RU2560980C2 - DIPEPTIDE-SYNTHESISING ENZYME CODING DNA (VERSIONS), Escherichia BACTERIA AND METHOD OF PRODUCING DIPEPTIDES USING SAME - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and provides a dipeptide-producing Escherichia bacterium, which is modified such that it contains DNA which encodes a protein with dipeptide-synthesising activity. The invention also relates to a method of producing a dipeptide or a salt thereof using said bacteria or using a protein with dipeptide-synthesising activity.

EFFECT: invention enables to obtain dipeptides efficiently.

16 cl, 28 dwg, 12 tbl, 14 ex

Description

FIELD OF THE INVENTION

The present invention relates to the biotechnological industry, more specifically to new dipeptide-synthesizing enzymes and methods for producing dipeptides, in particular dipeptides having acidic L-amino acid residues at the N-terminus.

State of the art

Dipeptides are used in pharmacology, the food industry, and in various other fields. For example, the Asp-Glu dipeptide was used to prepare a diuretic and natriuretic pharmaceutical composition (FR2662359 A1). Known pharmaceutical composition containing dipeptides having an agonistic effect on the subtypes of NR1 / NR2A and NR1 / NR2B of the NMDA receptor (JP 2009209131 A). The taste qualities of numerous dipeptides have been studied. For example, the Asp-Val dipeptide has an acidic taste (Sogame S. and Matsushita I., New Food Ind., 1996, 38 (12): 44-49 (Japanese)). An excellent salt flavor enhancer is known which is obtained using a glutamic acid-containing dipeptide such as Glu-Ala, Glu-Asp, Glu-Glu, Glu-Ile, Asp-Glu, His-Glu, Trp-Glu, etc. (WO2009113563 A1).

Various methods for producing dipeptides are known, including extraction from protein hydrolysates, chemical synthesis from protected and / or activated amino acids, and enzymatic synthesis methods involving peptidases and protected amino acids (Akabori S. et al., Bull. Chem. Soc. Japan, 1961, 34 : 739; Monter, B. et al., Biotechnol. Appl. Biochem., 1991, 14 (2): 183-191). A genetic construct encoding a peptide comprising the repeating amino acid sequence (Asp-Phe) n suitable for preparing benzylated and methylated derivatives of the Asp-Phe dipeptide (European Patent Application No. 0036258) has been described.

The preparation of dipeptides using chemical and / or chemical-enzymatic approaches requires the introduction and removal of protective groups for the functional groups of reacting amino acids and the isolation of the desired product from the racemic mixture. Thus, the described methods for the preparation of dipeptides are considered disadvantageous in terms of price, efficiency and the need for disposal of related chemicals, such as organic solvents, salts and the like.

Several approaches to the enzymatic preparation of dipeptides and their derivatives have been described, which include a method using a reversible proliniminopeptidase reaction having the ability to produce peptides from L-amino acids and their esters (RF patent No. 2279440); non-ribosomal peptide synthetase (NRPS) method (US Patent Nos. 5,795,738 and 5,652,116; Doekel S. and Marahiel MA, Chem. Biol., 2000, 7: 373-384; Dieckmann R. et al., FEBS Lett., 2001, 498: 42-45); a method using aminoacyl-m-PHK synthetase (Japanese patents Nos. 58-146539 (1983), 58-209992 (1983), and 59-106298 (1984)); and a method using a mutant protein having a peptide-synthesizing activity (Russian patent application 2007127719).

Enzymes belonging to the ATP-dependent carboxylate-amine / thiol-α-ligase superfamily have been widely used to produce dipeptides having an α-peptide bond between two L-amino acids. For example, when using the homology search function in SubtiList (http://genolist.pasteur.fr/SubtiList/), which is a database of the genomic DNA of Bacillus subtilis 168, and the amino acid sequence of the D-Ala-D-Ala ligase gene from Bacillus subtilis 168, a ywfE gene has been discovered that encodes an enzyme capable of synthesizing dipeptides having an L-amino acid at the N-terminus, such as, in particular, L-Ala, L-Gly, L-Met, L-Ser and L-Thr ( Tabata, K. et al., J. Bacteriol., 2005, 187 (15): 5195-5202; U.S. Patent Nos. 7,514,243 and 7,939,302). Despite the fact that YwfE protein (bacylisine synthetase, enzyme classification number (EC) 6.3.2.28) has an extremely broad substrate specificity, the enzyme does not bind high-charge amino acids such as L-Lys, L-Arg, L-Glu and L- Asp, and secondary amines such as L-Pro (Tabata K. et al., J. Bacteriol. 2005, 187 (15): 5195-5202). A protein encoded by the rhizocticin synthetase gene and having a dipeptide-synthesizing activity that utilizes the G-amino acids and G-β-Ala L-amino acids as substrates is described (US patent No. 7,939,294). As evidenced by the released phosphoric acid (Pi), as well as by time-of-flight mass spectroscopy (TOFMS) and NMR, the enzyme places L-Arg and L-Lys at the N-terminus of the dipeptide. Analysis based on the Hidden Markov Model (HMM) profile revealed five L-amino acid-α-ligases derived from Treponema denticola ATCC 35405, Photorhabdus luminescence subsp. laumondii TTO1, Streptococcus mutants UA159, Streptococcus pneumoniae TIGR4 and Actinobacillus pleuropneumoniae serological variant 1 str. 4074 capable of forming various peptide compounds from L-amino acids, as evidenced by the removal of phosphoric acid (Senoo A. et al., Biosci. Biotechnol. Biochem., 2010, 74 (2): 415-418). No dipeptide formation has been confirmed in a combination of L-Glu or L-Asp with other L-amino acids. A mutant protein having peptide-synthesizing activity, as was confirmed by high performance liquid chromatography (HPLC) using standard samples, forms dipeptides carrying L-Met at the N-terminus (Russian patent application No. 2007127719). Computer screening using the BLAST service at the NCBI (http://www.ncbi.nlm.nih.gov/BLAST/) and based on the amino acid sequence Lal from B. subtilis (BsLal) detected the RSp1486a protein from Ralstonia solanacearum, which is capable of form a dipeptide bond as confirmed by cleavage of phosphoric acid (Kino K. et al., Biochem. Biophys. Res. Comm., 2008, 371: 536-540; European Patent Application No. 1870454). Structural analysis using NMR confirmed the formation of dipeptides having L-Ser, L-Met, L-Gln, L-Phe, L-His, L-Ala and L-Cys at the N-terminus. Although inorganic phosphate was cleaved, as was proven, in a mixture containing RSp1486a and L-Asp with L-Phe, L-His, L-Met, L-Cys or L-Ala; or RSp1486a and L-Glu with L-Phe, L-His, L-Met, L-Cys, L-Ser or L-Ala, no structural analysis of the reaction products was performed. No additional removal of phosphoric acid above the background level was observed in the reaction mixture containing RSp1486a and L-Asp or L-Glu. It was found that the new open L-amino acid RizB gas from B. subtilis NBRC3134 synthesizes various hetero peptides and homo-oligomers of branched chain amino acids consisting of 2-5 amino acid residues (Kino K., Yakugaku Zasshi, 2010, 130 (11): 1463-1469 ) For example, mass spectroscopy (LC-ESI-MS analysis) proved the formation of L-Val dimer, trimer and tetramer in a mixture containing RizB, L-Val and L-Glu or L-Asp. No heteropeptides were found.

