain forms a colony which is larger than that of the unmodified strain or wild type strain of E. coli after 2 - 4 days incubation at 37°C on a plate with solid Adams medium at 37°C when the strain is cultivated on an agar medium containing the L-amino acid or its analog under an appropriate condition. The term "an appropriate condition" refers to temperature, pH, air supply or optional presence of essential nutrients or the like for the E. coli strain which is to be cultivated.
L-amino acid analogs are exemplified by 3,4-dihydroproline, DL-thiaisoleucine, DL-o-methylserine, 4-azaleucine, norleucine, L-o-fluorophenylalanine and DL-o-fluorophenylalanine, homoserine, 6-diazo-5-oxo-L-norleucine and DL-β-hydroxy-norvaline.
Above mentioned concentration of L-amino acid or its analog, under which the unmodified strain or the wild type strain of the bacterium cannot grow, varies very significantly (from 0.5 µg/ml for DL-thiaisoleucine to 9600 µg/ml for DL-o-methylserine) depending on the structure of used compound. For example, such concentration is generally 7 to 70 µg/ml, preferably 20 to 25 µg/ml in case of 3,4-dihydroproline; generally 0.5 to 5 µg/ml, preferably 0.9 to 1.1 in case of DL-thiaisoleucine; generally 1100 to 9600 µg/ml, preferably 3000 to 3500 in case of DL-o-methylserine; generally 15 to 150 µg/ml, preferably 40 to 50 µg/ml in case of 4-azaleucine; generally 150 to 1500 µg/ml, preferably 450 to 550 µg/ml in case of norleucine; generally 0.6 to 6 µg/ml, preferably 1.5 to 2 µg/ml in case of L-o-fluorophenylalanine; generally 2 to 20 µg/ml, preferably 5 to 7 µg/ml in case of DL-o-fluorophenylalanine; and generally 330 to 3300 µg/ml, preferably 900 to 1100 µg/ml in case of homoserine, generally 5 to 50µg/ml, preferably 12 to 18 in case of 6-diazo-5-oxo-L-norleucine, and generally 25 to 250µg/ml, preferably 70 to 90µg/ml in case of DL-β-hydroxy-norvaline
In the bacterium of the present invention, the activities of the proteins of the present invention are enhanced by transformation of the bacterium with DNA coding for protein as defined in (E) or (F) or by alteration of promoter sequence of the DNA on the chromosome of the bacterium. The DNA, which is used for modification of the bacterium of the present invention, is represented by the b1242 gene. The b1242 gene can be obtained by, for example, PCR using primers having nucleotide sequence shown in SEQ ID No: 9 and 10.
Analysis of complete genome sequence of Escherichia coli allowed to select the genes coding for proteins having 4 or more putative TMS. Proteins with known function and transporters described by
The DNA coding for substantially the same protein as the protein defined in (E) may be obtained by, for example, modification of nucleotide sequence coding for the protein defined in (E) using site-directed mutagenesis so that one or more amino acid residue will be deleted, substituted, inserted or added. Such modified DNA can be obtained by conventional methods using treatment with reagents and conditions generating mutations. Such treatment includes treatment the DNA coding for proteins of present invention with hydroxylamine or treatment the bacterium harboring the DNA with UV irradiation or reagent such as N-methyl-N'-nitro-N-nitrosoguanidine or nitrous acid.
The DNA includes variants which can be found in the different strains and variants of bacteria belonging to the genus Escherichia according to natural diversity. The DNA coding for such variants can be obtained by isolating the DNA, which hybridizes with b1242 gene or part of the genes under the stringent conditions, and which codes the protein enhancing L-amino acid production. The term "stringent conditions" referred to herein is a condition under which so-called specific hybrid is formed, and non-specific hybrid is not formed. For example, the stringent conditions includes a condition under which DNAs having high homology, for instance DNAs having homology no less than 70% to each other, are hybridized. Alternatively, the stringent conditions are exemplified by conditions which comprise ordinary condition of washing in Southern hybridization, e.g., 60°C, 1 x SSC, 0.1% SDS, preferably 0.1 x SSC, 0.1% SDS. As a probe for the DNA which codes for variants and hybridizes with b1242 gene, a partial sequence of the nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 5 respectively can also be used. Such a probe may be prepared by PCR using oligonucleotides produced based on the nucleotide sequence of SEQ ID NO: 11 as primers, and a DNA fragment containing the nucleotide sequence of SEQ ID NO: 11 as a template. When a DNA fragment in a length of about 300 bp is used as the probe, the conditions of washing for the hybridization consist of, for example, 50°C, 2 x SSC, and 0.1% SDS.
