RU2288268C2 - 6-phosphogluconolactonase from escherichia coli, dna fragment, microorganism belonging to escherichia genus as producer of l-amino acid and method for preparing l-amino acids - Google Patents

Abstract

FIELD: biotechnology, biochemistry, enzymes, amino acids, microbiology.

SUBSTANCE: invention relates to a method for producing and preparing L-amino acids, such as L-tryptophan, L-phenylalanine, L-tyrosine and L-histidine that are prepared by culturing the modified microorganism of genus Escherichia with enhanced activity of 6-phosphogluconolactonase. The claimed invention provides preparing indicated amino acids with the high degree of effectiveness.

EFFECT: enhanced yield of amino acids.

15 cl, 9 dwg, 5 tbl, 14 ex

Description

The field of technology.

The present invention relates to biotechnology, specifically to a gene derived from the bacterium Escherichia coli encoding 6-phosphogluconolactonase, and to a method for producing L-amino acids by fermentation. This gene contributes to an increase in the production of L-amino acids, namely aromatic L-amino acids, such as L-tryptophan, L-phenylalanine and L-tyrosine.

The prior art.

The pentose phosphate pathway (PPP) is an important part of central metabolism in most organisms. The synthesis of NADP-H occurs in the oxidative branch of the pentose phosphate pathway, and phosphorylated carbohydrates, intermediate compounds of the non-oxidative branch of the pentose phosphate pathway, are the precursors of the biosynthesis of nucleotides (ribose-5-phosphate), aromatic amino acids and vitamins (erythrozo-5-phosphate). Erythrose-4-phosphate (E4p) is the main precursor of the general pathway for the biosynthesis of aromatic L-acids. Thus, optimization of specific pathways of phosphoenolpyruvate (PEP) and E4p biosynthesis can positively affect the production of aromatic L-amino acids.

The oxidative branch of the pentose phosphate cycle includes three reactions. The first and third reactions are catalyzed by the well-studied enzymes glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and gluconate-6-phosphate dehydrogenase (EC 1.1.1.44), which are encoded by the zwf and gnd genes, respectively. The second reaction is the hydrolysis of 6-phosphogluconolactone to form 6-phosphogluconate (Escherichia coli and Salmonella, Second Edition, Editor in Chief: F.C. Neidhardt, ASM Press, Washington D.C., 1996). An enzyme that catalyzes this reaction has been found in many organisms, including, for example, humans (Collard, F., et al, FEBS Lett., 459: 2, 223-6 (1999)), Trypanosoma brucei (Duffieux, F. , et al, J. Biol. Chem., 275: 36, 27559-65 (2000)), Plasmodium berghei (Clarke, JL, et al, Eur. J. Biochem., 268: 7, 2013-9 (2001) ), Pseudomonas aeroginosa (Hager PW et al, J. Bacteriol., 182: 14, 3934-41 (2000)), Pseudomonas putida (Petruschka, L., et al, FEMS Microbiol. Lett., 215: 1, 89- 95 (2002)), although it is also known that this reaction can proceed spontaneously.

δ-6-Phosphogluconolactone, one of the reaction products catalyzed by glucose-6-phosphate dehydrogenase, is able to form the γ-6-phosphogluconolactone isomer by intramolecular rearrangement. Only δ-6-phosphogluconolactone can spontaneously hydrolyze to form 6-phosphogluconate, and the well-known 6-phosphogluconolactonase enzymes (EC 3.1.1.31) catalyze exactly the same reaction (Miclet E. et al., J Biol Chem., 276: 37, 34840 -46 (2001)). The E. coli pgl gene, hypothetically presumably encoding 6-phosphogluconolactonase, was mapped on the E. coli chromosome between att-λ and the chlD gene (in modern databases, the modC gene). Mutants of E. coli (pgl ^- ) have the phenotype "maltose-blue" - blue colonies on a medium with maltose (Kupor, SR and Fraenkel, DG, J. Bacteriol., 100: 3, 1296-1301 (1969)), which a distinctive feature of the strains accumulating maltodextrin (Adhya S. and Schwartz M., J Bacteriol, 108: 2, 621-626 (1971)).

But at present, neither the nucleotide sequence nor the exact location of the pgl gene on the E. coli chromosome is known. No enzymes with 6-phosphogluconolactonase activity were isolated from E. coli, and there are no publications linking an increase in the activity of 6-phosphogluconolactonase in the bacterial cell producing L-amino acids with an increase in the production of L-amino acids by this bacterium.

Description of the invention.

The aim of the present invention is to provide

6-phosphogluconolonctonases from E. coli, to increase the production of aromatic L-amino acids by producer strains, as well as a method for producing aromatic L-amino acids by said strains.

This goal was achieved by identifying the fact that the open reading frame (ORF) of ybhE in E. coli K-12 strain encodes 6-phosphogluconolactonase and enhancing the expression of ybhE ORF (pgl gene) can increase the production of L-tryptophan by the corresponding producer strains of L- tryptophan. Thus, the present invention has been completed.

The aim of the present invention is the provision of 6-phosphogluconolactonase selected from the group consisting of the following proteins:

(A) a protein comprising the amino acid sequence shown in sequence listing number 2 (SEQ ID NO: 2); and

(B) a protein comprising an amino acid sequence comprising deletions, substitutions, insertions, or addition of one or more amino acids in the amino acid sequence shown in sequence listing number 2 (SEQ ID NO: 2), which has at least 70% homology to the amino acid sequence shown under number 2 (SEQ ID NO: 2), and which has the activity of 6-phosphogluconolactonase.

Hereinafter, the proteins described in points (A) or (B) are referred to as “proteins of the present invention”.

Another objective of the present invention is the provision of DNA encoding the above-described 6-phosphogluconolactonase.

It is also an object of the present invention to provide an L-amino acid producing bacterium, wherein said bacterium is modified so that the activity of 6-phosphogluconolactonase is increased.

It is also an object of the present invention to provide the bacterium described above, wherein said bacterium belongs to the Enterobacteriaceae family and to a genus selected from the genera Escherichia, Erwinia, Providencia and Serratia.

It is also an object of the present invention to provide a bacterium as described above, in which the activity of 6-phosphogluconolactonase is increased by modifying the nucleotide sequence controlling the expression of the 6-phosphogluconolactonase gene in the chromosome of said bacterium, so that the expression of said gene is enhanced.

It is also an object of the present invention to provide a bacterium as described above in which the natural promoter of the gene described above is replaced by a stronger promoter.

It is also an object of the present invention to provide a bacterium as described above in which a 6-phosphogluconolactonase gene is obtained from a bacterium belonging to the genus Escherichia.

Another objective of the present invention is the provision of the above-described bacteria producing L-amino acids, in which the gene encoding 6-phosphogluconolactonase is selected from the group consisting of:

(a) a DNA comprising the sequence of nucleotides 1 to 996 shown in the list of sequences under the number 1 (SEQ ID NO: 1); or

(b) DNA that hybridizes under stringent conditions with the nucleotide sequence 1 to 996 listed in the sequence number 1 (SEQ ID NO: 1), or with a probe prepared on the basis of the specified nucleotide sequence, and encodes a protein having activity 6-phosphogluconolactonase.

It is also an object of the present invention to provide the bacteria described above, where stringent conditions are conditions under which washing is carried out at 60 ° C for 15 minutes, and the salt concentration corresponds to 1 × SSC and 0.1% SDS.

It is also an object of the present invention to provide a bacterium as described above, wherein the bacterium is modified such that expression of the open ybhE reading frame is enhanced.

It is also an object of the present invention to provide the bacteria described above, wherein the L-amino acid is an aromatic L-amino acid selected from the group of amino acids consisting of L-tryptophan, L-phenylalanine and L-tyrosine.

It is also an object of the present invention to provide a method for producing aromatic L-amino acids, comprising growing the above-described bacteria and isolating the L-amino acid accumulated therein from the culture fluid.

It is also an object of the present invention to provide a process as described above in which L-amino acids are aromatic amino acids selected from the group consisting of L-tryptophan, L-phenylalanine and L-tyrosine.

It is also an object of the present invention to provide a method as described above, wherein in said bacterium, expression of aromatic amino acid biosynthesis pathway genes is enhanced.

The described method for producing L-amino acids includes a method for producing L-tryptophan using the producer bacteria L-tryptophan, in which the activity of the protein according to the present invention is increased. The described method for producing L-amino acids also includes a method for producing L-phenylalanine using a producer bacterium L-phenylalnine, in which the activity of the protein according to the present invention is increased. The described method for producing L-amino acids also includes a method for producing L-tyrosine using L-tyrosine producing bacteria, in which the activity of the protein of the present invention is enhanced.

Brief Description of the Drawings

Figure 1 shows the structure of a natural fragment of bacterial DNA around an open reading frame (ORF) of ybhE.

Figure 2 shows the structure of a bacterial DNA fragment with delegated ybhE ORF.

Figure 3 shows the structure of a bacterial DNA fragment with delegated ybhA ORF.

Figure 4 shows the structure of a bacterial DNA fragment with delegated ybhD ORF.

Figure 5 shows the structure of the bacterial DNA fragment with the delegated pgi gene.

Figure 6 shows the structure of the bacterial DNA fragment with the deleted zwf-edd-eda operon.