There is currently no data describing the synthesis of dipeptides having acidic L-amino acid residues, such as L-Glu or L-Asp residues, at the N-terminus and any other L-amino acid or its derivative at the C-terminus, using L- amino acid α-ligase (Lal).

Disclosure of the invention

The purpose of the present invention is the provision of DNA encoding an L-amino acid-α-ligase (Lal) capable of synthesizing dipeptide (s) containing (s) acidic L-amino acids, such as L-Asp or L-Glu, at the N-terminus and any other L-amino acid or its derivative at the C-terminus, in a reaction mixture that contains high-energy (macroergic) molecules, such as adenosine 5′-triphosphate (ATP) or its salt.

Another objective of the present invention is the provision of bacteria of the genus Escherichia, belonging to the species Escherichia coli, which is modified so that it contains DNA encoding Lal, as described here.

Another objective of the present invention is the provision of a method for producing dipeptides having acidic L-amino acids, such as L-Asp or L-Glu, at the N-terminus and any other L-amino acid or its derivative at the C-terminus, in a reaction mixture that contains macroergic molecules such as adenosine 5′-triphosphate (ATP) or a salt thereof using the Lal enzyme as described herein, or Escherichia bacteria, which is modified to contain DNA encoding the Lal enzyme as described herein.

These goals were achieved thanks to the discovery of new bacterial L-amino acid-α-ligases (Lals) catalyzing the formation of dipeptides having acidic L-amino acids, such as L-Asp or L-Glu, at the N-terminus.

The purpose of the present invention is the provision of DNA encoding a protein having a dipeptide-synthesizing activity, characterized in that the DNA is selected from the group consisting of:

(A) DNA having the nucleotide sequence of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17;

(B) DNA hybridizing under stringent conditions with a nucleotide sequence complementary to that shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17, characterized in that said stringent conditions include washing one or more times in a solution containing a salt concentration of 1 × SSC, 0.1% SDS or 0.1 × SSC, 0.1% SDS, at 60 ° C or 65 ° C;

(C) DNA encoding a protein having the amino acid sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18;

(D) DNA encoding a variant of a protein having the amino acid sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18, but which contains a substitution, deletion, insertion, or addition of one or more amino acid residues, and having a dipeptide-synthesizing activity in accordance with the amino acid sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18;

(E) DNA encoding a protein having homology defined by | Log ₁₀ (E-value) |, not less than 128, not less than 142, not less than 162, not less than 175, not less than 182, not less than 196 or not less than 233 compared to the amino acid sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16 and 18 and having a dipeptide-synthesizing activity in accordance with the amino acid sequence of SEQ ID NOs: 2 , 4, 6, 8, 10, 12, 14, 16, and 18.

The purpose of the present invention is the provision of recombinant DNA for the expression of DNA, as described above, containing DNA, as described above.

The purpose of the present invention is the provision of dipeptide-producing bacteria belonging to the genus Escherichia and modified so as to contain recombinant DNA, as described above.

Another objective of the present invention is the provision of bacteria, as described above, characterized in that the bacterium belongs to the species Escherichia coli.

Another objective of the present invention is the provision of a bacterium, as described above, characterized in that the bacterium is modified so as to have weakened or inactivated one or more genes encoding proteins having peptidase activity.

Another objective of the present invention is the provision of bacteria, as described above, characterized in that the genes encoding the proteins having peptidase activity are selected from the group consisting of peRA, peRB, pepD, pepE, pepP, pepQ, pepN, pepT, iadA, iaaA (ybiK) and dapE.

The purpose of the present invention is the provision of a protein having a dipeptide-synthesizing activity, characterized in that the protein is selected from the group consisting of:

(F) a protein having the amino acid sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16 and 18;

(G) a variant of a protein having the amino acid sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16 and 18, but which contains a substitution, deletion, insertion or addition of one or more amino acid residues, and having a dipeptide - synthesizing activity in accordance with the amino acid sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16 and 18;

(H) a protein having homology defined by | Log ₁₀ (E-value) |, not less than 128, not less than 142, not less than 162, not less than 175, not less than 182, not less than 196 or not less than 233 compared to the amino acid sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16 and 18, and having a dipeptide synthesizing activity in accordance with the amino acid sequence of SEQ ID NOs: 2, 4 , 6, 8, 10, 12, 14 and 16.

The purpose of the present invention is the provision of a method for producing protein, as described above, including:

(a) growing the bacteria, as described above, in a growth medium for the production of said protein;

(b) the accumulation of the specified protein in bacteria and / or culture fluid and, if necessary,

(c) isolating said protein from a bacterium or culture fluid.