Transformation of bacterium with DNA coding for protein means introduction of the DNA into bacterium cell for example by conventional methods to increase expression of the gene coding for the protein of present invention and to enhance the activity of the protein in the bacterial cell.
Techniques for enhancement of gene expression includes methods increasing the gene copy number. Introduction of a gene into a vector that is able to function in a bacterium belonging to the genus Escherichia increases copy number of the gene. For such purposes multi-copy vectors can be preferably used. The multi-copy vector is exemplified by pBR322, pMW119, pUC19, pET22b or the like.
Besides, enhancement of gene expression can be achieved by introduction of multiple copies of the gene into bacterial chromosome by, for example, method of homologous recombination or the like.
In case that expression of two or more genes is enhanced, the genes may be harbored together on the same plasmid or separately on different plasmids. It is also acceptable that one of the genes is harbored on a chromosome, and the other gene is harbored on a plasmid.
On the other hand, enhancement of gene expression can be achieved by alteration of promoter sequence of the gene. Alteration of promoter sequence of a gene includes introducing mutation in the inherent promoter sequence of the gene so that the expression of the gene is enhanced (
The bacterium of the present invention can be obtained by introduction of the aforementioned DNAs into bacterium inherently having ability to produce L-amino acid. Alternatively, the bacterium of present invention can be obtained by imparting ability to produce L-amino acid to the bacterium already harboring the DNAs. For the parent strain which is to be enhanced in activities of the proteins of the present invention, L-threonine producing bacteria belonging to the genus Escherichia such as strains VL2054 (VKPM B-8067), VNIIGenetika 472T23 (
Further, for the parent strain which is to be enhanced in activity of the proteins of the present invention, L-arginine producing bacteria belonging to the genus Escherichia such as strains AJ11531 and AJ11538 (
The bacterium of the present invention may be further enhanced expression of one or more genes which are involved in L-amino acid biosynthesis. Such genes are exemplified by threonine operon, which preferably comprises a gene encoding aspartate kinase - homoserine dehydrogenase of which feedback inhibition by L-threonine is desensitized (
The method of the present invention includes method for producing L-threonine, comprising steps of cultivating the bacterium of the present invention in a culture medium, to allow L-threonine to be produced and accumulated in the culture medium, and collecting L-threonine from the culture medium. Also the method of present invention includes method for producing L-valine, comprising steps of cultivating the bacterium of the present invention in a culture medium, to allow L-valine to be produced and accumulated in the culture medium, and collecting L-valine from the culture medium.
In the present invention, the cultivation, the collection and purification of L-amino acid from the medium and the like may be performed in a manner similar to the conventional fermentation method wherein an amino acid is produced using a microorganism. A medium used for culture may be either a synthetic medium or a natural medium, so long as the medium includes a carbon source and a nitrogen source and minerals and, if necessary, appropriate amounts of nutrients which the microorganism requires for growth. The carbon source may include various carbohydrates such as glucose and sucrose, and various organic acids. Depending on the mode of assimilation of the used microorganism, alcohol including ethanol and glycerol may be used. As the nitrogen source, various ammonium salts such as ammonia and ammonium sulfate, other nitrogen compounds such as amines, a natural nitrogen source such as peptone, soybean-hydrolysate and digested fermentative microorganism are used. As minerals, potassium monophosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, calcium chloride, and the like are used.
The cultivation is performed preferably under aerobic conditions such as a shaking culture, and stirring culture with aeration, at a temperature of 20 to 40 °C, preferably 30 to 38 °C. The pH of the culture is usually between 5 and 9, preferably between 6.5 and 7.2. The pH of the culture can be adjusted with ammonia, calcium carbonate, various acids, various bases, and buffers. Usually, a 1 to 5-day cultivation leads to the 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 target L-amino acid can be collected and purified by ion-exchange, concentration and crystallization methods.