Figure 7 shows the structure of a bacterial DNA fragment with an artificial promoter region (P _{tac *} ) before the pgl gene (ybhE ORF).

On Fig shows the separation in the gel protein (His) ₆ -YbhE and its purification in the gel. A. Coarse extract of cells of strain BL21 (DE3) [pET-HTybhE]. Lanes 1, 2, 9 — protein marker of molecular weight; lanes 4, 3 — total protein from strains after induction with IPTG and without it; lanes 6, 5 — soluble fraction from the strains after induction with and without IPTG; lanes 8, 7, - insoluble fraction from the strains after induction using IPTG and without it. B. Lane 1 — total protein from strain BL21 (DE3) [pET-HTybhE], lanes 2, 3, 4, 6 — increasing concentrations of purified protein (His) ₆ -YbhE; lane 5 is a protein marker of molecular weight.

The best way of carrying out the invention.

According to the present invention, 6-phosphogluconolactonase is an enzyme derived from a bacterium belonging to the genus Escherichia and having 6-phosphogluconolactonase activity. The term “6-phosphogluconolactonase activity” means an activity that catalyzes the hydrolysis of 6-phosphogluconolactone to 6-phosphogluconate. The activity of 6-phosphogluconolactonase can be measured using the method described, for example, in Kupor, S.R. and Fraenkel, D.G. (J. Bacteriol, 100: 3, 1296-1301 (1969)).

An example of a 6-phosphogluconolactonase derived from a bacterium belonging to the genus Escherichia is a protein selected from the group consisting of the following proteins:

(A) a protein comprising the amino acid sequence shown in sequence listing number 2 (SEQ ID NO: 2); and

(B) a protein comprising an amino acid sequence comprising deletions, substitutions, insertions, or the addition of one or more amino acids to the amino acid sequence listed in sequence number 2 (SEQ ID NO: 2), which has at least 70% homology to the amino acid sequence shown under number 2 (SEQ ID NO: 2), and which has the activity of 6-phosphogluconolactonase.

As a gene encoding 6-phosphogluconolactonase from Escherichia coli (EC 3.1.1.31), the pgl gene is claimed, which includes the open reading frame ybhE ORF (nucleotide numbers 797809 to 798804 in sequence with accession number NC_000913.1 in GenBank; gi: 16128735). The open reading frame of ybhE is located on the chromosome of E. coli K 12 strain between the open reading frame (ORF) of ybhA and ybhD.

Thus, the pgl gene can be obtained by PCR (polymerase chain reaction; according to White, T.J. et al., Trends Genet., 5, 185 (1989)) using primers synthesized based on the nucleotide sequence of this gene.

An example of a pgl gene from Escherichia coli is DNA selected from the group consisting of:

(a) DNA, which includes the sequence of nucleotides 1 to 996 shown in the list of sequences under the number 1 (SEQ ID NO: 1); or

(b) DNA that hybridizes under stringent conditions with the nucleotide sequence 1 to 996 listed in the sequence number 1 (SEQ ID NO: 1), or with a probe prepared on the basis of the specified nucleotide sequence, and encodes a protein having activity 6-phosphogluconolactonase.

The DNA encoding the proteins of the present invention includes DNA encoding a protein containing deletions, substitutions, insertions, or additions of one or more amino acids at one or more positions in protein (A), provided that the protein does not lose its activity. Despite the fact that the number of “several” amino acids may vary depending on the position and type of amino acid residue in the three-dimensional structure of the specified protein, this amount can vary from 2 to 30, preferably from 2 to 20, and more preferably from 2 to 10 amino acid residues for protein (A). A protein according to the present invention containing the deletions, substitutions, insertions or additions of one or more amino acids described above must have at least 70% homology to the protein represented in the sequence list under number 2 (SEQ ID NO: 2). The percent homology is measured by comparing the studied sequence with the listed sequence number 2 (SEQ ID NO: 2) along the entire length of the sequence and determining the number of similar amino acid residues. The protein of the present invention should have at least 70% homology to the protein shown in sequence listing number 2 (SEQ ID NO: 2), more preferably at least 80% homology, even more at least 90% homology, and most preferred is a homology of at least 95% of the protein shown in sequence listing number 2 (SEQ ID NO: 2).

The percentage of protein or DNA homology can also be estimated using known calculation methods such as BLAST, FASTA or CrustalW. BLAST (Basic Local Alignment Search Tool) is a heuristic search algorithm used in the software products blastp, blastn, blastx, megablast, tbiastn, and tbiastx; these programs attribute search results using statistical methods developed by Karlin, Samuel, and Stephen F. Altschul ("Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes". Proc. Natl. Acad. Sci. USA 1990, 87: 2264-68; "Applications and statistics for multiple high-scoring segments in molecular sequences." Proc. Natl. Acad. Sci. USA, 1993, 90: 5873-7). The FASTA search method is described by W.R. Pearson ("Rapid and Sensitive Sequence Comparison with FASTP and FASTA", Methods in Enzymology, 1990 183: 63-98). The ClustalW method is described by Thompson J.D., Higgins D.G. and Gibson T.J. ("CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice", Nucleic Acids Res. 1994, 22: 4673-4680).

In order for protein activity to remain, substitutions, deletions, insertions or additions of one or more amino acid residues in the protein should be a conservative mutation (s). An example of a conservative mutation is a conservative substitution. Examples of conservative substitutions include replacing Ala with Ser or Thr, replacing Arg with Gln, His or Lys, replacing Asn with Glu, Gln, Lys, His or Asp, replacing Asp with Asn, Glu or Gln, replacing Cys with Ser or Ala, replacing Gln with Asn, Glu, Lys, His, Asp or Arg, replacing Glu with Asn, Gln, Lys or Asp, replacing Gly with Pro, replacing His with Asn, Lys, Gln, Arg or Tyr, replacing Ile with Leu, Met, Val or Phe, replace Leu with Ile, Met, Val or Phe, replace Lys with Asn, Glu, Gln, His or Arg, replace Met with Ile, Leu, Val or Phe, replace Phe with Trp, Tyr, Met, Ile or Leu, replacing Ser with Thr or Ala, replacing Thr with Ser or Ala, replacing Trp with Phe or Tyr, replacing Tyr with His, Phe or Trp, and replacing Val with Met, Ile or Leu.

DNA encoding almost the same proteins as the protein described in paragraph (A) can be obtained, for example, by modifying the nucleotide sequence of the DNA encoding the protein described in paragraph (A), for example, using the site-directed mutagenesis method, such that one or more amino acid residues can be delegated, replaced, inserted or added. DNA modified as described above can be obtained by conventionally known processing methods to produce mutations. Such methods include treating the DNA encoding the protein of the present invention with hydroxylamine or treating the bacterium containing said DNA with UV light or with a reagent such as N-methyl-N'-nitro-N-nitrosoguanidine or nitrous acid.

DNA encoding the proteins of the present invention also includes DNA, which can be obtained from various strains and variants of bacteria belonging to the genus Escherichia due to natural diversity. DNA encoding such proteins can be obtained by isolating DNA that hybridizes with the pgl gene or part thereof under stringent conditions and encodes a protein having 6-phosphogluconolactonase activity. The term "stringent conditions", mentioned here, means the conditions under which the so-called specific hybrids are formed, and non-specific ones are not formed. It is difficult to clearly describe such conditions in any numerical terms. For example, stringent conditions include conditions under which DNA with a high degree of homology hybridizes, for example, DNA with a homology of at least 70%, preferably at least 80%, more preferably at least 90%, more preferably at least 95%, each relative to a friend. On the other hand, an example of stringent conditions is the conditions under which DNA hybridizes with each other, at a salt concentration corresponding to standard washing conditions for Southern hybridization, for example, 1 × SSC, 0.1% SDS, preferably 0.1 × SSC, 0.1% SDS at 60 ° C. The duration of washing depends on the type of filter used for blotting and, as a rule, recommended by the manufacturer. For example, the recommended duration of washing a Hybond ^TM N + nylon filter (Amersham) under harsh conditions is 15 minutes. Preferably, washing is carried out two or three times.

Part of the nucleotide sequence numbered 1 can be used as a probe for DNA encoding the proteins described above, and which hybridizes with the pgl gene. A probe of this kind can be obtained by PCR using oligonucleotides obtained on the basis of the nucleotide sequence as primers number 1, and a DNA fragment containing the nucleotide sequence under number 1, as a matrix. When a DNA fragment with a length of about 300 base pairs is used as a probe, the washing conditions during hybridization correspond, for example, to 50 ° С, 2 × SSC, and 0.1% SDS.

Transformation of a bacterium using DNA encoding a protein means introducing said DNA into a bacterial cell, for example, by conventional methods to increase the expression of said gene encoding a protein of the present invention and increase the activity of said protein in a bacterial cell.

The bacterium according to the present invention is a bacterium belonging to the genus Escherichia, a producer of L-amino acids with increased protein activity, which leads to an increase in the production of the target L-amino acid. Preferably, the bacterium according to the present invention is a bacterium belonging to the genus Escherichia, an aromatic L-amino acid producer having increased protein activity according to the present invention. More preferably, the bacterium according to the present invention is a bacterium belonging to the genus Escherichia, a producer of aromatic L-amino acids, such as a bacterium-producer of L-tryptophan, modified so that the activity of 6-phosphogluconolactonase is increased. Even more preferably, if the bacterium according to the present invention contains DNA with the pgl gene (ybhE ORF), the sequence of control of the expression of the specified gene in the chromosome of this bacterium is modified so that the ability of this bacterium to produce L-tryptophan is enhanced.