The purpose of the present invention is the provision of a method for producing a dipeptide or its salt, including:

(a) the reaction of L-amino acids, or derivatives of L-amino acids, or their salts under acceptable conditions in the presence of a protein, as described above;

(b) the accumulation of the specified dipeptide or its salt in an acceptable solvent and, if necessary,

(c) isolating said dipeptide or salt thereof from an acceptable solvent.

The purpose of the present invention is the provision of a method for producing a dipeptide or its salt, including:

(a) growing bacteria, as described above, in a nutrient medium;

(b) the accumulation of the specified dipeptide in bacteria and / or culture fluid and, if necessary,

(c) isolating said dipeptide from a bacterium or culture fluid.

The purpose of the present invention is the provision of a method as described above, characterized in that said L-amino acids or their derivatives are selected from the group consisting of L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L -glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine and the lower alkyl ester of L-phenylalanine.

The purpose of the present invention is the provision of a method as described above, characterized in that the dipeptide is represented by the formula

R1-R2,

characterized in that R1 is an acid L-amino acid residue or derivative of an acid L-amino acid residue and R2 is an L-amino acid residue or derivative of an L-amino acid residue, said L-amino acid residue selected from the group consisting of L-alanine residue, L arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine , L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine and the lower alkyl ester of L-phenylalanine.

The purpose of the present invention is the provision of a method as described above, wherein R1 is a residue of L-aspartic acid or a residue of L-glutamic acid and R2 is a residue of L-glutamic acid, L-isoleucine, L-phenylalanine, L-tryptophan, L -valine or lower alkyl ester of L-phenylalanine.

The purpose of the present invention is the provision of a method as described above, wherein R1 is a residue of L-aspartic acid or a residue of L-glutamic acid and R2 is a residue of L-glutamic acid, L-isoleucine, L-phenylalanine, L-tryptophan, L -valine or lower alkyl ester of L-phenylalanine.

The purpose of the present invention is the provision of a method as described above, characterized in that the lower alkyl ester of L-phenylalanine is methyl, ethyl or propyl ester of L-phenylalanine.

A description of the present invention is given below.

Brief Description of the Figures

Figure 1 shows a diagram of a ligation reaction catalyzed by L-amino acid-α-ligases (Lals). R _A and R _B are side chain groups that may be of the same or different types. ATP means adenosine 5′-triphosphate, ADP means adenosine 5′-diphosphate and Pi means inorganic phosphate, phosphoric acid or its salt.

Figure 2 shows the activity of BBR47_51900 in the ligation of the same canonical L-amino acids.

Figure 3 shows the activity of BBR47_51900 in the ligation of two different canonical L-amino acids, where one L-amino acid is L-Glu. Cnt: control (L-Glu).

Figure 4 shows the activity of BBR47_51900 in the ligation of two different canonical L-amino acids, where one L-amino acid is L-Asp. Cnt: control (L-Asp).

Figure 5 shows the activity of Staur_4851 in ligation of two different canonical L-amino acids, where one L-amino acid is L-Asp.

Figure 6 shows the activity of Staur_4851 in the ligation of L-Asp and L-Phe, determined by TLC. 1 - (Tris-HCl pH 9.0 50 mM, MgCl ₂ 10 mM, L-Asp 10 mM, L-Phe 10 mM, ATP 10 mM, Staur_4851 2 μg); 2 - (Tris-HCl pH 8.0 50 mM, MgCl ₂ 10 mM, L-Asp 10 mM, L-Phe 10 mM, ATP 10 mM, Staur_4851 2 μg); 3 - (Tris-HCl pH 8.0 50 mM, MgCl ₂ 10 mM, L-Asp 20 mM, L-Phe 0 mM, ATP 10 mM, Staur_4851 2 μg); 4 - (Tris-HCl pH 8.0 50 mM, MgCl ₂ 10 mM, L-Asp 0 mM, L-Phe 20 mM, ATP 10 mM, Staur_4851 2 μg); 5 - (Tris-HCl pH 8.0 50 mM, MgCl ₂ 10 mM, L-Asp 10 mM, L-Phe 10 mM, ATP 0 mM, Staur_4851 2 μg); 6 - (Tris-HCl pH 8.0 50 mM, MgCl ₂ 10 mM, L-Asp 10 mM, L-Phe 10 mM, ATP 10 mM, Staur_4851 0 μg).

Figure 7 shows the activity of BBR47_51900 in the ligation of L-Asp and L-Phe, as determined by LC-QTOF / MS / MS. SP: sample; ST: standard (αAsp-Phe and βAsp-Phe).

Figure 8 shows the activity of BBR47_51900 in the ligation of L-Asp and L-Val, as determined by LC-QTOF / MS / MS. SP: sample; ST: standard (αAsp-Val).

Figure 9 shows the activity of BBR47_51900 in the ligation of L-Glu and L-Val, as determined by LC-QTOF / MS / MS. SP: sample; ST: standard (αGlu-Val and γGlu-Val).

Figure 10 shows the alignment of BBR47_51900 and Staur_4851 (ClustalW, in PIR format).

Figure 11 shows the data obtained using the HMMsearch program based on alignment of BBR47_51900 and Staur_4851 (the first 49 hits are presented).

Figure 12 shows a distribution chart of | Log ₁₀ (E-value) | values obtained using the HMMsearch program using the alignment BBR47_51900 and Staur_4851 (see Figure 10). The following hits are marked by solid arrows: 1 - BBR47_51900, 2 - Staur_4851, 3 - DES, 4 - ALL, 5 - BMY, 13 - BTH, 17 - BUR, 47 - AME, 49 - SFL.

Figure 13 shows the alignment of BBR47_51900, Staur_4851, DES and ALL (ClustalW, in PIR format).

Figure 14 shows the output obtained using the HMMsearch program based on alignment BBR47_51900, Staur_4851, DES and ALL (the first 65 hits are presented).

