The method of obtaining l-leucine (options), escherichia coli 505/pacyc-tyr b producing l-leucine

 

The invention relates to the microbiological industry. Perform receiving L-leucine using bacteria belonging to the genus Escherichia, which produces L-valine, L-isoleucine and L-hemolysin in the quantity < 1% of the amount of produced L-leucine, due to the inactivation of the ilvE gene encoding the branched-chain amino acid aminotransferase, and which produces an increased amount of L-leucine as a result of increased activity of transaminases aromatic amino acids encoded by the genome of tyrB. The invention improves the production of L-leucine by reducing the production of L-valine, L-isoleucine and L-hemolysin. 3 N. C.p. f-crystals, 5 C.p. f-crystals, 1 Il., 1 tab.,

Prerequisites to the creation of inventions

The technical field

The present invention relates to the microbiological industry, in particular to a method of production of amino acids. More specifically the present invention relates to a method for producing L-leucine using bacteria belonging to the genus Escherichia, in which the amount of produced L-valine, L-isoleucine and L-homoserine less than 1% of the amount of produced L-leucine.

Prior tov microorganisms, obtained from natural sources, or their mutants, specially modified to increase the productivity of L-amino acids.

There are various strains of bacteria belonging to the genus Escherichia, which are used for the production of L-leucine fermentation. It strains resistant to L-Latino and its analogs such as 4-isoleucin or 5,5,5-triptorelin (U.S. patent 567446331),-2-titillans and-hydroxylation (U.S. patent 5763231), L-valine, 4-isoleucin, 3-hydroxylation and L-leucine (RF patent 2140450); strains that requires lipoic acid for growth (U.S. patent 6214591); strains with increased activity of enzymes involved in the biosynthesis of L-leucine, such as ilvE gene (U.S. patent 5120654); strains in which the target enzyme is absent sensitivity to inhibition produced L-leucine by the type of feedback, such as isopropylmalate synthase (European patent EP 1067191).

Almost all known strains - producers of L-leucine at the same time produce L-valine and small amounts of L-isoleucine. For example, a strain of E. coli J 11478 (U.S. patent 5763231) simultaneously produces 1.9 g/l of L-leucine and 0.09 g/l L-valine (the number of L-valine is 4.7% of the number of L-latyninoj fluid. In addition, by-products of L-valine and L-isoleucine reduces the yield of L-leucine, since both amino acids are derived from a common precursor - 2-ketoisovaleric.

Previously it was shown that some non-natural amino acids, such as Norvaline, homosalate and norleucine, can be formed in Serratia marcescens using enzymes of the biosynthesis of L-leucine from-ketobutyrate,-keto-methylvalerate andketomalonate, respectively (Kisumi, M., Sugiura, M. and I. Chibata, J. Biochem. 1976, 80(2) 333-9).

Description of the invention

The aim of the present invention to provide a bacteria - bacteria producing L-leucine, which produces L-valine, L-isoleucine and L-hemolysin in the quantity < 1% of the amount of produced L-leucine.

This goal is achieved by inactivation of the ilvE gene encoding the branched-chain amino acid aminotransferase.

Inactivation of the gene ilvE decreases the production of L-leucine, as this aminotransferase involved in the formation of L-leucine from its keto-predecessor - 2-keto-methylpentanoate. Another aminotransferase, which may participate in the production of L-leucine is transaminase levels of CIN in the case of inactivation ilvE gene was increased activity of the enzyme, encoded tyrB gene, for example, by transforming bacteria mnogostadiinoi a plasmid containing the gene tyrB.

Thus was accomplished the present invention.

Thus, the present invention provides a bacterium belonging to the genus Escherichia, producing L-leucine, which produces L-valine, L-isoleucine and L-hemolysin in the quantity < 1% of the amount of produced L-leucine. Further, the present invention provides a bacterium belonging to the genus Escherichia, producing L-leucine, which produces L-valine, L-isoleucine and L-hemolysin in the quantity < 1% of the amount of produced L-leucine, and in which the production of L-leucine enhanced by increasing the activity of the enzyme encoded by the genome of tyrB.

Further, the present invention provides a method of obtaining L-leucine fermentation, including the stage of growth of the indicated bacteria in a nutrient medium to produce and accumulate L-leucine in the medium and excretion of L-leucine from the culture fluid.