Figure 1 shows the construction of plasmid pΔlacZ.
The present invention will be more concretely explained below with reference to Examples. In the Examples an amino acid is of L-configuration unless otherwise noted.
For cloning of the b2682 and b2683 genes vector pΔlacZ was used. Vector pΔlacZ is a derivative of the vector pET-22b(+) (Novagen, Madison, WI, USA). pET-22b(+) was treated by BglII and XbaI and ligated with polymerase chain reaction (PCR) fragment of plasmid pMB9-lac (
The initial material for cloning of E. coli b2682 and b2683 putative reading frames (b2682 and b2683 genes) was the PCR fragment, which was obtained using DNA from E. coli strain TG1 as a template. For synthesis of this fragment two primers having sequence depicted in SEQ ID Nos: 1 and 2 were used. PCR was carried out on "Perkin Elmer GeneAmp PCR System 2400" under the following conditions: 40 sec. at 95 °C, 40 sec. at 47 °C, 40 sec. at 72 °C, 30 cycles. Thus, the 1158 bp linear DNA fragment contained b2682 and b2683 genes was obtained. This PCR fragment was treated by XbaI and BamHI restrictases and inserted into multicopy vector pΔlacZ previously treated by the same restrictases.
Resulted plasmid with the PCR fragment was named pYGAZH and carried both gene b2682 and b2683 under the control of the lactose promoter (Plac UV5).
Similarly, the initial material for cloning of E. coli b1242 putative reading frame (b1242 gene) was the PCR fragment, which was obtained using DNA from E. coli strain TG1 as a template. For synthesis of this fragment two primers having sequence depicted in SEQ ID Nos: 9 and 10 were used. Resulted plasmid with the PCR fragment was named pYCHE and carried b1242 gene under the control of the lactose promoter (P1ac UV5). The initial material for cloning of E. coli b3434 putative reading frame (b3434 gene) was the PCR fragment, which was obtained using DNA from E. coli strain TG1 as a template. For synthesis of this fragment two primers having sequence depicted in SEQ ID Nos: 13 and 14 were used. Resulted plasmid with the PCR fragment was named pYHGN and carried b3434 gene under the control of the lactose promoter (Plac UV5).
E. coli strain TG1(pYGAZH), TG1(pYCHE), TG1(pYHGN) and TG1 strain having a vector without insertion (control strain) were grown overnight on LB medium supplemented with ampicilline (100 µg/ml). The night cultures of all strains were diluted at 25 times in fresh LB medium supplemented with ampicilline (100 µg/ml) and IPTG (0.5 mM) and were incubated 2 hours at 37 °C with aeration. The log phase cultures were diluted in 0,9% solution of NaCl and about 1000 cells were seeded on plates with solid Adams medium supplemented with ampicilline (100 µg/ml), IPTG (0.5 mM) and amino acid or its analog. After 2 - 4 days incubation at 37 °C the differences in colony size or colony number between the TG1 strain with hybrid plasmid and control TG1 strain were registered. The results of experiments are presented in Table 1.
R - more colonies or colony size
S - less colonies or colony size compare to the control strain
The threonine producing strain VL2054 was transformed by the plasmid pYGAZH carried the b2682 and b2683 genes under the control of Plac UV5 promoter. Obtained strain was named VL2054(pYGAZH). The strain VL2054 is derivative of the strain VKPM B-3996 and carried on its chromosome:
- a) the integrated threonine operon under the control of PR promoter
- b) wild type rhtA gene
- c) the inactivated chromosomal gene encoding transhydrogenase (tdh gene) and inactivated kanamycin resistant gene (kan) gene in the Tn5 (tdh::Tn5, Kan5)
- d) mutation ilvA442·
The strain VL2054 has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (
The 5 colonies of each strain VL2054, strain VL2054(pΔlacZ) as a control strain contained plasmid without insertion and VL2054(pYGAZH) were suspended in 2 ml of minimal medium ((NH4)2SO4 - 11 g/l; NaCl - 0.4 g/l; MgSO4 - 0.4 g/l; K2HPO4 - 1 g/l; FeSO4 - 10 mg/l; MnSO4 - 10 mg/l; thiamin - 0.1 mg/l; yeast extract - 0.5 g/l; glucose - 40 g/l; ampicilline - 300 mg/l if necessary) in 20-ml test tubes and were incubated overnight with aeration at 32 °C. The 0.2 ml of each night culture was transferred to the three 20-ml test tubes with 2 ml of fresh medium for fermentation with or without IPTG and cultivated at 32 °C for 48 or 72 hours with rotary shaker.