Figure 15 shows a distribution chart of | Log ₁₀ (E-value) | values obtained using the HMMsearch program using the alignments BBR47_51900, Staur_4851, DES, and ALL (see Figure 13). The following hits are marked by solid arrows: 1 - BBR47_51900, 2 - DES, 3 - EVERYTHING, 4 - Staur_4851, 5 - BMY, 8 - BTH, 18 - BUR, 33 - AME, 62 - SFL.

Figure 16-1 shows the aligned BBR47_51900, Staur_4851 and DES in alignment BBR47_51900, Staur_4851, DES, ALL and BMY (ClustalW, in PIR format).

Figure 16-2 shows the aligned ALL and BMY in alignment BBR47_51900, Staur_4851, DES, ALL and BMY (ClustalW, in PIR format).

Figure 17 shows the output obtained using the program HMMsearch based on alignment BBR47_51900, Staur_4851, DES, ALL and BMY (the first 65 hits are presented).

Figure 18-1 shows the aligned Staur_4851, BBR47_51900 and ALL in alignment BBR47_51900, Staur_4851, DES, ALL, BMY and BTN (ClustalW, in PIR format).

Figure 18-2 shows the aligned DES, BMY, and VTN in alignment BBR47_51900, Staur_4851, DES, ALL, BMY, and VTN (ClustalW, in PIR format).

Figure 19 shows the data obtained using the HMMsearch program based on alignment of BBR47_51900, Staur_4851, DES, ALL, BMY and BTN (the first 73 hits are presented).

Figure 20-1 shows the aligned Staur_4851 and BBR47_51900 in alignment BBR47_51900, Staur_4851, DES, ALL, BMY, BTN and BUR (ClustalW, in PIR format).

Figure 20-2 shows the aligned ALL and DES in alignment BBR47_51900, Staur_4851, DES, ALL, BMY, BTN and BUR (ClustalW, in PIR format).

Figure 20-3 shows aligned BMY and BTN in alignment BBR47_51900, Staur_4851, DES, ALL, BMY, VTN and BUR (ClustalW, in PIR format).

Figure 20-4 shows the aligned BUR in alignment BBR47_51900, Staur_4851, DES, ALL, BMY, BTN and BUR (ClustalW, in PIR format).

Figure 21 shows the data obtained using the HMMsearch program based on alignment of BBR47_51900, Staur_4851, DES, ALL, BMY, BTN and BUR (the first 104 hits are presented).

Figure 22-1 shows the aligned Staur_4851 and BBR47_51900 in alignment BBR47_51900, Staur_4851, DES, ALL, BMY, BTN, BUR and AME (ClustalW, in PIR format).

Figure 22-2 shows aligned ALL and DES in alignment BBR47_51900, Staur_4851, DES, ALL, BMY, BTN, BUR, and AME (ClustalW, in PIR format).

Figure 22-3 shows aligned BMY and BTN in alignment BBR47_51900, Staur_4851, DES, ALL, BMY, VTN, BUR and AME (ClustalW, in PIR format).

Figure 22-4 shows aligned BUR and AME in alignment BBR47_51900, Staur_4851, DES, ALL, BMY, BTH, BUR and AME (ClustalW, in PIR format).

Figure 23 shows the output obtained using the HMMsearch program based on alignment BBR47_51900, Staur_4851, DES, ALL, BMY, BTH, BUR and AME (the first 65 hits are presented).

Figure 24-1 shows the aligned Staur_4851 and BBR47_51900 in alignment BBR47_51900, Staur_4851, DES, ALL, BMY, BTH, BUR, AME and SFL (ClustalW, in PIR format).

Figure 24-2 shows aligned ALL and DES in alignment BBR47_51900, Staur_4851, DES, ALL, BMY, BTH, BUR, AME, and SFL (ClustalW, in PIR format).

Figure 24-3 shows aligned BMY and BTH in alignment BBR47_51900, Staur_4851, DES, ALL, BMY, BTH, BUR, AME, and SFL (ClustalW, in PIR format).

Figure 24-4 shows aligned BUR and AME in alignment BBR47_51900, Staur_4851, DES, ALL, BMY, BTH, BUR, AME and SFL (ClustalW, in PIR format).

Figure 24-5 shows the aligned SFL in alignment BBR47_51900, Staur_4851, DES, ALL, BMY, BTH, BUR, AME and SFL (ClustalW, in PIR format).

Figure 25 shows the data obtained using the HMMsearch program based on alignment of BBR47_51900, Staur_4851, DES, ALL, BMY, BTH, BUR, AME and SFL (the first 38 hits are presented).

Figure 26 shows an analysis of isofunctional Lals using the profile of HMMs (Models 1 to 7). *: E-value = 0 (Figures 21, 23 and 25). BBR means BBR47_51900 and STA means Staur_4851.

Figure 27 shows TLC analysis of specific aspartate-peptide-hydrolyzing (DP3-hydrolyzing) activity in E. coli 4-5Δ strains. For calibration, a solution of (1 μl) 5-fluorotryptophan (standard) was used: (1) 3 mM, (2) 2 mM, (3) 1 mM, and (4) 0.5 mM. An aliquot (1 μl) of the reaction mixture containing Mn ²⁺ (5, 6) or Zn ²⁺ (7, 8) was placed on a TLC plate. 5FT - 5-fluorotryptophan, DP3 - L-Asp-L-5-fluorotryptophan dipeptide, Asp - L-aspartate.

Figure 28 shows a DP3 toxicity study design in view of the specific aspartate-peptide-hydrolyzing activity in E. coli 1-5Δ strains. The E. coli strain is grown in the presence of the DP3 dipeptide. In a strain containing peptidases (E. coli P ⁺ ), DP3 is hydrolyzed, resulting in the formation of L-aspartate and 5-fluorotryptophan (5FT). 5FT is toxic to the cell, which thus leads to stunted growth. The DP3 dipeptide is stable and does not affect the cell growth of a strain deficient in peptidases and a strain with low peptidase activity (E. coli P ^- ).