The term "bacterium-producer of L-amino acids" means a bacterium having the ability to accumulate this L-amino acid in a nutrient medium, under the conditions when the bacterium according to the present invention is grown in the specified nutrient medium. The ability to produce L-amino acids can be given or improved by selection. As used herein, the term “L-amino acid producing bacterium” also means a bacterium that is capable of producing an L-amino acid and causes the L-amino acid to accumulate in the medium in quantities greater than the natural or parent strain, and preferably means that the microorganism is capable of to produce and accumulate in the fermentation medium the target L-amino acid in concentrations of not less than 0.5 g / l, more preferably not less than 1.0 g / l.

L-amino acids include: L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine (cystine), L-glutamic acid, L-glutamine, L-glycine, L-histidine, L- isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine and L-valine, and preferably include aromatic L-amino acids such as L-tryptophan, L-phenylalanine and L-tyrosine.

The Enterobacteriaceae family includes bacteria belonging to the genus Escherichia, Erwinia, Providencia, and Serratia. The genus Escherichia is preferred. The term "bacterium belonging to the genus Escherichia" means that the bacterium belongs to the genus Escherichia in accordance with the classification known to the person skilled in the field of microbiology. As an example of a microorganism belonging to the genus Escherichia used in the present invention, the bacterium Escherichia coli (E. coli) may be mentioned.

The term "modified so that the activity of 6-phosphogluconolactonase is increased" means that the specific activity becomes higher than that of an unmodified strain, for example, a natural strain. For example, in the case when the number of 6-phosphogluconolactonase molecules in the cell is increased, when the specific activity of the 6-phosphogluconolactonase molecule is increased and so on. In addition, a strain of Escherichia coli K-12 may be mentioned as a natural strain that can serve as an object for comparison. As a result of the increase in the intracellular activity of 6-phosphogluconolactonase, the effect of increasing the amount of accumulated L-amino acid is observed.

An increase in the activity of 6-phosphogluconolactonase in the bacterial cell can be achieved by increasing the expressed amount of the gene encoding 6-phosphogluconolactonase. An example of a gene encoding 6-phosphogluconolactonase can be a gene isolated from bacteria belonging to the genus Escherichia.

The bacterium of the present invention includes a bacterium in which the activity of the protein of the present invention is increased by replacing the sequence that controls the expression of the DNA encoding the protein described in (A) or (B) in the bacterial chromosome with a stronger one. Enhanced gene expression can be achieved by placing the DNA of the present invention under the control of a stronger promoter instead of the natural one. The term "natural promoter" means a DNA fragment in a natural organism located in front of an open reading frame (ORF) of a gene and facilitating transcription of this gene. The strength of the promoter is determined by the frequency of the act of starting RNA synthesis. Methods for evaluating promoter strength and examples of strong promoters are described by Deuschle, U., Kammerer, W., Gentz, R., Bujard, H. (Promoters in Escherichia coli: a hierarchy of in vivo strength indicates alternate structures. EMBO J., 1986 5, 2987-2994).

Enhanced translation can be achieved by introducing into the DNA of the present invention a more efficient Ribosome Binding Site (RBS) instead of the native RBS sequence. The RBS sequence is the region located in front of the start codon of mRNA that interacts with 16S RNA of the ribosome (Shine J. and Dalgarno L., Proc. Natl. Acad. Sci. USA, 1974, 71, 4, 1342-6). The term "natural RBS sequence" means an RBS sequence in a naturally occurring organism. An example of an effective RBS sequence is the RBS sequence of gene 10 from T7 phage (Olins P. O. et al, Gene, 1988, 73, 227-235).

Methods for producing chromosomal DNA, hybridization, PCR, obtaining plasmid DNA, cutting and ligation of DNA, transformation, selection of oligonucleotides as primers and the like can be conventional methods well known to those skilled in the art. These methods are described, for example, in Sambrook, J., and Russell D., "Molecular Cloning A Laboratory Manual, Third Edition", Cold Spring Harbor Laboratory Press (2001) and the like.

The bacterium of the present invention can be obtained by introducing the above DNA into a bacterium already possessing the ability to produce L-amino acids. On the other hand, a bacterium according to the present invention can be obtained by giving a bacterium already containing said DNA the ability to produce L-amino acids.

As the parent strain, in which the activity of the protein according to the present invention will be increased, strains belonging to the genus Escherichia, L-tryptophan producers such as E. coli strains JP4735 / pMU3028 (DSM10122) and JP6015 / pMU91 (DSM10123) can be used. tryptophanyl-tRNA synthetase deficient encoded by the mutant trpS gene (US Pat. No. 5,756,345); E. coli strain SV164 (pGH5) with the serA gene allele encoding the feedback-removed serine inhibition enzyme (US Pat. No. 6,180,373); E. coli strains AGX17 (pGX44) (NRRL B-12263) and AGX6 (pGX50) aroP (NRRL B-12264) enzyme tryptophanase deficient (US patent 4371614); E. coli strain AGX17 / pGX50, pACKG4-pps, in which the ability to produce phosphoenolpyruvate (WO 9708333, US patent 6319696) and the like are enhanced.

As the parent strain, in which the activity of the protein according to the present invention will be increased, strains belonging to the genus Escherichia, producers of L-phenylalanine, such as strain AJ12739 (tyrA :: Tn10, tyrR) (VKPM B-8197) can be used; strain HW1089 (ATCC accession number 55371) containing the pheA34 gene (US patent 5354672); mutant strain MWEC101-b (South Korean patent 8903681); strains NRRL B-12141, NRRL B-12145, NRRL B-12146 and NRRL B-12147 (US patent 4407952) and the like. Also, as the parent strain for the subsequent increase in the activity of the protein according to the present invention, L-phenylalanine producing bacteria belonging to the genus Escherichia, E. coli K-12 strains [W3110 (tyrA) / pPHAB (FERM BP-3566) can be used ), E. coli K-12 [W3110 (tyrA) / pPHAD] (FERM BP-12659), E. coli K-12 [W3110 (tyrA) / pPHATerm] (FERM BP-12662) and E. coli K-12 [W3110 (tyrA) / pBR-aroG4, pACMAB], also called AJ 12604 (FERM BP-3579) (European patent 488424 B1).

The authors of the present invention previously showed that the yddG gene, which encodes a membrane protein that is not involved in the biosynthesis of any of the L-amino acids, in cases where the allele of a natural gene is amplified on a multi-copy vector, imparts resistance to L-phenylalanine and several amino acid analogs to microorganisms. In addition, the yddG gene can increase the production of L-phenylalanine or L-tryptophan, in cases where additional copies of this gene are introduced into the cells of the corresponding producer strain (patent application RF 2002121670, WO 03044192). Therefore, it is desirable that the bacterium producing L-tryptophan be further modified to enhance expression of the open reading frame of yddG.

Genes important for L-tryptophan biosynthesis include the trpEDCBA operon genes, genes common to the biosynthesis of all aromatic amino acids - such as aroF, aroG, aroH, aroB, aroD, aroE, aroK, aroL, aroA and aroC, L biosynthesis genes -serine, such as serA, serB and serC, and the like.

As the parent strain, in which the activity of the protein according to the present invention will be increased, strains belonging to the genus Escherichia, producers of L-tyrosine, such as E. coli strains, in which the ability to produce phosphoenolpyruvate is increased or the activity of enzymes of the total aromatic amino acid biosynthesis pathways (European Patent Application EP 0877090).

A method for producing an L-amino acid according to the present invention includes the steps of growing a bacterium according to the present invention in a nutrient medium in order to produce and accumulate the L-amino acid in the nutrient medium, isolating the L-amino acid from the culture fluid. Also, the method according to the present invention includes a method for producing L-tryptophan and includes the steps of growing the bacteria according to the present invention in a nutrient medium in order to produce and accumulate L-tryptophan in a nutrient medium, isolating L-tryptophan from the culture fluid. Also, the method according to the present invention includes a method for producing L-phenylalnine, and includes the steps of growing the bacteria according to the present invention in a culture medium in order to produce and accumulate L-phenylalnine in a culture medium, isolating L-phenylalnine from the culture fluid. Also, the method according to the present invention includes a method for producing L-tyrosine and includes the steps of growing the bacteria according to the present invention in a culture medium for the purpose of production and accumulation of L-tyrosine in a culture medium, the allocation of L-tyrosine from the culture fluid.

According to the present invention, the cultivation, isolation and purification of L-amino acids, preferably aromatic amino acids, such as L-tryptophan, L-phenylalnine or L-tryptophan, from a culture or similar liquid can be carried out in a manner similar to traditional fermentation methods in which the amino acid is produced using a microorganism.

The nutrient medium used for growing can be either synthetic or natural, provided that the medium contains sources of carbon, nitrogen, mineral additives and, if necessary, the appropriate amount of nutrient additives necessary for the growth of microorganisms. Carbon sources include various carbohydrates such as glucose and sucrose, as well as various organic acids. Depending on the nature of the assimilation used by the microorganism, alcohols such as ethanol and glycerin may be used. Various inorganic ammonium salts, such as ammonia and ammonium sulfate, other nitrogen compounds, such as amines, natural nitrogen sources, such as peptone, soybean hydrolyzate, microorganism fermentolizate, can be used as a nitrogen source. As mineral additives, potassium phosphate, magnesium sulfate, sodium chloride, iron sulfate, manganese sulfate, calcium chloride and the like can be used. Some nutritional supplements may, if necessary, be added to the culture medium. For example, if tyrosine (tyrosine auxotrophy) is required for growth by the microorganism, an appropriate amount of tyrosine can be added to the growth medium.