Thus, the present invention provides the following:

(1). The bacterium belonging to the genus Escherichia, producing L-leucine, which produces L-valine, L-isoleucine and L-hemolysin in number, munisipality and L-hemolysin in number, < 1% of the amount of produced L-leucine due to inactivation of the gene HvE or reduce the activity of the proteins encoded by ilvE gene.

(3). The bacterium according to (2) in which the activity of the proteins encoded by the genome of tyrB, increased.

(4). The bacterium according to (3) in which the activity of the proteins encoded by the genome of tyrB, enhanced by the transformation specified bacteria DNA, containing the gene tyrB.

(5). The bacterium according to (4), in which the transformation is done using mnogoopytnogo vector.

(6). The method of obtaining L-leucine, which includes stages:

- growing bacteria in accordance with (1) - (5) in a nutrient medium to produce and accumulate L-leucine in the medium, and

- selection of L-leucine from the culture fluid.

(7). The method according to (7), in which the bacterium is modified to increase the expression of genes of the biosynthesis of L-leucine.

1. The bacterium according to the present invention

The bacterium according to the present invention is a bacterium belonging to the genus Escherichia, producing L-leucine, which produces L-valine, L-isoleucine and L-hemolysin in the quantity < 1% of the amount of produced L-leucine.

The term “bacteria producing L-leucine”, ISOLITE more than the wild-type strain or a parent strain of E. coli, such as E. coli strain K-12, and preferably means that the microorganism is able to produce and accumulate in the medium is not less than 0.5 g/l, more preferably not less than 1.0 g/l of L-leucine.

The term “bacterium belonging to the genus Escherichia” means that the bacterium belongs to the genus Escherichia according to the classification known to a specialist in the field of Microbiology. As examples of the microorganism belonging to the genus Escherichia, used in the present invention, may be mentioned the bacterium Escherichia coli (E. coli).

The term “produces L-valine, L-isoleucine and L-hemolysin in the quantity < 1% of the amount of produced L-leucine”, means that the number of L-valine, L-isoleucine and L-hemolysin, which is present in the medium after cultivation of bacteria - bacteria producing L-leucine, significantly lower in comparison with the quantity of the main product, L-leucine. The number of L-valine, L-isoleucine and L-hemolysin significantly lower in comparison with L-leucine in the case when, for example, the amount of each of these compounds as L-valine, L-isoleucine and L-hemolysin, less than 1% of the amount of produced L-leucine. Preferably, this oznacza for example, thin-layer chromatography (TLC) or HPLC.

The term “inactivation ilvE gene” means that the target gene is modified so that the modified gene encodes a mutant (inactive) enzyme with not detektivami known methods by level of activity or the modified gene is not able to encode any enzyme. IlvE gene encodes the branched-chain amino acid transaminase (309 amino acid residues), which catalyzes the amination reaction-ketocarbofuran and their salts. Specified transaminase levels of branched chain amino acids converts, for example,-keekaroo in L-leucine,-ketoisovaleric in L-valine,-keto--methylvalerate in L-isoleucine. IlvE gene (nucleotides with 3950107 on 3951036 sequence number NC 000913.1 in GenBank gi:16131628) is located between genes and ilvM ilvD. Inactivation of the gene can be produced by traditional methods, such as mutagenic treatment with UV-radiation or treatment nitrosoguanidine (N-methyl-N’-nitro-N-nitrosoguanidine), site-specific mutagenesis, and the disintegration of the gene using homologous recombination or/and insti protein, encoded by ilvE gene” means that the ilvE gene sequence encoding a protein, or a sequence that regulates gene expression ilvE, were modified so that the specific activity of the enzyme decreased. The decreased activity of the specified protein can be carried out by traditional methods, such as mutagenic treatment with UV-radiation or treatment nitrosoguanidine (N-methyl-N’-nitro-N-nitrosoguanidine) or site-specific mutagenesis and subsequent selection of bacteria with the desired phenotype. Bacteria containing the specified protein mutation type “leaky”, can also be used in the present invention. Protein containing a mutation type “leaky”, is mutant protein, which changes the sequence does not lead to a complete loss of its activity (Lewin Century, Genes VII, Oxford Press, 2000, p. 16).