Description of sequences

SEQ ID NO: 1 shows the BBR47_51900 gene

SEQ ID NO: 2 shows the protein BBR47_51900

SEQ ID NO: 3 shows the Staur_4851 gene

SEQ ID NO: 4 shows Staur_4851 protein

SEQ ID NO: 5 shows the DES gene

SEQ ID NO: 6 shows DES protein

SEQ ID NO: 7 shows the BUR gene

SEQ ID NO: 8 shows a BUR protein

SEQ ID NO: 9 shows the ALL gene

SEQ ID NO: 10 shows ALL protein

SEQ ID NO: 11 shows the VTN gene

SEQ ID NO: 12 shows the protein BTN

SEQ ID NO: 13 shows the AME gene

SEQ ID NO: 14 shows AME protein

SEQ ID NO: 15 shows the SFL gene

SEQ ID NO: 16 shows SFL protein

SEQ ID NO: 17 shows the BMY gene

SEQ ID NO: 18 shows BMY protein

SEQ ID NO: 19 shows an XbaI-EcoRI fragment containing BBR47_51900

SEQ ID NO: 20 shows an XbaI-EcoRI fragment containing Staur_4851

SEQ ID NO: 21 shows an XbaI-EcoRI fragment containing DES

SEQ ID NO: 22 shows an XbaI-EcoRI fragment containing BUR

SEQ ID NO: 23 shows an XbaI-EcoRI fragment containing ALL

SEQ ID NO: 24 shows an XbaI-EcoRI fragment containing BTN

SEQ ID NO: 25 shows an XbaI-EcoRI fragment containing AME

SEQ ID NO: 26 shows an XbaI-EcoRI fragment containing SFL

SEQ ID NO: 27 shows an XbaI-EcoRI fragment containing BMY

SEQ ID NO: 28 shows primer P1

SEQ ID NO: 29 shows primer P2

SEQ ID NO: 30 shows primer P3

SEQ ID NO: 31 shows primer P4

SEQ ID NO: 32 shows primer P5

SEQ ID NO: 33 shows primer P6

SEQ ID NO: 34 shows primer P7

SEQ ID NO: 35 shows primer P8

SEQ ID NO: 36 shows primer P9

SEQ ID NO: 37 shows primer P10

SEQ ID NO: 38 shows primer P11

SEQ ID NO: 39 shows primer P12

SEQ ID NO: 40 shows primer P13

SEQ ID NO: 41 shows primer P14

SEQ ID NO: 42 shows primer P15

SEQ ID NO: 43 shows primer P16

SEQ ID NO: 44 shows primer P17

SEQ ID NO: 45 shows primer P18

SEQ ID NO: 46 shows primer P19

SEQ ID NO: 47 shows primer P20

SEQ ID NO: 48 shows primer P21

SEQ ID NO: 49 shows primer P22

SEQ ID NO: 50 shows primer P23

SEQ ID NO: 51 shows primer P24

SEQ ID NO: 52 shows primer P25

SEQ ID NO: 53 shows primer P26

SEQ ID NO: 54 shows primer P27

SEQ ID NO: 55 shows primer P28

SEQ ID NO: 56 shows primer P29

SEQ ID NO: 57 shows primer P30

SEQ ID NO: 58 shows primer P31

SEQ ID NO: 59 shows primer P32

SEQ ID NO: 60 shows primer P33

SEQ ID NO: 61 shows primer P34

SEQ ID NO: 62 shows primer P35

SEQ ID NO: 63 shows primer P36

SEQ ID NO: 64 shows primer P37

SEQ ID NO: 65 shows primer P38

SEQ ID NO: 66 shows primer P39

SEQ ID NO: 67 shows primer P40

SEQ ID NO: 68 shows an optimized gene encoding BBR47_51900 from Brevibacillus brevis NBRC 100599

SEQ ID NO: 69 shows an optimized gene encoding Staur_4851 from Stigmatella aurantiaca

BEST MODE FOR CARRYING OUT THE INVENTION

1. Enzyme

The term “enzyme” can mean an L-amino acid-α-ligase (Lal) having the ability to attach amino acids in a high-energy molecular-dependent (macroergic) manner to form a peptide bond between amino acid residues.

The enzyme according to the present invention may be an L-amino acid-α-ligase selected from the group consisting of BBR47_51900 (hypothetical protein), Staur_4851 (arginine succinate lyase 2-like protein), DES (pyridoxalphosphate-dependent enzyme), BUR (putative lyase) , ALL (argininosuccinate lyase domain protein), BTN (hypothetical protein YBT020_25570), AME (conservative hypothetical protein), SFL (protein with unknown function DUF201) and BMY (argininosuccinate lyase domain protein), which is not limited to the aforementioned proteins.

The nucleotide sequence of the gene (NCBI sequence listing: YP_002774671.1; nucleotides complementary to the nucleotides at position: 5464162 through 5465418; gene identifier: 7721040).

The nucleotide sequence of the gene (NCBI sequence listing: YP_002774671.1; nucleotides complementary to nucleotides at position 5464162 to 5465418; gene identifier: 7721040) from Brevibacillus brevis NBRC 100599 (NCBI taxon identifier: 358681) and the amino acid sequence BBR47_5 shown in code BB0047_5 SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

The nucleotide sequence of the gene (NCBI sequence listing: AD072629.1; nucleotides complementary to the nucleotides at position: 5973963 to 5975216; gene identifier: 9878344) from Stigmatella aurantiaca DW4 / 3-1 (NCBI taxon identifier: 378806) and the amino acid sequence Staur_4851, encoded the genome represented in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

The nucleotide sequence of the gene (GenBank accession number EGK06810.1, GI: 332967701) from Desmospora sp. 8437 (taxon identifier NCBI: 997346) and the DES amino acid sequence encoded by this gene are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

The nucleotide sequence of the gene (GenBank accession number EBA51208.1, GI: 134251129) from Burkholderia pseudomallei 305 (NCBI taxon identifier: 425067) and the amino acid sequence BUR encoded by this gene are presented in SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

The nucleotide sequence of the gene (GenBank accession number EEK72190.1, GI: 228615090) from Bacillus cereus AH621 (NCBI taxon identifier: 526972) and the ALL amino acid sequence encoded by this gene are presented in SEQ ID NO: 9 and SEQ ID NO: 10, respectively.