The cultivation is preferably carried out under aerobic conditions, such as mixing the culture fluid on a rocking chair, shaking with aeration, at a temperature in the range from 20 to 42 ° C, preferably in the range from 37 to 40 ° C. The pH of the medium is maintained in the range from 5 to 9, preferably from 6.5 to 7.2. The pH of the medium can be adjusted by ammonia, calcium carbonate, various acids, bases and buffer solutions. Typically, growing for 1 to 5 days leads to the accumulation of the target L-amino acid in the culture fluid.

After growth, solid residues, such as cells, can be removed from the culture fluid by centrifugation or filtration through a membrane, and then the target L-amino acid can be isolated and purified by ion exchange chromatography, concentration and crystallization.

Examples

In more detail, the present invention will be explained below with reference to the following Examples, not limiting the scope of the present invention.

Example 1. Identification of the pgl gene from E. coli and comparison of nucleotide sequences.

Kupor and Fraenkel mapped the pgl mutation between the chlD genes (currently known as modC) and bioA on the E. coli chromosome (Kupor, S.R. and Fraenkel, D.G., J. Bacteriol., 100: 3, 1296-1301 (1969)). This corresponds to 17.18 and 17.40 minutes of the E. coli genetic map. Eight open reading frames encoding proteins with unknown functions are located in this area. Further, the E. coli Stock Center Database determined the pgl mutation between minutes 17.20 and 17.22. These coordinates correspond quite accurately to the coordinates of the open reading frame ybhE, located between the open reading frames ybhA and ybhD (Figure 1).

A search using the BLAST program showed that YbhE protein has a large number of homologs with unknown functions in various organisms, such as Shigella flexmeri (98.8% similarity), Salmonella typhi (92.8% similarity), Yersinia pestis (68.4% similarity) , several homologs with known functions, for example, the heme domain of Cytochrome D1 from Bacillus anthracis (28% identity), 3-carboxymeximate cyclase from Pseudomonas fluorescens, predicted by automatic computer analysis (28% identity), muconate cycloisomerase from Trichosporon beigelii, and 26% one homologue from Bacillus cereus described in databases ak 6 fosfoglyukonatlaktonaza under accession number NP_833107, but without reference to the printed experimental work.

Using the NCBI Conserved Domain Search program, three overlapping conservative protein domains were detected. Two of them belong to the conservative protein family with unknown function, and one domain belongs to the 3-carboxy-muccinate cyclase family.

When searching with the BLAST program within the E. coli proteome, no homologues of the described 6-phosphogluconate lactonases, for example, from Pseudomonas putida, were found.

In order to establish whether the ORF, denoted as ybhE on the E. coli chromosome, the pgl gene encoding 6-phosphogluconate lactonase, sequentially destroyed the open reading frames of ybhA, ybhE and ybhD, and the obtained mutants were tested for the maltose blue phenotype " (see below).

Example 2. The division of the open reading frame (ORF) ybhE. Replacing ybhE ORF with a DNA fragment containing the chloramphenicol resistance gene (Cm ^R ).

For deletion of ybhE ORF in the chromosome of E. coli strain BW25113 [pKD46], instead of the natural ybhE ORF, a DNA fragment was integrated containing a chloramphenicol resistance marker (Cm ^R ) encoded by the cat gene according to the method described by Datsenko KA and Wanner BL (Proc. Natl Acad. Sci. USA, 2000, 97, 6640-6645). This method is also referred to as “integration using the Red system” and / or “integration using the Red system”. The nucleotide sequence of the original natural region of ybhE ORF and the amino acid sequence encoded by the indicated ORF are presented in the sequence list under numbers 1 and 2, respectively (SEQ ID NO: 1 and 2). The Escherichia coli strain BW25113 containing the recombinant plasmid pKD46 can be obtained from E. coli Genetic Stock Center, Yale University, New Haven, USA, strain number is CGSC7630.

The DNA fragment containing the Cm ^R marker can be obtained by PCR, using the commercially available plasmid pACYC184 (accession number X06403 in GenBank / EMBL, Fermentas, Lithuania) as well as P1 primers (SEQ ID NO: 3) and P2 (SEQ ID NO: 4). Primer P1 contained 36 nucleotides homologous to the 5 ′ end of the ybhE ORF frame and primer P2 contained 36 nucleotides homologous to the 3 ′ end of the ybhE ORF frame. These ybhE gene sequences were included in primers P1 and P2 for further integration of the resulting fragment into the chromosome.

PCR was performed using a TennoHybaid PCR Express Amplifier. The reaction mixture with a total volume of 50 μl contained 5 μl of 10x PCR buffer (Fermentas, Lithuania) supplemented with MgCl ₂ to a final concentration in the reaction mixture of 15 mM, 200 μM of each dNTP triphosphate, 25 pM of each primer used and 1 unit of Taq- polymerases (Fermentas, Lithuania). About 5 ng of plasmid DNA was added to the reaction mixture as a template for PCR amplification. The PCR temperature profile was as follows: the initial stage of denaturation for 5 min at 95 ° C, followed by 25 cycles of denaturation at 95 ° C for 30 seconds, annealing at 55 ° C for 30 seconds, chain extension at 72 ° C for 30 seconds the final stage of polymerization at 72 ° C for 7 minutes.

Then, the amplified DNA fragment was purified by agarose gel electrophoresis, isolated from the gel using GenElute Spin Columns (Sigma, USA) and planted with ethanol. The nucleotide sequence of the synthesized DNA fragment is presented in SEQ ID NO: 5.

The obtained DNA fragment, purified as described above, was used for electroporation and integration using the Red system into the bacterial chromosome of E. coli strain BW25113 [pKD46]. The recombinant plasmid pKD46 (Datsenko, K.A., Wanner, BL, Proc. Natl. Acad. Sci. USA, 2000, 97, 6640-6645) with a heat-sensitive replicon was used as a donor of phage λ genes responsible for the functioning of the Red recombination system .

BW25113 [pKD46] cells were grown overnight at 30 ° C in LB liquid medium containing ampicillin (100 μg / ml), then diluted 1: 100 with SOB medium (yeast extract, 5 g / l; NaCl, 0.5 g / l; Tryptone, 20 g / l, KCl, 2.5 mM; MgCl ₂ , 10 mM) containing ampicillin (100 μg / ml) and arabinose (10 mM) (arabinose was used to induce a plasmid encoding the genes of the phage homologous recombination system λ), and grown at 30 ° C until the optical density of the bacterial culture reaches OD ₆₀₀ = 0.4-0.7. Cells from 10 ml of the grown bacterial culture were washed 3 times with deionized water, cooled on ice, then resuspended in 100 μl of water. 10 μl (100 ng) of the DNA fragment dissolved in deionized water was added to the suspension of bacterial cells. Electroporation was carried out using a Bio-Rad device (B), USA (No. 165-2098, version 2-89) in accordance with the manufacturer's recommendations.

After electroporation, 1 ml of SOC medium Sambrook et al, "Molecular Cloning A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press (1989)) was added to the cell suspension, grown for 2 hours at 37 ° C, and then plated on plates with L-agar containing chloramphenicol (25 μg / ml). Colonies grown on these plates for 24 hours were tested by PCR for the presence of the Cm ^R marker, which replaced the natural ybhE frame, using the primers P3 (SEQ ID NO: 6) and P4 (SEQ ID NO: 7). For this purpose, freshly grown colonies were resuspended in 20 μl of water and 1 μl of the resulting suspension was used as a template for PCR. The PCR temperature profile was as follows: the initial stage of DNA denaturation - for 10 min at 95 ° С, then 30 denaturation cycles at 95 ° С for 30 seconds, annealing at 55 ° С for 30 seconds, chain extension at 72 ° С within 1 minute; the final stage of polymerization at 72 ° C for 7 minutes.

Only a few Cm ^R colonies tested contained the desired 1279 bp DNA fragment confirming the replacement of the natural frame of ybhE ORF with a Cm ^R DNA marker. One of the obtained strains was cured of the heat-sensitive plasmid pKD46 by growing the strain at 37 ° C. The strain obtained by all of the above procedures was named E. coli BW25113-ΔybhE.

The structure of the bacterial DNA fragment with the destroyed ybhE ORF frame is shown in FIG. 2.

Example 3. Deletion of the open reading frames ybhA ORF and ybhD ORF. Replacing the ybhA and ybhD ORF framework with DNA fragments containing the chloramphenicol resistance gene (Cm ^R ).

For deletion of the ybhA ORF and ybhD ORF frameworks, DNA fragments containing the chloramphenicol resistance marker (Cm ^R ) encoded by the cat gene were independently integrated into the chromosome of E. coli strain BW25113 [pKD46] instead of the natural framework of ybhA ORF and ybhD ORF in Example 2.