After cultivation the plasmid stability and optical absorbance of the medium at 540 nm were determined by conventional methods. Accumulated amount of threonine in the medium was determined by thin layer chromatography (TLC). Liquid phase composition for TLC was as follows: isopropanol - 50 ml, acetone - 50 ml, NH4OH (30 %) - 12 ml, H2O - 8 ml. The results are shown in Table 2. As it is seen, the hybrid plasmid pYGAZH improved the threonine accumulation by the threonine producing strain VL2054.
The valine producing strain H-81 was transformed by the plasmid pYGAZH carried the b2682 and b2683 genes under the control of Plac UV5 promoter. The strain H-81 has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (
The 5 colonies of each strain H-81, H-81(pΔlacZ) as a control strain contained plasmid without insertion and H-81(pYGAZH) were suspended in 2 ml of minimal medium ((NH4)2SO4 - 18 g/l, K2HPO4 - 1.8 g/l, MgSO4 - 1.2 g/l, thiamin - 0.1 mg/l, yeast extract - 0.5 g/l, glucose - 60 g/l, ampicilline - 300 mg/l, if necessary) in 20-ml test tubes and were incubated overnight with aeration at 32 °C. The 0.2 ml of each night culture was transferred to the three 20-ml test tubes with 2 ml of fresh medium for fermentation with or without IPTG and cultivated at 32 °C for 48 or 72 hours with rotary shaker.
After cultivation the plasmid stability and optical absorbance of the medium at 540 nm were determined by conventional methods. Accumulated amount of valine in the medium was determined by TLC. Liquid phase composition for TLC was as follows: isopropanol - 80 ml, ethylacetate - 80 ml, NH4OH (30 %) - 15 ml, H2O - 45 ml. The results are shown in Table 3. As it is seen, the hybrid plasmid pYGAZH improved the valine accumulation by the valine producing strain H-81.
The cells of wild type strain E. coli K12 (VKPM B-7) was treated with a mutagen, N-methyl-N'-nitro-N-nitrosoguanidine (0.1 mg/ml), for 20 min at 37°C, washed and plated on minimal agar medium M9 supplemented with 1.25 mg/ml tryptone, 10 mg/ml L-proline and 0.05 mg/ml 2,3,5-triphenyltetrazolium chloride. Most colonies arisen after 3 day of incubation at 37°C were colored red. A few colonies, which could not oxidize L-proline, were white. One of such colonies was used as a parent for obtaining mutants resistant to proline analogs (3,4-dehydroxyproline and azetidine-2-carboxylate) which were added into M9 agar medium in concentration of 2 mg/ml each.
Some of mutants arisen could produce L-proline. The best L-proline producer 702 was treated with a P1 bacteriophage grown on cells of the strain TG1 in which the gene ilvA was disrupted by the insertion of chloramphenicol (Cm) resistance (Cmr) gene. One of obtained Cm resistant transductant, 702ilvA, which turned to be L-isoleucine auxotroph, was much more effective L-proline producer than the L-isoleucine prototrophic parent strain 702 (Table 4). The fermentation medium contained 60 g/l glucose, 25 g/l ammonium sulfate, 2 g/l KH2PO4, 1 g/l MgSO4, 0.1 mg/l thiamine, 50 mg/l L-isoleucine and 25 g/l chalk (pH 7.2). Glucose and chalk were sterilized separately. 2 ml of the medium was placed into test tubes, and inoculated with one loop of the tested microorganisms, and the cultivation was carried out at 37°C for 2 days with shaking.