The nucleotide sequence of the gene (GenBank accession number ADY24341.1, GI: 324329081) from Bacillus thuringiensis subsp. finitimus (strain YBT-020) (NCBI taxon identifier: 930170) and the BTH amino acid sequence encoded by this gene are shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively.

The nucleotide sequence of the gene (GenBank accession number ABR48216.1, GI: 149949688) from Alkaliphilus metalliredigens QYMF (NCBI taxon identifier: 293826) and the amino acid sequence AME encoded by this gene are presented in SEQ ID NO: 13 and SEQ ID NO: 14, respectively.

The nucleotide sequence of the gene (accession number in GenBank ADWO 1942.1, GI: 320007092) from Streptomyces flavogriseus ATCC 33331 (NCBI taxon identifier: 591167) and the amino acid sequence SFL encoded by this gene are presented in SEQ ID NO: 15 and SEQ ID NO: 16, respectively.

The nucleotide sequence of the gene (NCBI sequence listing ZP_04160564.1, GI: 229002475) from Bacillus mycoides Rock3-17 (NCBI taxon identifier: 526999) and the amino acid sequence BMY encoded by this gene are presented in SEQ ID NO: 17 and SEQ ID NO: 18 , respectively.

Due to the fact that there may be some differences in DNA sequences between genera or species and strains of these genera, the genes encoding BBR47_51900, Staur_4851, DES, BUR, ALL, BTN, AME, SFL and BMY are not limited to the genes presented in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, or 17, but may include genes that are variant nucleotide sequences or homologous to the nucleotide sequences of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15 or 17 encoding protein variants BBR47_51900, Staur_4851, DES, BUR, ALL, BTN, AME, SFL and BMY. In addition, genes encoding BBR47_51900, Staur_4851, DES, BUR, ALL, BTN, AME, SFL, and BMY may be variants of nucleotide sequences.

The term "variant nucleotide sequence" may mean a nucleotide sequence that encodes a "variant protein".

The term "nucleotide sequence variant" may mean a nucleotide sequence that encodes a "protein variant" using any synonymous amino acid codons in accordance with the standard genetic code table (see, for example, Lewin B., Genes VIII, 2004, Pearson Education, Inc. , Upper Saddle River, NJ 07458).

The term "variant nucleotide sequence" can also mean a nucleotide sequence that hybridizes under stringent conditions with a nucleotide sequence complementary to the sequence shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, or 17, or probe, which can be synthesized based on these nucleotide sequences, provided that it encodes a functional L-amino acid-α-ligase. "Harsh conditions" means those conditions under which a specific hybrid is formed, for example a hybrid having an identity of at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, not less than 98% or not less than 99%, and a nonspecific hybrid is not formed, for example, a hybrid having a homology lower than that indicated above. A practical example of harsh conditions can be a single or multiple washing, or, alternatively, two or three washing at a salt concentration of 1 × SSC (standard sodium citrate or standard sodium chloride) and 0.1% SDS (sodium dodecyl sulfate) or, in otherwise, 0.1 × SSC and 0.1% SDS at 60 ° C or 65 ° C. The duration of washing depends on the type of membrane used for blotting and, as a rule, is as recommended by the manufacturer. For example, the recommended washing time for a Hybond ™ -N + positive charge nylon membrane (GE Healthcare) under severe conditions is 15 minutes. Rinsing can be done two or three times. As a probe, a portion of the sequence complementary to that shown in SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, or 17 can be used. A similar probe can be obtained using the PCR method (polymerase chain reaction. White TJ et al., Trends Genet., 1989, 5: 185-189) using oligonucleotide primers prepared based on the sequences given in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, or 17, and a DNA fragment containing the nucleotide sequence as a template. The recommended probe length should be> 50 bp, it can be selected depending on hybridization conditions and is usually from 100 to 1000 bp For example, when using a DNA fragment of about 300 bp in length as a probe washing conditions can be as follows: 2 × SSC, 0.1% SDS at 50 ° C, or at 60 ° C, or at 65 ° C. On the other hand, stringent conditions may be hybridization at 6 × SCC at 45 ° C followed by one, two or multiple washings at 0.2 × SCC and 0.1% SDS at temperatures from 50 to 65 ° C.

Genes encoding the proteins BBR47_51900, Staur_4851, DES, BUR, ALL, BTN, AME, SFL and BMY are known (see description above), therefore genes encoding variants of the proteins BBR47_51900, Staur_4851, DES, BUR, ALL, BTN, AME, SFL and BMY can be obtained using PCR using primers synthesized based on the nucleotide sequence of genes encoding BBR47_51900, Staur_4851, DES, BUR, ALL, BTN, AME, SFL and BMY. Genes encoding the proteins BBR47_51900, Staur_4851, DES, BUR, ALL, BTN, AME, SFL and BMY or their variants of proteins from other microorganisms can be obtained in a similar way.

The term "protein variant" may mean a protein that contains one or more changes in the sequence when compared with SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16 and 18, whether they are substitutions, divisions, inserts and / or additions of amino acid residues, provided that the activity corresponding to the activity of the proteins BBR47_51900, Staur_4851, DES, BUR, ALL, BTN, AME, SFL and BMY, respectively, is preserved, or the tertiary structure of the proteins BBR47_51900, Staur_4851, DES, BUR, ALL , BTN, AME, SFL, and BMY are not changed significantly compared to the wild-type protein or unmodified protein. The number of changes in protein variants depends on the position or type of amino acid residues in the tertiary structure of the protein. It can be, but is not strictly limited, from 1 to 45, or from 1 to 30, or from 1 to 15, or from 1 to 10, or from 1 to 5 in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16 and 18.