To obtain DNA fragments necessary for electroporation and destruction of the ybhA ORF and ybhD ORF frames by PCR, two pairs of primers P5 (SEQ ID NO: 8) and P6 (SEQ ID NO: 9), and P7 (SEQ ID NO: 10) and P8 (SEQ ID NO: 11), respectively. Primer P5 contained 36 nucleotides homologous to the 3 ′ end of the ybhA ORF frame. Primer P6 contained 36 nucleotides homologous to the 5 ′ end of the ybhA ORF frame. Primer P7 contained 36 nucleotides homologous to the 3 ′ end of the ybhD ORF frame. And the P8 primer contained 36 nucleotides homologous to the 5 ′ end of the ybhD ORF frame. These sequences were included in primers P5, P6, P7 and P8 for further integration of the resulting fragments into the chromosome.

The nucleotide sequences of the synthesized DNA fragments are presented in SEQ ID NO: 12 and SEQ ID NO: 13, respectively. The nucleotide sequences of the replaced natural fragments with the ybhA ORF and ybhD ORF frames are presented in the GenBank database with inventory number NC_000913.1 (nucleotide numbers 796836 to 797654 and 798845 to 799777, gi: 16128734 and gi: 33347481, respectively). The structures of the bacterial DNA fragments with the destroyed ybhA ORF and ybhD ORF frames are shown in FIG. 3 and FIG. 4, respectively.

After electroporation, the obtained colonies were checked by PCR for the presence of the Cm ^R marker: P9 primers (SEQ ID NO: 14) and P10 (SEQ ID NO: 15) were used for the destroyed ybhD ORF framework; P11 primers were used for the destroyed ybhD ORF framework (SEQ ID NO: 16) and P12 (SEQ ID NO: 17).

In the first case, several Cm ^R colonies tested contained the desired 1424 bp DNA fragment, confirming the replacement of the natural ybhA ORF framework with the Cm ^R gene. In the second case, several Cm ^R colonies tested contained the desired 1386 bp DNA fragment, confirming the replacement the natural framework of ybhD ORF on the Cm ^R gene. In both cases, one of the obtained strains was cured of the heat-sensitive plasmid pKD46 by growing the strain at 37 ° C, and the resulting strains were named E. coli BW25113-ΔybhA and BW25113-ΔybhD, respectively.

Example 4. Testing the mutants ybhE ^- , vbhA ^- and ybhD ^- for the phenotype "maltose blue".

Each of the three mutant strains obtained was tested for the maltose blue phenotype (blue colonies on maltose medium) by the method described in Kupor, SR and Fraenkel, DG (J. Bacteriol., 100: 3, 1296-1301 (1969)) . The strains were replicated by replicating on plates with minimal M9 medium containing 0.8% maltose. After 6 hours, 5 ml of the solution containing 0.01 M I ₂ and 0.03 M KI were poured into the incubated plates, after which the color of each replicated colony was evaluated as “blue” or “not blue”.

One of the obtained strains BW25113-ΔybhE was rated as "blue", while the other strains BW25113-ΔybhA, BW25113-ΔybhD and BW25113 (as a control) were counted as "not blue".

Example 5. Construction of strains with double mutation - divisions pgi and ybhE or ybhD. Comparative characteristics of the growth of such strains on media with different carbon sources.

In the mutant strain deficient in phosphoglucose isomerase (pgi ^- ), growth on glucose is slowed down, since only the oxidative branch of the pentose phosphate cycle works. The double mutant, also deficient in phosphogluconolactonase (pgl), which catalyzes the second stage of the oxidative branch of the pentose phosphate cycle, should grow even slower on glucose, since the conversion of 6-phosphogluconolactone to gluconate-6-phosphate in this case is possible only due to spontaneous hydrolysis. This means that if the ybhE ORF frame is indeed the pgl gene, then the double mutant pgi, ybhE will grow more slowly than the natural strain and the pgi mutant. To verify this assumption, a double mutant pgi, ybhE, was created.

A mutation was introduced into the pgi gene by replacing the corresponding natural site of the bacterial chromosome in E. coli strain BW25113 [pKD46] with a DNA fragment containing the kanamycin resistance gene (Km ^R ), as described in Example 2. The nucleotide sequence of the replaced natural pgi shadow fragment represented in the GenBank database, inventory number NC_000913.1 (nucleotide numbers 4231337 to 4232986; gi: 16131851).

DNA fragments containing the Km ^R gene were synthesized by PCR, using the commercially available plasmid pUC4KAN (accession number X06404 in GenBak / EMBL, Fermentas, Lithuania) as primers, as well as primers P13 (SEQ ID NO: 18) and P14 (SEQ ID NO: 19). Primer P13 contains 36 nucleotides homologous to the 3 ′ end of the pgi gene, and primer P14 contains 36 nucleotides homologous to the 5 ′ end of the pgi gene. These pgi gene sequences were included in primers P13 and P14 for further integration of the resulting fragment into the bacterial chromosome.

PCR was performed as described in Example 2.

Then, the amplified DNA fragment was purified by agarose gel electrophoresis, isolated from the gel using GenElute Spin Columns (Sigma, USA) and planted with ethanol. The nucleotide sequence of the synthesized DNA fragment is presented in SEQ ID NO: 20.

The obtained DNA fragment, purified as described above, was used for electroporation and integration using the Red system into the bacterial chromosome of E. coli strain BW25113 [pKD46] as described above in Example 2, except that the cells were plated after electroporation on plates with L-agar containing kanamycin at a concentration of 50 μg / ml.

Colonies grown on these plates for 24 hours were tested by PCR for the presence of the Km ^R marker, which replaced the natural pgi gene, using primers P15 (SEQ ID NO: 21) and P16 (SEQ ID NO: 22). For this purpose, freshly grown colonies were resuspended in 20 μl of water and 1 μl of the resulting suspension was used as a template for PCR. The PCR conditions are described in Example 2. Several tested Km ^R colonies contained the desired 1286 bp DNA fragment confirming the replacement of the natural pgi gene with DNA with a Km ^R marker. One of the obtained strains was cured of the heat-sensitive plasmid pKD46 by growing the strain at 37 ° C. The strain obtained by all of the above procedures was named E. coli BW25113-Δpgi.

The structure of the obtained bacterial DNA fragment with the delegated pgi gene is shown in Figure 5.

The deletion of the pgi gene was transferred by Fraenkel transduction (J. Bacteriol. 93 (1967), 1582-1587) into the E. coli strain MG1655, followed by selection on kanamycin plates. The resulting strain was named MG-Δpgi. Then, mutations in the framework of ybhE ORF and ybhD ORF were also transferred by transduction from strains BW25113-ΔybhE and BW25113-ΔybhD described in Examples 2 and 3 to the resulting strain, followed by selection on plates with chloramphenicol. The resulting strains were named MG-Δpgi-ΔybhE and MG-Δpgi-ΔybhD, respectively.

Both strains obtained, together with MG1655 and MG1655-Δpgi, were replicated by replicating onto plates with minimal M9 medium containing glucose or gluconate as a carbon source. After 24 hours of incubation of the plates, a visual comparison was made of the growth of these strains. Strain MG-Δpgi-ΔybhE grew worse compared to other strains on a plate with glucose, but practically did not grow on a plate with gluconate.

Example 6. Construction of a plasmid containing the pgi gene from Pseudomonas putida, and complementation of this mutation ybhE.

The pgl gene has been described for several organisms. Among them is also known the 6-phosphogluconolactonase gene from Pseudomonas putida, an organism that is a close relative of E. coli. Experiments on the cloning of several genes from P. putida to E. coli are described, leading to the complementation of the corresponding mutations in E. coli (Ramos-Gonzalez, MI and Molin, S., J. Bacteriol., V 180, 13, p. 3421, 1998).

The pgl gene from P. putida was cloned using primers 17 (SEQ ID No.23) and 18 (SEQ ID No.24). Primer P17 contains a sequence complementary to the nucleotide sequence of the pgl gene from P. putida from 1 to 19 p. This primer also contains the E. coli lacZ ribosome binding site (RBS) located in front of the complementary sequence described above, as well as the SacI restriction enzyme recognition site introduced at the 5 ′ end of the primer. Primer P18 contains a sequence complementary to the sequence of the pgl gene from P. putida from 709 to 729 bp, and the EcoRI restriction enzyme recognition site introduced at the 5'-end of the primer.

Chromosomal DNA of P. putida strain KT2440 (ATCC 47054) (Bagdasarian, M. & Timmis, K.N., Current Topics of Microbiology and Immunology, eds. Goebel, W. & Hofschneider, PH (Springer, Berlin), p. 47- 67 (1981)) was isolated in a standard way. PCR was performed on a Perkin Elmer GeneAmp PCR System 2400 thermocycler under the following conditions: 40 sec. at 95 ° С, 40 sec. at 53 ° С, 40 sec. at 72 ° C, 25 cycles with Taq polymerase (Fermentas). The obtained PCR fragment containing the pgl gene from P. putida with the Rac of the lacZ gene was digested with restriction enzymes SacI and EcoRI and cloned into a multi copy pUC19 vector pretreated with the same restriction enzymes. Thus, the plasmid pUC19-pgl was obtained.

Strain BW25113-ΔybhE was transformed with the obtained plasmid pUC19-pgl. The transformed culture was plated on plates with minimal medium containing maltose and ampicillin 100 μg / ml, after which they were treated with the solution described above and tested for the maltose blue phenotype. The transformants did not have a "maltose blue" phenotype, in contrast to the control strain BW25113-ΔybhE.