The strains 702 and 702ilvA have been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) under the accession number VKPM B-8011 and VKPM B-8012, respectively, since July 25, 2000.
The proline producing strain E. coli 702ilvA was transformed by the plasmid pYGAZH carried the b2682 and b2683 genes under the control of Plac UV5 promoter.
The 5 colonies of each strain 702ilvA, 702ilvA(pΔlacZ) as a control strain contained plasmid without insertion and 702ilvA(pYGAZH) were suspended in 2 ml of minimal medium ((NH4)2SO4 - 18 g/l, K2HPO4 - 1.8 g/l, MgSO4 - 1.2 g/l, thiamin - 0.1 mg/l, yeast extract - 0.5 g/l, glucose - 60 g/l, isoleucine - 50 mg/l, ampicilline - 300 mg/l, if necessary) in 20-ml test tubes and were incubated overnight with aeration at 32 °C. The 0.2 ml of each night culture was transferred to the three 20-ml test tubes with 2 ml of fresh medium for fermentation with or without IPTG and cultivated at 32 °C for 40 hours with rotary shaker.
After cultivation the plasmid stability and optical absorbance of the medium at 540 nm were determined by conventional methods. Accumulated amount of proline in the medium was determined by TLC. Liquid phase composition for TLC was as follows: ethanol - 80 ml, NH4OH (30 %) - 5 ml, H2O - 25 ml. The results are shown in Table 5. As it is seen, the hybrid plasmid pYGAZH improved the proline accumulation by the proline producing strain 702ilvA.
The cells of wild type strain E. coli K12 (VKPM B-7) was treated with a mutagen, N-methyl-N'-nitro-N-nitrosoguanidine (0.05 mg/ml), for 20 min at 37°C, washed 4 times with physiological solution and plated on minimal agar medium M9 supplemented with 4.0 mg/ml DL-4-azaleucine. The plates were incubated for 5 days at 37°C. Colonies appeared on the plates were picked up and purified by streaking on the L-agar plates. One of the obtained mutant resistant to DL-4-azaleucine was used for induction of double L-isoleucine and L-valine auxotrophy. The numerous amount of double auxotrophs, requiring L-isoleucine and L-valine for growth, were obtained. It was shown that double L-isoleucine and L-valine auxotrophy was caused by mutation in the ilvE gene. Among the obtained double auxotrophs, the best L-leucine producer, strain 505 producing 1.8 g/l of L-leucine, has been selected. The fermentation medium contained 60 g/l glucose, 25 g/l ammonium sulfate, 2 g/l KH2PO4, 1 g/l MgSO4, 0.1 mg/l thiamine, 100 mg/l L-isoleucine, 100 mg/l L-valine and 25 g/l chalk (pH 7.2). Glucose and chalk were sterilized separately. 2 ml of the medium was placed into test tubes, and inoculated with one loop of the tested microorganisms, and the cultivation was carried out at 37°C for 2 days with shaking.
The strain E. coli 505 has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (
The leucine producing strain E. coli 505 was transformed by the plasmid pYGAZH carried the b2682 and b2683 genes under the control of Plac UV5 promoter.
The 20 colonies of each strain 505, 505(pΔlacZ) as a control strain contained plasmid without insertion and 505(pYGAZH) were transferred by one loop of culture in 20-ml test tubes with L-broth with or without ampicilline and were incubated overnight with aeration at 32 °C. The 0.1 ml of each night culture was transferred into the 20-ml test tubes (inner diameter 22 mm), suspended in 2 ml of medium for fermentation with or without IPTG and cultivated at 32 °C for 72 hours with rotary shaker.
After cultivation the plasmid stability was determined by conventional method. Accumulated amount of leucine in the medium was determined by TLC. Liquid phase composition for TLC was as follows: isopropanol - 80 ml, ethylacetate - 80 ml, NH4OH (30 %) - 25 ml, H2O - 50 ml. The results are shown in Table 6. As it is seen, the hybrid plasmid pYGAZH improved the leucine accumulation by the leucine producing strain 505.