Also, the bacterium according to the present invention is a bacterium belonging to the genus Escherichia, producing L-leucine, which produces L-valine, L-isoleucine and L-hemolysin in the quantity < 1% of the amount of produced L-leucine, and in which the activity of the proteins encoded by the genome of tyrB, increased.

The term “activity of the proteins encoded by the genome of tyrB, raised” means, cruet the aromatic amino acid transaminase (397 amino acid residues), which, using glutamate as a donor of amino groups, catalyzes transamination-ketoacids, such as phenylpyruvate and 4-hydroxyphenylpyruvate, with the formation of phenylalanine and tyrosine, respectively. But the term “activity” is used here, means the activity of transformingKetoprofen in L-leucine using glutamate as a donor of amino groups (Escherichia coli and Salmonella, Second Edition, Editor in Chief: F. C. Neidhardt, ASM Press, Washington D. C., 1996). TyrB gene (nucleotides with 4264693 on 4265886 in the sequence NC_000913.1 in the database GenBank gi:16131880) is located between the genes of air and aphA.

Methods of increasing the activity of the protein according to the present invention, in particular the methods of increasing the number of molecules of a specified protein in the cell, the methods include increasing the number of copies of the gene and methods for modifying the sequence that regulates the expression of DNA that encodes this protein, or a sequence of enhacer”, but are not limited to.

The term “transformation of bacteria with DNA containing the tyrB gene” means the introduction of a specified DNA in a bacterial cell, for example, using traditional methods to increase the number of copies of the gene. The number of copies of the gene can be Ovelia obtained recombinant DNA in a microorganism. Examples of vectors used for the introduction of recombinant DNA, are plasmid vectors such as pMW118, pBR322, pUC19, pET22b, pACYC184, and the like, phage vectors, such as 11059, 1BF101, M13mp9, phage Mu (lined patent application of Japan No. 2-109985) and the like, and the transposon (Berg, D. E. and Berg, C. M., Bio/Technol, 1,417 (1983)), such as Mu, Tn10, Tn5, and the like. In addition, amplification of gene expression can be achieved by integration of the gene into a bacterial chromosome by the method of homologous recombination or the like.

Methods for modifying the sequence that regulates the expression or sequence of enhacer” can be used in conjunction with methods to increase the number of copies of the gene.

For removing microorganism belonging to the genus Escherichia and having increased expression of a specified gene, the desired gene can be obtained by PCR (polymerase chain reaction) based mainly on the already available information about genes of E. coli. For example, tyrB gene can be cloned from the chromosomal DNA of the strains E. coli K12 or E. coli MG 1655 using the method of PCR. Chromosomal DNA required for this purpose can also be obtained from any other strain of E. coli.

Changing the sequence of the governing Exo present invention, under the control of a strong promoter. As strong promoters are known, for example, lac promoter, trp promoter, trc promoter, PL promoter of phage lambda. On the other hand, the promoter can be enhanced, for example, by introducing mutations in the indicated promoter to increase the level of transcription of a gene located after the promoter. Further, it is known that substitution of several nucleotides in the area between the binding site of the ribosome (RBS) and start codon, and in particular, in the sequence immediately before the start-codon, has a significant impact on transliruemie mRNA (Gold et al., Annu. Rev. Environ, 35, 365-403, 1981; Hui et al., EMBO J., 3, 623-629, 1984).

Moreover, an “enhancer” can be added to increase the transcription level of the specified gene. Introduction DNA containing either the gene or the promoter in the chromosomal DNA, as described, for example, in patent application laid Japan No. 1215280(1989).

The bacterium according to the present invention can be further improved by increasing the expression of one or more genes involved in the biosynthesis of L-leucine. Examples of such genes are genes latinoware operon, leu operon, preferably the gene encoding isopropenylacetate (leuA gene, nucleotides with 81958 aniu L-leucine by the type of feedback (European patent EP 1067191). Lazenby operon also includes leuB genes (gi:16128067), leuC (gi:16128066) and leuD (gi:16128065) (nucleotides with 80867 on 81961, 79464 on 80864 and 78848 on 79453 in the sequence NC_000913.1 in GenBank, respectively).