Examples of substitution, deletion, insertion and / or addition of one or more amino acid residues can be a conservative mutation (s) such that the activity and characteristics of a protein variant are preserved and are similar to proteins BBR47_51900, Staur_4851, DES, BUR, ALL, BTN, AME, SFL and BMY. An example of a conservative mutation is a conservative substitution. Conservative substitutions may be reciprocal substitutions between Phe, Trp and Tyr, if the substitution site is an aromatic amino acid; between Ala, Leu, Ile and Val, if the substitution site is a hydrophobic amino acid; between Glu, Asp, Gln, Asn, Ser, His and Thr if the substitution site is a hydrophilic amino acid; between Gln and Asn if the substitution site is a polar amino acid; between Lys, Arg and His, if the substitution site is a basic amino acid; between Asp and Glu, if the substitution site is an acidic amino acid; and between Ser and Thr, if the substitution site is an amino acid having a hydroxyl group. Examples of conservative substitutions include replacing Ser or Thr with Ala, replacing Asn, Glu or Gln with Asp, replacing Ser or Ala with Cys, replacing Asn, Glu, Lys, His, Asp or Arg with Gln, replacing Asn, Gln, Lys or Asp with Glu, replacing Pro with Gly, replacing Asn, Lys, Gln, Arg or Tyr with His, replacing Leu, Met, Val or Phe with Ile, replacing Ile, Met, Val or Phe with Leu, replacing Asn, Glu, Gln, His or Arg with Lys, replacing Ile, Leu, Val or Phe with Met, replacing Trp, Tyr, Met, Ile or Leu with Phe, replacing Thr or Ala with Ser, replacing Ser or Ala with Thr, replacing Phe or Tyr with Trp replacing His, Phe or Trp with Tyr and replacing Met, Ile or Leu with Val. These changes in the protein variant can occur in regions of the protein that are not critical to the function of the protein. This is because some amino acids have a high homology to each other, such that the tertiary structure or activity of the protein is not affected due to such changes. Thus, protein variants encoded by the genes represented in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, or 17 may have similarity or identity of at least 40%, at least 50%, not less than 60%, not less than 70%, not less than 80%, not less than 90%, not less than 95%, not less than 96%, not less than 97%, not less than 98%, or not less than 99% with respect to the amino acid sequences represented in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18, as long as the functionality of the proteins BBR47_51900, Staur_4851, DES, BUR, ALL , BTN, AME, SFL and BMY, respectively, remains unchanged. Also, protein variants encoded by the genes represented in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, or 17 can have homology, which can be determined using | Log ₁₀ (E-value) | calculated using the HMMsearch program when the hidden profile of the Markov model (the profile hidden Markov model, profile HMM; HMM profile) is built using the aforementioned program (Finn RD et al., HMMER web server: interactive sequence similarity searching, Nucleic Acids Res ., 2011, 39 (Web Server issue): W29-37), as described below in Example 6, at least 128, at least 142, at least 162, at least 175, at least 182, at least than 196, or not less than 233 rel to the complete amino acid sequences given in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18, as long as the functionality of the proteins BBR47_51900, Staur_4851, DES, BUR, ALL, BTN, AME , SFL and BMY, respectively, remain unchanged.

Examples of substitutions, deletions, insertions and / or additions of one or more amino acid residues may also include non-conservative (s) mutation (s), provided that this (these) mutation (s) is compensated (s) by one or more secondary mutations in one or several positions of the amino acid sequence so that the activity and characteristics of the protein variant remain unchanged and are similar to the proteins BBR47_51900, Staur_4851, DES, BUR, ALL, BTN, AME, SFL and BMY.

The degree of homology of the protein or DNA can be determined using several well-known approaches, for example, the computer algorithms BLAST and FASTA and the ClustalW method. The BLAST algorithm (Basic Local Alignment Search Tool, www.ncbi.nlm.nih.gov/BLAST/), which allows hierarchical searches, is embedded in the programs blastp, blastn, blastx, megablast, tblastn and tblastx; these programs assign significance to found objects using the statistical methods described in Samuel K. and Altschul S.F. ("Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes" Proc. Natl. Acad. Sci. USA, 1990, 87: 2264-2268; "Applications and statistics for multiple high-scoring segments in molecular sequences" (Proc. Natl. Acad. Sci. USA, 1993, 90: 5873-5877). The BLAST algorithm computes three parameters: the number of amino acid residues, identity, and similarity. The FASTA search algorithm is described in Pearson W.R. (“Rapid and sensitive sequence comparison with FASTP and FASTA”, Methods Enzymol., 1990, 183: 63-98). The ClustalW method is described in Thompson J.D. et al. (“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).

The term "activity of L-amino acid-α-ligase (Lal)" may mean the activity of an enzyme that catalyzes the reaction of a compound of amino acids in a macroergic molecular-dependent manner with the formation of a peptide bond between amino acid residues. The reaction product catalyzed by Lal may be a dipeptide, tripeptide or peptide with a linear or branched structure, consisting of more than three amino acid residues or their derivatives. The reaction scheme catalyzed by Lal can be described as shown in Figure 1, without limiting the type of amino acids or their derivatives and reaction conditions that are used in the following non-limiting examples of the present invention. Lal activity can be determined by the method described in Example 3 or in Tabata K. et al., J. Bacterial., 2005, 187 (15): 5195-5202. The term "activity of L-amino acid-α-ligase (Lal)" may be equivalent, in particular, to the term "dipeptide-synthesizing activity".

In addition, when the amino acid sequence that contains the substitution, deletion, insertion and / or addition of one or more amino acid residues in the amino acid sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18, residual activity L-amino acid-α-ligase can be maintained at 10% or more, at 30% or more, at 50% or more, at 70% or more, and at 90% or more of the activity of a protein having the amino acid sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18.

The term "isofunctional protein" can mean a protein that has the activity of L-amino acid-α-ligase (Lal), as described above. For example, an isofunctional protein can synthesize a dipeptide having an acidic L-amino acid residue, such as an L-Glu or L-Asp residue, at the N-terminus and any other L-amino acid or its derivative at the C-terminus.