The plasmidless threonine and leucine deficient strain E. coli C600 was used as a parental strain. At first, the Leu+ variants of E. coli C600 strain was obtained by transduction of phage P1 grown on E. coli K-12 strain. Then, after treatment with N-methyl-N'-nitro-N-nitrosoguanidine (NTG) the mutant strain 44 resistant to 8 g/l of L-homoserine has been obtained. The strain 44 is L-threonine-deficient, resistant to high concentrations of L-homoserine. The strain 44 has been deposited in Russian National Collection of Industrial Microorganisms (VKPM) under the accession number VKPM B-2175.
Then, the strains, which are the mutants resistant to a methionine analog, norleucine, was induced from strain 44 by mutagenesis using NTG. The cells of night culture grown in L-broth were spun down and resuspended in physiological solution (0.9% NaCl) containing 50 µg/ml of NTG. After 30 min of exposure with NTG at 37° C the cells were spun down, washed 4 times with physiological solution and plated on the minimal agar medium M9, containing 0.5 mg/ml of threonine and 2.5 mg/ml or 5.0 mg/ml of norleucine. The plates were incubated for 5 days at 37°C. Colonies appeared on the plates were picked up and purified by streaking on the L-agar plates. The best L-methionine producer among them was strain 218. Test-tube cultivation of the novel strain 218 carried out at 32°C for 3 days with shaking leads to accumulation in the culture medium about 1 g/l of L-methionine. As a fermentation medium was used minimal medium M9 containing glucose (4%), ammonia sulfate (2.5%), threonine (0.5 g/l), calcium carbonate (25 g/l). Glucose and chalk were sterilized separately.
The strain 218 has been deposited in Russian National Collection of Industrial Microorganisms (VKPM) under the accession number VKPM B-8125 since May 14, 2001, and transferred from the original deposit to international deposit based on Budapest Treaty on February 1, 2002.
Further, the phage P1 mediated deletion of ppc gene has been introduced into strain 218 followed by integration of pycA gene from Bacillus subtilis (
The strain E. coli 73 has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (
The methionine producing strain E. coli 73 was transformed by the plasmid pYGAZH carried the b2682 and b2683 genes under the control of Plac UV5 promoter.
The 5 colonies of each strain 73, 73(pΔlacZ) as a control strain contained plasmid without insertion and 73(pYGAZH) were suspended in 2 ml of minimal medium ((NH4)2SO4 - 18 g/l, K2HPO4 - 1.8 g/l, MgSO4 - 1.2 g/l, thiamin - 0.1 mg/l, yeast extract - 10 g/l, glucose - 60 g/l, threonine - 400 mg/l, ampicilline - 300 mg/l, if necessary) in 20-ml test tubes and were incubated overnight with aeration at 32 °C. The 0.2 ml of each night culture was transferred to the three 20-ml test tubes with 2 ml of fresh medium for fermentation with or without IPTG and cultivated at 32 °C for 48 hours with rotary shaker.
After cultivation the plasmid stability and optical absorbance of the medium at 540 nm were determined by conventional methods. Accumulated amount of methionine in the medium was determined by TLC. Liquid phase composition for TLC was as follows: isopropanol - 80 ml, ethylacetate - 80 ml, NH4OH (30 %) - 15 ml, H2O - 45 ml. The results are shown in Table 7. As it is seen, the hybrid plasmid pYGAZH improved the methionine accumulation by the methionine producing strain 73.
The threonine producing strain VL2054 was transformed by the plasmid pYCHE carried the b1242 gene under the control of Plac UV5 promoter. Obtained strain was named VL2054(pYCHE).