As the source of the strain, which is inactivated transaminase activities of branched chain amino acids encoded by ilvE gene, and increased activity of transaminases aromatic amino acids encoded by the genome of tyrB can be used by bacteria belonging to the genus Escherichia, such as E. coli K12, E. coli W1660 and the like. Can also be used as a parental strain of the bacterium belonging to the genus Escherichia, the producers of L-leucine, such as H-9068 (ATSS 21530), N-9070 (FERM BP-4704) and N-9072 (FERM BP-4706), resistant to 4-isoleucine or 5,5,5-triptorelin (U.S. patent 5744331), strains of E. coli containing isopropylpalmitate, whose sensitivity to Latino type of feedback lost (European patent ER), a strain of E. coli AJ11478, resistant-2-titillans and-hydroxylysine (U.S. patent 5763231) and the like.

Methods of obtaining plasmid DNA, digestion and ligation of DNA, transformation, selection of an oligonucleotide as a nucleating and similar aimer, in the book of Sambrook, J., Fritsch, E. F., and Maniatis, T., "Molecular Cloning A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press (1989).

2. The method according to the present invention

The method according to the present invention is a method of obtaining L-leucine, including the stage of growth of bacteria according to the present invention in a nutrient medium to produce and accumulate L-leucine in the medium, and excretion of L-leucine from the culture fluid.

According to the present invention, the cultivation, isolation and purification of L-leucine from the culture or similar fluid may be carried out in a manner similar to traditional methods of fermentation, in which the amino acid is produced using a microorganism.

The nutrient medium used for cultivation, can be both synthetic and natural, provided that the medium contains sources of carbon, nitrogen, mineral supplements and, if necessary, the appropriate amount of nutritional supplements necessary for growth of microorganisms. The carbon sources include various carbohydrates such as glucose and sucrose, and various organic acids. Depending on the nature of assimilation used microorganism can appearancesa ammonium salts, such as ammonia and ammonium sulfate, other nitrogen compounds such as amines, a natural nitrogen sources such as peptone, soybean hydrolysate, fermentolizat microorganisms. As mineral additives can be used potassium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, calcium chloride and similar compounds. As vitamins can be used thiamin and yeast extract.

The cultivation is carried out preferably under aerobic conditions, such as mixing the culture fluid on the rocking chair, stirring with aeration, at a temperature in the range from 20 to 40°C, preferably in the range from 30 to 38°C. the pH of the environment is maintained within the range of from 5 to 9, preferably from 6.5 to 7.2. pH can be adjusted by ammonia, calcium carbonate, various acids, bases and buffer solutions. Usually growing within 1 to 5 days, resulting in the accumulation of the target L-amino acid in the culture fluid.

After cultivation, solids such as cells can be removed from the culture fluid by centrifuging or by filtration through a membrane, and then L-leucine can be isolated and purified by methods of ion-exchange chromatography is Lina

The best way of carrying out the invention

In more detail the present invention will be explained below with reference to examples.

Example 1. Getting bacteria belonging to the genus Escherichia, producer of L-leucine.

To implement this approach, cells natural strain E. coli K-12 (VKPM B-7) were treated with the mutagen N-methyl-N’-nitro-N-nitrosoguanidine (0.05 mg/ml) for 20 min at 37°C, washed 4 times with saline and sown on minimal M9 medium with agar containing 4.0 mg/ml DL-4-isoleucine. The cups were incubated for 5 days at 37°C. Colonies appearing on the plates were selected and purified on cups in L-agar by applying the strips. The best of the obtained mutants that are resistant to DL-4-isoleucine, 55 mutant was produced 2.1 g/l of L-leucine and 0.8 g/l L-valine (table, see below). This strain of E. coli 55 was selected and used to produce a double auxotrophy of L-isoleucine and L-valine. There were a large number of double auxotrophs requiring L-isoleucine and L-valine for growth. Among the obtained dual auxotrophs was selected as the best producer of L-leucine - strain 505, producing 1.3 g/l of L-leucine. The specified strain did not produce L-valine and L-isoleucine, but double auxotrophy led to women ilvE. This was confirmed by the fact that the introduction of plasmids containing the ilvE gene, strain 505 fully complementable double auxotrophy of L-isoleucine and L-valine. Moreover, the measurement of the enzymatic activity of the branched-chain amino acid aminotransferase encoded by ilvE gene from strain 505, using a 2-ketoisovaleric as substrate showed her lack of any activity. The measurement conditions of the enzymatic activity described Paste R. H. and Kohlhaw, G. (Nonidentity of the aspartate and the aromatic aminotransferase components oftransaminase A in E. coli. J. Bacteriology, 1972, 112(1), p.365-371).