Thus, a cloned copy of the pgl gene from Pseudomonas putida complements the ybhE mutation in E. coli, further confirming our hypothesis that the ybhE ORF frame is the coding region of the pgl gene.

Example 7. Measurement of the activity of 6-phosphogluconolactonase in the mutant strain of ybhE.

Overnight cultures of strains BW25113 and BW25113-ΔybhE were diluted 50 times with minimal glucose-containing M9 medium. Cells were grown until the culture reached an optical density of OD ₅₄₀ = 1. Cell extracts were prepared from 3 ml of culture. Cells were washed with physiological saline, resuspended in 400 μl of calcium phosphate buffer (pH 7.0), and sonicated. The supernatant fraction obtained after centrifugation was used for measurements without further dilution.

To measure the activity of 6-phosphogluconolactonase, the technique described by Collard, F. et al. (FEBS Letters, 459, 223-226 (1999)). Lactone was synthesized directly in the reaction mixture, incubating 50 μM glucose-6-phosphate (Sigma, USA) in the presence of 0.2 mM NADP, 25 mM HEPES (pH 7.1), 2 mM MgCl ₂ and 1.75 units of yeast glucose-6-phosphate dehydrogenase (Sigma, USA) at 30 ° C (total volume of the reaction mixture is 1 ml). When the optical density of the reaction mixture at A ₃₄₀ reached a plateau, a mixture of 0.5 u / ml 6-phosphogluconate dehydrogenase (Sigma, United States) with previously obtained supernatant fractions was added to the reaction mixture, after which the optical density at A _{340 was} measured for 10 minutes. The amount of protein was measured by the method described in Bradford, MM (Anal. Biochem. 72, 248-254 (1976)). The data obtained are presented in Table 1. Activity was measured in relative units, calculated in mg of total protein.

Table 1 Strain The activity of 6-phosphogluconate lactonase (for various extract concentrations)  BW25113 4.0 6.1 5.4 BW25113-ΔybhE 0.3 0.2 Spontaneous hydrolysis 0.3

As can be seen from the Table, the activity of 6-phosphogluconolactonase in the strain mutant for ybhE is at least an order of magnitude lower than in the natural strain and is comparable with the level of spontaneous hydrolysis.

Example 8. The division of the operon zwf-edd-eda. Replacing the zwf-edd-eda genes with DNA fragments containing the kanamycin resistance gene (Km ^R ).

In order to obtain a strain with enhanced expression of the ybhE gene, it was decided to integrate using the Red system (see Example 9) a constitutive promoter derived from P _tac between ybhE RBS and the natural promoter of this gene.

But the attempt to modify the chromosome in this way of the natural strain MG1655 was unsuccessful. We cannot explain the toxic effect of increased expression of the pgl gene (ybhE), but we assume that this is due to an increase in the activity of phosphogluconolactonase, which leads to an imbalance of the pentose phosphate pathway (Pentose-Phosphate Pathway (PPP)) and the associated possible accumulation of some toxic compounds (or lack of certain substances necessary for the cell to survive). Thus, we decided to completely block the pentose phosphate cycle due to the deletion of the zwf gene encoding the first enzyme of this pathway.

The deletion of the zwf-edd-eda operon was performed by the method described in Example 5 for the pgi gene. The nucleotide sequence of the natural zwf-edd-eda operon is shown in sequence with accession number NC_000913.1 in the GenBank database (nucleotide numbers from 1932863 to 1934338, gi: 16129805; from 1930817 to 1932628, gi: 16129804 and from 1930139 to 1930780, gi 16129803 for the zwf, edd and eda genes, respectively). DNA fragments containing the Km ^R gene were synthesized using PCR primers P19 (SEQ ID NO: 25) and P20 (SEQ ID NO: 26). Primer P19 contains 36 nucleotides complementary to the 3 ′ end of the eda gene. Primer P20 contains 36 nucleotides complementary to the 5 ′ end of the zwf gene. The nucleotide sequence of the synthesized DNA fragment is presented in SEQ ID NO: 27.

Colonies grown for 24 hours were tested for the presence of the Km ^R marker instead of the zwf-edd-eda operon using PCR primers P21 (SEQ ID NO: 28) and P22 (SEQ ID NO: 29). Several tested Km ^R colonies contained the desired 1287 bp DNA fragment confirming the replacement of the natural zwf-edd-eda operon with DNA with the Km ^R marker. One of the obtained strains was cured of the heat-sensitive plasmid pKD46 by growing the strain at 37 ° C and the resulting strain was named E. coli BW25113-Δzwf-edd-eda. The structure of a bacterial DNA fragment containing a deletion of the zwf-edd-eda operon is shown in FIG. 6.

Example 9. Replacement of the natural region of DNA in front of the ybhE gene located on the E. coli chromosome with a new regulatory element containing an artificial P _{tac *} promoter.

For further integration in front of the pgl (ybhE) gene of artificial promoters of different strengths P _{tac *} , the E. coli strain BW25113-Δzwf-edd-eda was again transformed with the plasmid pKD46. The resulting kanamycin and ampicillin resistant strain was named E. coli BW25113-Δzwf-edd-eda [pKD46}. Since plasmid pKD46 is thermosensitive, further selection of transformants was carried out at a temperature of 30 ° C.

It is widely known that in mutants with a modified “-35” region of the promoter recognized by the complex of E. coli RNA polymerase and factor σ ⁷⁰ , the efficiency of transcription initiation is significantly changed. Therefore, among the promoters obtained by randomizing the promoter region, promoters with different strengths can be obtained. Thus, this general approach can be used to fine-tune the expression level of the target gene. The authors of the present invention previously obtained a library of modified P _tac promoters of various strengths (from now on, such modified P _tac promoters will be marked with an asterisk). These promoters differ from each other in the sequence of 4 central nucleotides of the “-35” region. In this work, we used two promoters P _{tsc *} with different strengths. Based on the activity values of β-galactosidase expressed under the control of the corresponding promoter, they were named P _tac-3900 (normal P _tac ) and P _tac-10000 (with central nucleotides TTGC instead of natural TGACs).

Further, each of these artificial P _{tac *} promoters was integrated upstream of the coding region of the pgl gene on the chromosome of E. coli strain BW25113-Δzwf-edd-eda [pKD4-6] as described above (see Example 2). In addition, a DNA fragment with the chloramphenicol resistance gene (Cm ^R ) was also integrated in front of the promoter region (see FIG. 7).

The construction of the aforementioned artificial DNA fragments integrated into the corresponding regions of the bacterial chromosomes was carried out in several stages. At the first stage, a DNA fragment containing the BglII restriction enzyme recognition site was synthesized by PCR before the corresponding P _{tac *} promoter.

Chromosomal DNAs from E. coli MG1655 strains containing the artificial promoters P _tac-3900 and P _tac-10000 integrated into the chromosome were used for PCR as a template. PCR was performed using primers P23 (SEQ ID NO: 30) and P24 (SEQ ID NO: 31) for the promoter P _tac-3000 and P _tac-10000, respectively, and primer P25 (SEQ ID NO: 32) for both promoters. Both primers P23 and P24 contain a BglII restriction enzyme recognition site inserted at the 5'-end. Primer P25 contains 11 nucleotides (including RBS) in front of the pgl gene and the first 25 nucleotides of the coding region of the pgl gene. The above sequences were introduced into P25 primer for further integration into the bacterial chromosome.

PCR was performed using a TermoHybaid PCR Express Amplifier. The reaction mixture with a total volume of 50 μl contained 5 μl of 10x PCR buffer (Fermentas, Lithuania) supplemented with MgCl ₂ to a final concentration in the reaction mixture of 15 mM, 200 μM of each dNTP triphosphate, 25 pM of each primer used and 1 unit of Taq- polymerases (Fermentas, Lithuania). Approximately 0.5 μg of plasmid DNA was added to the reaction mixture as a template for PCR amplification. The PCR temperature profile was as follows: the initial stage of denaturation for 5 min at 95 ° C followed by 25 cycles of denaturation at 95 ° C for 30 seconds, annealing at 53 ° C for 30 seconds, chain extension at 72 ° C for 30 seconds the final stage of polymerization at 72 ° C for 7 minutes.

Next, the second stage of constructing the target DNA fragment was carried out. The Cm ^R gene was amplified by PCR, using the commercially available plasmid pACYC184 (accession number X06403 in the GenBank / EMBL database, Fermentas, Lithuania) and primers P26 (SEQ ID NO: 33) and P27 (SEQ ID NO : 34). Primer P26 contains the BglII restriction enzyme recognition site necessary for the further attachment of this DNA fragment to the previously obtained fragment containing the P _{tac *} promoter. Primer P27 contains 46 nucleotides complementary to nucleotides 58 to 12 located in front of the start codon of the pgl gene (ybhE) on the E. coli chromosome; this complementary region is necessary for further integration of the resulting fragment into the bacterial chromosome.

Amplified DNA fragments were concentrated by agarose gel electrophoresis, isolated from the gel using GenElute Spin Columns (Sigma, USA) and planted with ethanol. Then, both obtained DNA fragments were treated with BglII endonuclease and then ligated using T4 DNA ligase (Maniatis T., Fritsch EF, Sambrook, J .: Molecular Cloning: A Laboratory Manual. 2 ^nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1989).