The 5 colonies of each strain VL2054, strain VL2054(pΔ1acZ) as a control strain contained plasmid without insertion and VL2054(pYCHE) were suspended in 2 ml of minimal medium ((NH4)2SO4 - 11 g/l; NaCl - 0.4 g/l; MgSO4 - 0.4 g/l; K2HPO4 - 1 g/l; FeSO4 - 10 mg/l; MnSO4 - 10 mg/l; thiamin - 0.1 mg/l; yeast extract - 0.5 g/l; glucose - 40 g/l; ampicilline - 300 mg/l if necessary) in 20-ml test tubes and were incubated overnight with aeration at 32 °C. The 0.2 ml of each night culture was transferred to the three 20-ml test tubes with 2 ml of fresh medium for fermentation with or without IPTG and cultivated at 32 °C for 45 hours with rotary shaker. Fermentation medium composition:
After cultivation the plasmid stability and optical absorbance of the medium at 540 nm were determined by conventional methods. Accumulated amount of threonine in the medium was determined by thin layer chromatography (TLC). Liquid phase composition for TLC was as follows: isopropanol - 50 ml, acetone - 50 ml, NH4OH (30 %) - 12 ml, H2O - 8 ml. The results are shown in Table 8. As it is seen, the hybrid plasmid pYCHE improved the threonine accumulation by the threonine producing strain VL2054.
The valine producing strain H-81 was transformed by the plasmid pYCHE carried the b1242 gene under the control of Plac UV5 promoter.
The 5 colonies of each strain H-81, H-81(pΔlacZ) as a control strain contained plasmid without insertion and H-81(pYCHE) were suspended in 2 ml of minimal medium ((NH4)2SO4 - 18 g/l, K2HPO4 - 1.8 g/l, MgSO4 - 1.2 g/l, thiamin - 0.1 mg/l, yeast extract - 0.5 g/l, glucose - 60 g/l, ampicilline - 300 mg/l, if necessary) in 20-ml test tubes and were incubated overnight with aeration at 32 °C. The 0.2 ml of each night culture was transferred to the three 20-ml test tubes with 2 ml of fresh medium for fermentation with or without IPTG and cultivated at 32 °C for 45 hours with rotary shaker.
After cultivation the plasmid stability and optical absorbance of the medium at 540 nm were determined by conventional methods. Accumulated amount of valine in the medium was determined by TLC. Liquid phase composition for TLC was as follows: isopropanol - 80 ml, ethylacetate - 80 ml, NH4OH (30 %) - 15 ml, H2O - 45 ml. The results are shown in Table 9. As it is seen, the hybrid plasmid pYCHE improved the valine accumulation by the valine producing strain H-81.
The arginine producing strain 382 was transformed by the plasmid pYHGN carried the b3434 gene under the control of Plac UV5 promoter. The strain 382 has been deposited in the Russian National Collection of Industrial Microorganisms (VKPM) (
The 5 colonies of each strain 382, 382(pΔlacZ) as a control strain contained plasmid without insertion and 382(pYHGN) were suspended in 2 ml of minimal medium ((NH4)2SO4 - 25.0 g/l, K2HPO4 - 2.0 g/l, MgSO4 7H2O- 1.0 g/l, thiamin - 0.2 mg/l, yeast extract - 5 g/l, glucose - 60 g/l, ampicilline - 100 mg/l, if necessary) in 20-ml test tubes and were incubated overnight with aeration at 32 °C. The 0.2 ml of each night culture was transferred to the three 20-ml test tubes with 2 ml of fresh medium for fermentation with or without IPTG and cultivated at 32 °C for 72 hours with rotary shaker.
After cultivation the plasmid stability and optical absorbance of the medium at 540 nm were determined by conventional methods. Accumulated amount of arginine in the medium was determined by TLC. Liquid phase composition for TLC was as follows: isopropanol - 80 ml, ethylacetate - 40 ml, NH4OH (30 %) - 25 ml, H2O - 50 ml. The results are shown in Table 10. As it is seen, the hybrid plasmid pYHGN improved the arginine accumulation by the arginine producing strain 382.
The proline producing strain E. coli 702ilvA was transformed by the plasmid pYHGN carried the b3434 gene under the control of Plac UV5 promoter.