Strain 505 was deposited in the Russian national collection of industrial microorganisms (VKPM) (Russia, 113545 Moscow, 1st Road travel, 1) may 14, 2001, under inventory number VKPM B-8124.

Example 2. Cloning tyrB gene from E. coli plasmid pACYC185.

A fragment of the chromosome of E. coli strain K-12 (VKPM B-7) containing tyrB gene was amplified by PCR using nucleating 1 (SEQ ID NO:1) and 2 (SEQ ID NO:2) shown in the List of sequences. Seed 1 and 2 (24-tier) contain sequences of sites Wamn and HindIII, respectively, attached to their 5’-ends. Then VMN - indIII DNA fragment length 1.7 T. p. A. was Legerova on the corresponding sites of plasmid pACYC184 (Chang, A. C. Y. and Cohen, S. N.,ACYC184, Nucleic Acids Res., 16, 355, 1988) to obtain the plasmid pACYC-tyrB. Plasmid pACYC-tyrB was introduced into the cells of the strain E. coli by the method of transformation was thus obtained strain 505/pACYC-tyrB.

Example 3. The effect of amplification tyrB gene on the production of L-leucine

One loop of each of the strains 55, 505, 505/pACYC-tyrB was transferred into 20 ml test tubes with L-broth and the culture was incubated overnight with aeration at 32°C. 0.1 ml of each overnight culture was transferred into a 20 ml test tube (inner diameter 22 mm), diluted in 2 ml of culture medium for fermentation and were grown at 32°C for 48 hours on a rotary shaker. The nutrient medium for fermentation contained 60 g/l glucose, 25 g/l ammonium sulfate, 2 g/l KN2RHO41 g/l MgSO4, 0.1 mg/l thiamine, 5 g/l yeast extract Difco and 25 g/l of chalk (pH 7.2). Glucose and chalk were sterilized separately.

After growing the traditional method was determined the stability of plasmids. The number of L-leucine, accumulated in the culture fluid was determined by TLC. The mobile phase for TLC had the following composition: isopropanol - 80 ml, ethyl acetate: 80 ml, NH4OH (30%) - 25 ml, H2O - 50 ml.

As can be seen from Tabanou production of L-leucine. The tyrB gene amplification increased the accumulation of L-leucine strain 505 - producer of L-leucine.

Claims

1. The method of obtaining L-leucine, including the stage of growth of bacteria Escherichia coli in a nutrient medium and a selection from the culture fluid obtained and accumulated in it L-leucine, characterized in that as a producer - L-leucine using the bacterium Escherichia coli, which produces L-valine, L-isoleucine and L-hemolysin in the quantity < 1% of the amount of produced L-leucine, due to inactivation of the gene ilvE or reduce the activity of the proteins encoded by ilvE gene.

2. The method according to p. 1, characterized in that the bacterium additionally modified so that, in the bacteria enhanced the expression of genes of the biosynthesis of L-leucine.

3. The method of obtaining L-leucine, including the stage of growth of bacteria Escherichia coli in a nutrient medium and a selection from the culture fluid obtained and accumulated in it L-leucine, characterized in that as a producer - L-leucine using the bacterium Escherichia coli, which produces L-valine, L-isoleucine and L-hemolysin in the quantity < 1% of the amount of produced L-leucine, due to inactivation of the gene is ' series of raised.

4. The method according to p. 3, characterized in that the activity of the proteins encoded by the genome of tyrB, enhanced by transformation of the bacterium with a DNA fragment containing a gene tyrB.

5. The method according to p. 4, characterized in that the transformation is done using mnogoopytnogo vector.

6. The method according to p. 5, characterized in that the bacterium additionally modified so that, in the bacteria enhanced the expression of genes of the biosynthesis of L-leucine.

7. The method according to p. 5, characterized in that as a producer of L-leucine using a strain of Escherichia coli 505/resus-tyr.

8. The strain Escherichia coli 505/pACYC-tyrB producing L-leucine.



 

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