The ligation product was amplified by PCR using the primers P25 and P27. The reaction mixture with a total volume of 50 μl contained 5 μl of 10x PCR buffer (Sigma, USA), 200 μM of each dNTP triphosphate, 25 pM of each primer used, and 1 unit of AccuTaq LA polymerase (Sigma, USA). About 50 ng of plasmid DNA was added to the reaction mixture as a template for PCR amplification. The PCR temperature profile was as follows: the initial stage of denaturation for 5 min at 95 ° C, followed by 25 cycles of denaturation at 95 ° C for 30 seconds, annealing at 55 ° C for 30 seconds, chain extension at 72 ° C for 4 minutes the final stage of polymerization at 72 ° C for 7 minutes.

The nucleotide sequence of the obtained DNA fragment is presented in the sequences SEQ ID NO: 35 and 36 for the promoters P _tac-3900 and P _tac-10000, respectively.

The obtained DNA fragments, purified as described above, were used for electroporation and Red-dependent integration into the bacterial chromosome of E. coli strain BW25113Δzwf-β-eda [pKD46], as described in Example 2.

Colonies grown for 24 hours in a medium with chloramphenicol were tested for the presence of the Cm ^R marker in front of the pgl gene using PCR primers P27 (SEQ ID NO: 34) and P10 (SEQ ID NO: 15). The same colonies were also tested by PCR for the presence of the promoter region P _{tac *} in front of the pgl gene - primers P23 (SEQ ID NO: 30) and P24 (SEQ ID NO: 31) for P _tac-3900 and P _tac-10000, respectively P10 (SEQ ID NO: 15). For this purpose, freshly grown colonies were resuspended in 20 μl of water and 1 μl of the resulting suspension was used as a template for PCR. The PCR temperature profile was as follows: the initial stage of DNA denaturation - for 10 minutes at 95 ° C, then 30 cycles of denaturation at 95 ° C for 30 seconds, annealing at 54 ° C for 30 seconds, chain extension at 72 ° C within 1 minute; the final stage of polymerization at 72 ° C for 7 minutes. Several tested Cm ^R colonies contained the desired 1192 bp DNA fragments. and 124 bp, confirming the presence of both the entire constructed DNA fragment before the pgl gene and a hybrid regulatory element containing the P _{tac *} promoter in the tested chromosomes of E. coli strains. In both cases, one of the obtained strains was cured of the heat-sensitive plasmid pKD46 by growing the strain at 37 ° C and the obtained strains were named E. coli BW25113-Ptac-3900-ybhE and BW25113-Ptac-10000-ybhE, respectively. The structure of the constructed bacterial DNA fragment in front of the pgl gene is shown in FIG. 7.

Example 10. Measurement of the activity of 6-phosphogluconolactonase in strains with enhanced expression of the pgl gene.

The activity of 6-phosphogluconolactonase in strains BW25113-P _tac-3900- ybhE and BW25113-P _tac-10000- ybhE was measured by the method described in Example 7. The obtained data are shown in Table 2. When measuring activity, the level of spontaneous hydrolysis was subtracted.

table 2 Strains The activity of 6-phosphogluconate lactonase, relative units BW25113 5.6 BW25113-P _tac-3900 -ybhE 21.1 BW25113-P _tac-10000 -ybhE 54.0

Thus, enhanced expression of the pgl gene leads to an increase in the activity of 6-phosphogluconolactonase.

Example 11. The effect of enhancing pgl gene expression on tryptophan production.

An E. coli L-tryptophan producing strain SV164 [pMW-P _lacUV5 -serA5-fruR, pYDDG2] was used as a parent strain to evaluate the effect of enhancing pgl gene expression on tryptophan production. Strain SV164 is described in detail in US Pat. No. 6,180,373. Strain SV164 [pMW-P _lacUV5 -serA5-fruR, pYDDG2] is derived from strain SV164 and further comprises plasmids pMW-P _lacUV5 -serA5-fruR and pYDDG2. Plasmid pMW-P _tacUV5 -serA5-fruR contains the mutant gene serA5, which encodes a protein that is not inhibited by serine by feedback. Amplification of the serA5 gene is necessary to increase the pool of serine, the precursor of L-tryptophan (US patent 6180373). The plasmid pYDDG2 was constructed on the basis of the pAYCTER3 vector (WO 03/044192) and contains the yddG gene, which encodes a transmembrane protein (a possible exporter) that positively affects the production of L-tryptophan. The pAYCTER3 vector is a derivative of the pAYC32 vector, which is a low copy and very stable vector constructed on the basis of the RSF1010 plasmid and containing a streptomycin resistance marker (Christoserdov AY, Tsygankov YD, Broad-host range vectors derived from a RSF 1010 Tnl plasmid, v Plasmid, 1986 .16, p. 161-167). The pAYCTER3 vector was obtained by introducing into the pAYC32 plasmid a polylinker from plasmid pUC19 and the strong terminator rrnB instead of the promoter.

To assess the effect of enhanced expression of the pgl gene under the control of P _{tac *} promoters on the tryptophan production, DNA fragments from the chromosome of the aforementioned E. coli strains BW25113-P _tac-3900- ybhE and BW25113-Р _tac-10000 -ybhE using P1 transduction Miller, JH (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press, Plainview, NY) were transferred to E. coli tryptophan producer strain S. Coli SV164 [pMW-P _lacUV5 -serA5-fruR]. Then, plasmid pYDDG2 was introduced into the obtained transductants and strain SV164 [pMW-P _lacUV5 -serA5-fruR].

Strains SV164 [pMW-P _lac -serA5-fruR, pYDDG2],

S164-P _tac-3900 -ybhE [pMW-P _lacUV5 -serA5-fruR, pYDDG2] and

SV164-P _tac-10000 -ybhE [pMW-P _lacUV5 -serA5-fruR, pYDDG2] were grown overnight on a rotary shaker at 37 ° C in 3 ml of nutrient broth containing ampicillin (100 μg / ml) and streptomycin (50 μg / ml). 0.3 ml of the resulting cultures were inoculated into 20 × 200 mm test tubes from 3 ml of fermentation medium containing these antibiotics, and grown at 37 ° C for 40 hours on a rotary shaker at 250 rpm. The composition of the medium for fermentation is presented in Table 3.

Table 3 Fraction Component Final concentration BUT Glucose / sucrose 40 g / l MgSO ₄ × 7H ₂ O 0.3 g / l MnSO ₄ × 5H ₂ O 5 mg / l Memeno 0.058 g / l of total nitrogen (NH ₄ ) ₂ SO ₄ 15 g / l KH ₂ PO ₄ 0.268 g / l NaCl 0.143 g / l L-methionine 0.086 g / l L-phenylalanine 0.286 g / l L-tyrosine 0.286 g / l Kcl 0.286 g / l FeSO ₄ × 7H ₂ O 5 mg / l Sodium citrate 667 mg / l CaCl ₂ × 2H ₂ O 4.29 mg / l Sterilization at 116 ° C for 30 minutes B Thiamine HCl 2.5 mg / l Na ₂ MoO ₄ × 2H ₂ O 0.15 mg / l H ₃ BO ₃ 2.5 mg / l CoCl ₂ × 6H ₂ O 0.7 mg / l CuSO ₄ × 5H ₂ O 0.25 mg / l ZnSO ₄ × 7H ₂ O 0.3 mg / l Sterilization at 110 ° C for 30 minutes FROM Pyridoxine 45 mg / l Filtration

The pH value of 7.1 in fraction A was adjusted with NH ₄ OH. Each fraction was sterilized separately.

After growth, the amount of L-tryptophan accumulated in the medium was determined by thin layer chromatography (TLC). We used 10 × 15 cm TLC plates coated with Sorbfil silica gel layers without a 0.11 mm fluorescent indicator (Sorbpolymer Joint-Stock Company, Krasnodar, Russia). Sorbfil plates were exposed in the liquid phase: propan-2-ol: ethyl acetate: 25% aqueous ammonia solution: water = 16: 16: 3: 9 (volume fractions). A solution (2%) of ninhydrin in acetone was used as an imaging agent. The data obtained are presented in Table 4.

Table 4 Strain OD ₆₀₀ Tryptophan concentration, g / l SV164 [pMW-P _lacUV5 -serA5-fruR, pYDDG2] 7.5 4.20 SV164-P _tac-3900- ybhE [pMW-P _lacUV5 -serA5-fruR, pYDDG2] 7.5 4.61 SV164-P _tac-10000- ybhE [pMW-P _lacUV5 -serA5-fruR, pYDDG2] 7.5 4.92

As can be seen from Table 4, increased expression of the pgl gene positively affects the ability of strain SV164 [pMW-P _tacUV5- serA5-fruR, pYDDG2] to produce tryptophan.

Example 12. Isolation of YbhE protein with His ₆ terminal group and determination of its activity.

All the results described in Examples 1-7 can only serve as indirect evidence that ybhE ORF is the pgl gene encoding the functionally active 6-phosphogluconolactonase protein in E. coli. On the other hand, it is likely that ybhE ORF encodes, for example, a positive expression regulator of another unknown gene, which, in turn, is 6-phosphogluconolactonase. Thus, the final conclusion regarding the nature of ybhE ORF can be made only on the basis of direct measurement of the biological activity of the protein product.