The 5 colonies of each strain 702ilvA, 702ilvA(pΔlacZ) as a control strain contained plasmid without insertion and 702ilvA(pYHGN) were suspended in 2 ml of minimal medium ((NH4)2SO4 - 18 g/l, K2HPO4 - 1.8 g/l, MgSO4 - 1.2 g/l, thiamin - 0.1 mg/l, yeast extract - 0.5 g/l, glucose - 60 g/l, isoleucine - 50 mg/l, ampicilline - 300 mg/l, if necessary) in 20-ml test tubes and were incubated overnight with aeration at 32 °C. The 0.2 ml of each night culture was transferred to the three 20-ml test tubes with 2 ml of fresh medium for fermentation with or without IPTG and cultivated at 32 °C for 40 hours with rotary shaker.
After cultivation the plasmid stability and optical absorbance of the medium at 540 nm were determined by conventional methods. Accumulated amount of proline in the medium was determined by TLC. Liquid phase composition for TLC was as follows: ethanol - 80 ml, NH4OH (30 %) - 5 ml, H2O - 25 ml. The results are shown in Table 11. As it is seen, the hybrid plasmid pYHGN improved the proline accumulation by the proline producing strain 702ilvA.
- <110> Ajinomoto Co., Inc.
- <120> METHOD FOR PRODUDCING L-AMINO ACID USING BACTERIA BELONGING TO THE GENUS ESCHERICHIA
- <130> EPA-53844
- <140>
- <150>
RU 2001103865
<151> 2001-02-13 - <150>
RU 2001104998
<151> 2001-02-26 - <150>
RU 2001104999
<151> 2001-02-26 - <150>
RU 2001117632
<151> 2001-06-28 - <150>
RU 2001117633
<151> 2001-06-28 - <160> 16
- <170> Patentln Ver. 2.0
- <210> 1
<211> 26
<212> DNA
<213> Artificial Sequence - <220>
<223> Description of Artificial Sequence:primer - <400> 1
ggtctagaca atcgttaagc gtacac 26 - <210> 2
<211> 26
<212> DNA
<213> Artificial Sequence - <220>
<223> Description of Artificial Sequence:primer - <400> 2
ccggatccga tatagtaacg acagtg 26 - <210> 3
<211> 738
<212> DNA
<213> Escherichia coli - <220>
<221> CDS
<222> (1) .. (735) - <400> 3
- <210> 4
<211> 245
<212> PRT
<213> Escherichia coli - <400> 4
- <210> 5
<211> 336
<212> DNA
<213> Escherichia coli - <220>
<221> CDS - <222> (1) .. (333)
- <400> 5
- <210> 6
<211> 111
<212> PRT
<213> Escherichia coli - <400> 6
- <210> 7
<211> 37
<212> DNA
<213> Artificial Sequence - <220>
<223> Description of Artificial Sequence:primer - <400> 7
cctttggtac cagatctgcg ggcagtgagc gcaacgc 37 - <210> 8
<211> 34
<212> DNA
<213> Artificial Sequence - <220>
<223> Description of Artificial Sequence:primer - <400> 8
ctgtttctag atcctgtgtg aaattgttat ccgc 34 - <210> 9
<211> 28
<212> DNA
<213> Artificial Sequence - <220>
<223> Description of Artificial Sequence:primer - <400> 9
ggtctagata tggctaacat tatccggc 28 - <210> 10
<211> 28
<212> DNA
<213> Artificial Sequence - <220>
<223> Description of Artificial Sequence:primer - <400> 10
ccggatccaa acggagcatg gcagctcc 28 - <210> 11
<211> 648
<212> DNA
<213> Escherichia coli - <220>
<221> CDS
<222> (1).. (645) - <400> 11
- <210> 12
<211> 215
<212> PRT
<213> Escherichia coli - <400> 12
- <210> 13
<211> 28
<212> DNA
<213> Artificial Sequence - <220>
<223> Description of Artificial Sequence:primer - <400> 13
ggtctagagt ccgcggcaat tatcaggg 28 - <210> 14
<211> 29
<212> DNA
<213> Artificial Sequence - <220>
<223> Description of Artificial Sequence:primer - <400> 14
ccagatctgg tagttgtgac gctaccggg 29 - <210> 15
<211> 594
<212> DNA
<213> Escherichia coli - <220>
- <221> CDS
<222> (1).. (591) - <400> 15
- <210> 16
<211> 197
<212> PRT
<213> Escherichia coli - <400> 16