For this purpose, ybhE ORF was expressed using the T7 phage expression system, E. coli strain BL21 (DE3) as a recipient strain containing the T7 phage polymerase RNA gene in the chromosome and plasmid vector pET-22b (+) with a late phage promoter T7 and an efficient ribosome binding site (RBS) of the T7 phage 10 gene. For convenience of subsequent protein purification, 6 histidine codons were introduced at the 5'-end, ybhE ORF, immediately after the ATG initiator codon.

For cloning of ybhE ORF with the addition of 6 histidine codons in the T7 phage expression system, PCR method was used with primers P28 (SEQ ID NO: 37) and P29 (SEQ ID NO: 38) and chromosomal DNA of E. coli strain MG1655 as a template. Primer P28 contains an NdeI restriction site, an ATG codon linked to 6 additional histidine codons before the second ybhE ORF codon. Primer P29 contains the BamHI restriction site at the 5 ′ end, necessary for further cloning. The amplified DNA fragment was isolated, digested with restriction enzymes NdeI and BamHI and ligated into plasmids pET-22b (+), pretreated with the same restriction enzymes. The design of the obtained plasmid pET-HT-ybhE was confirmed by determining the nucleotide sequence.

Then, BL21 (DE3) cells containing the T7 phage polymerase RNA gene in the chromosome under the control of the lactose promoter were transformed with the plasmid pET-HT-ybhE. The overnight culture obtained from one colony was diluted 50 times with LB medium and grown to an optical density of OD ₆₀₀ ~ 1.0, followed by the addition of IPTG (1 mM) for induction by T7 ybhE ORF phage RNA polymerase in the resulting recombinant plasmid. After incubation for 2 hours, cells were taken in a volume of 20 ml. The cell extract was obtained by sonication in a buffer containing 20 mM Tris-HCl, pH 8.0 and 2 mM PMSF. The samples were then centrifuged for 20 minutes at 16,000 g and 4 ° C and histidine supplemented protein was isolated from the supernatant using HiTrap Chelating Columns (Amersham Bioscience) according to the manufacturer's recommendations.

After 2 hours after the induction of the T7 phage expression system in the culture in the logarithmic growth phase, an accumulation of protein with electrophoretic mobility corresponding to the mobility of the YbhE protein with the addition of 6 histidine residues (molecular weight ≈37 kDa) was observed. The amount of this protein was about 15% of the total number of cell polypeptides. The specified protein was found mainly in the soluble phase (see Fig.8A).

The resulting His ₆ -YbhE protein was purified on Ni-NTA columns. Determination of the level of recombinant protein synthesis and control of the purification process was carried out using SDS-PAGE electrophoresis in accordance with the method described by Laemmli UK (Nature, 227, 680-685 (1970)). As can be seen from Figb, the purity of the obtained protein was more than 90%, while the protein showed 6-PGL activity in a standard test using latonase (Collard, F. et al, FEBS Letters, 459, 223-226 (1999)); Example 7). It is interesting to note that the measured specific activity of 6-PGL in purified His ₆ -YbhE protein (780 U / mg) was close to the previously described activity of a similarly modified human His ₆ -6-PGL protein (Collard, F. et al, FEBS Letters, 459, 223-226 (1999)).

Thus, it was concluded that ybhE ORF with an unknown function from E. coli is actually the pgl gene encoding 6-PGL.

Example 13. The effect of enhancing pgl gene expression on phenylalanine production.

Strain E. coli AJ12739, a phenylalanine producer, was used as a starting strain to evaluate the effect of enhancing pgl gene expression on phenylalanine production. Strain AJ12739 was deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) (Russia, 113545 Moscow, 1st Dorozhniy proezd, 1) on November 6, 2001 under accession number VKPM V-8197.

Chromosomal DNA fragments from strains BW25113-P _tac-3900- ybhE and BW25113-P _tac-10000 -ybhE were transferred to E. coli strain AJ12739, a producer of phenylalanine P1 by transduction to obtain strains AJ12739 P _tac-3900- ybhE P and _{AJ12 10,000} respectively. Each of these strains was grown at 37 ° C for 18 hours in a nutrient broth containing 25 mg / ml chloramphenicol; 0.3 ml of the resulting culture was transferred to 3 ml of fermentation medium containing 25 mg / ml chloramphenicol in a 20 × 200 tube mm and incubated at 34 ° C for 24 hours on a rotary shaker. After growing, the amount of phenylalanine accumulated in the medium was determined by TLC. We used 10 × 15 cm TLC plates coated with a 0.11 mm layer of Sorbfil silica gel without a fluorescent indicator (Sorbpolymer Company, Krasnodar, RF). Sorbfil plates were exposed with a mobile phase of the following composition: propan-2-ol: ethyl acetate: aqueous ammonia (25%): water = 40: 40: 7: 16 (v / v). As a reagent for visualization, a 2% solution of ninhydrin in acetone was used.

The composition of the medium for fermentation (g / l):

Glucose 40.0 (NH ₄ ) ₂ SO ₄ 16.0 K ₂ HPO ₄ 0.1 MgSO ₄ 7H ₂ O 1.0 FeSO ₄ 7H ₂ O 0.01 MnSoO ₄ 5H ₂ O 0.01 Thiamine HCl 0.0002 Yeast extract 2.0 Tyrosine 0.125 CaCO ₃ 20.0

Glucose and magnesium sulfate were sterilized separately. CaCO _{3 was} sterilized at 180 ° C for 2 hours. pH was maintained at around 7.0. The antibiotic was added to the culture medium after sterilization.

The results are presented in Table 5.

Table 5 Strain E. coli OD ₆₀₀ The amount of phenylalanine, g / l AJ12739 18.2 ± 0.1 0.65 ± 0.4 AJ12739 P _tac-3900 -ybhE 17.0 ± 0.3 0.9 ± 0.1 AJ12739 P _tac-10000 -ybhE 16.3 ± 0.2 1.3 ± 0.1

As can be seen from Table 5, increased expression of the pgl gene increased the ability of strain AJ12739 to produce phenylalanine.

Although the invention has been described in detail with reference to specific Examples, it will be apparent to those skilled in the art that various changes may be made and equivalent replacements may be made, and such changes and replacements are not outside the scope of the present invention. Each of the above documents cited in the description is part of the present description.

Claims (15)

1. 6-Phosphogluconolactonase selected from the group consisting of the following proteins: (A) a protein comprising the amino acid sequence shown in sequence listing number 2 (SEQ ID NO: 2); and (B) a protein comprising an amino acid sequence containing deletions, substitutions, insertions, or addition of one or more amino acids in the amino acid sequence shown in sequence listing number 2 (SEQ ID NO: 2), which has at least 70% homology in relative to the amino acid sequence shown under number 2 (SEQ ID NO: 2), and which has the activity of 6-phosphogluconolactonase. 2. A DNA fragment encoding 6-phosphogluconolactonase according to claim 1. 3. A bacterium belonging to the genus Escherichia is a producer of an L-amino acid selected from the group consisting of aromatic L-amino acid and L-histidine, characterized in that said bacterium is modified in such a way that it increases the activity of 6-phosphogluconolactonase according to claim .one. 4. The bacterium according to claim 3, characterized in that said 6-phosphogluconolactonase is obtained from a bacterium belonging to the genus Escherichia. 5. The bacterium according to claim 3, characterized in that said aromatic L-amino acid is an L-amino acid selected from the group consisting of L-tryptophan, L-phenylalanine and L-tyrosine. 6. The bacterium according to claim 3, characterized in that the activity of 6-phosphogluconolactonase is increased by modifying the sequence that controls the expression of the 6-phosphogluconolactonase gene on the chromosome of the bacterium in such a way that the expression of the specified gene is enhanced. 7. The bacterium according to claim 6, characterized in that the natural promoter of the specified gene is replaced by a stronger promoter. 8. The bacterium according to claim 6, characterized in that the gene encoding 6-phosphogluconolactonase is selected from the group consisting of: (a) DNA comprising the sequence of nucleotides 1 to 996 shown in the list of sequences under the number 1 (SEQ ID NO: 1); (b) DNA that hybridizes under stringent conditions with the nucleotide sequence 1 to 996 listed in the sequence number 1 (SEQ ID NO: 1), or with a probe prepared on the basis of the specified nucleotide sequence, and encodes a protein having activity 6-phosphogluconolactonase. 9. The bacterium of claim 8, characterized in that the harsh conditions are the conditions under which washing is carried out at 60 ° C, the salt concentration corresponds to 1 × SSC and 0.1% SDS, and the washing time is 15 minutes 10. The bacterium according to claim 3, characterized in that said bacterium is modified in such a way that expression of the open reading frame of yddG is enhanced. 11. A method for producing an L-amino acid selected from the group consisting of an aromatic L-amino acid and L-histidine, comprising the steps of growing a bacterium according to claim 3 in a nutrient medium and isolating the accumulated L-amino acid from the culture fluid. 12. The method according to claim 11, characterized in that said aromatic L-amino acid is L-tryptophan. 13. The method according to claim 11, characterized in that said aromatic L-amino acid is L-phenylalanine. 14. The method according to claim 11, characterized in that in said bacterium the expression of aromatic L-amino acid biosynthesis genes is enhanced. 15. The method according to claim 11, characterized in that in said bacterium the expression of L-histidine biosynthesis genes is enhanced.

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