2. Bacteria

The term "dipeptide producing bacterium" may mean a bacterium of the Enterobacteriaceae family, such as a bacterium belonging to the genus Escherichia, which is capable of producing and causing the dipeptide to accumulate in the culture fluid when the bacterium is grown (cultivated) in a nutrient medium. A bacterium may be capable of producing a dipeptide in the medium or bacterial cells and cause the dipeptide to accumulate to such an extent that the dipeptide is released (extracted) from the culture fluid or bacterial cells when the bacterium is grown in a nutrient medium.

A bacterium may have the ability to produce a dipeptide initially in accordance with its natural characteristics, or may be modified so that it receives the ability to produce a dipeptide using mutagenesis or recombinant DNA technology.

A bacterium belonging to the Enterobacteriaceae family may be from the genus Enterobacter, Erwinia, Escherichia, Klebsiella, Morganella, Pantoea, Photorhabdus, Providencia, Salmonella, Yersinia, etc. and may have the ability to produce a dipeptide. More specifically, bacteria belonging to the Enterobacteriaceae family can be used according to the taxonomy used in the NCBI database (National Center for Biotechnology Information) (http://www.ncbi.nlni.nih.gov/Taxonomy/Browser/wwwtax. cgi? id = 543). Examples are bacteria belonging to the genus Escherichia, Enterobacter or Pantoea.

The selection of bacterial strains belonging to the genus Escherichia that can be modified in the present invention is not limited in any way, however, as examples, bacteria of the genus Escherichia described in Neidhardt et al. (Bachmann, BJ, Derivations and genotypes of some mutant derivatives of E. coli K-12, p. 2460-2488. In FC Neidhardt et al. (Ed.), E. coli and Salmonella: cellular and molecular biology, 2 ^nd ed. ASM Press, Washington, DC, 1996) may be included among the bacteria of the present invention. As a specific example, E. coli strains such as E. coli W3110 (ATCC 27325), E. coli MG1655 (ATCC 47076), etc., which originate from the original wild-type strain, i.e. E. coli K-12 strain. This strain can be obtained, in particular, from the American Type Culture Collection (American Type Culture Collection, (ATCC) "(PO Box 1549, Manassas, VA 20108, United States of America). Each strain is assigned an individual registration number, and strains can be ordered according to the registration number (see link www.atcc.org). Registration numbers of strains are in the list of the catalog of the American Type Culture Collection, American Type Culture Collection, ATCC.

Examples of Enterobacter bacteria include Enterobacter agglomerans, Enterobacter aerogenes, etc. Examples of Pantoea bacteria include Pantoea ananatis, etc. Recently, some Enterobacter agglomerans species have been reclassified as Pantoea agglomerans, Pantoea ananatis, or Pantoea stewartii based on analysis of the 16S rRNA nucleotide sequence and other evidence. Bacteria belonging to the genus Enterobacter or Pantoea can be used in accordance with the present invention, as they belong to the Enterobacteriaceae family. Genetic engineering strains of Pantoea ananatis, such as strain Pantoea ananatis AJ13355 (PERM BP-6614), strain AJ13356 (PERM BP-6615), strain AJ13601 (PERM BP-7207) and their derivatives can be used in accordance with this invention. These strains were classified as Enterobacter agglomerans upon isolation, and they were deposited as Enterobacter agglomerans. However, they were later classified as Pantoea ananatis based on analysis of the 16S rRNA nucleotide sequence and other evidence.

The dipeptide producing bacterium of the present invention can be modified to have weakened or inactivated one or more genes encoding one or more types of proteins having peptidase or proteolytic activity, so that the activity of the peptidases (s) is reduced. For example, genes encoding peptidases, such as peA (KEGG, Kyoto Encyclopedia of Genes and Genomes, incoming No. b4260), pepB (KEGG, incoming No. b2523), pepD (KEGG, incoming No., can be attenuated and / or inactivated. b0237), pepE (KEGG, incoming No. b4021), pepP (KEGG, incoming No. b2908), pepQ (KEGG, incoming No. b3847), pepN (KEGG, incoming No. b0932), pepT (GenBank accession No. AAC74211 ), iadA (KEGG, incoming No. b4328), iaaA (ybiK) (KEGG, incoming No. b0828), dapE (KEGG, incoming No. b2472), etc.

The dipeptide producing bacterium described above can also be modified in such a way as to have weakened or inactivated one or more genes of one or more types encoding a protein (s) that (e) has (a) dipeptide permease (dpp) activity, wherein the peptide activity permease (permease) is reduced. For example, one or more dipeptide permease genes can be attenuated and / or inactivated, such as dppA (KEGG, incoming No. b3544), dppB (KEGG, incoming No. b3543), dppC (KEGG, entering No. b3542), dppD (KEGG, entering No. b3541), dppF (KEGG, incoming No. b3540), etc. The deletion of the whole operon of the dpp genes (dppA, dppB, dppC, dppD and dppF) may also be preferred in the dipeptide producing bacterium.

The dipeptide producing bacterium described above can also be modified so as to have one or more genes of one or more types attenuated or inactivated, encoding for proteins involved in the biosynthesis of aromatic amino acids, the activity of such proteins being reduced. For example, one or more genes can be attenuated and / or inactivated, such as tyrR (KEGG, entry No. b1323), tryA (KEGG, entry No. b2600), etc.

The term “attenuated gene encoding a peptidase” or “attenuated gene encoding a protein” is equivalent to the term “peptidase encoded by a gene with impaired expression” or the term “protein encoded by a gene with attenuated expression”, respectively. Subsequently, the term “peptidase” can be replaced by the term “protein” as described above (for example, a protein having dipeptide permease activity (dpp) or a protein involved in the biosynthesis of aromatic amino acids) to interpret the term “attenuated gene encoding a protein” , etc. Thus, such replacement terms may be referred to in order to clarify the present invention.

The term “peptidase encoded by a gene with attenuated expression” may mean that the amount of peptidase in a modified bacterium in which the expression of a gene encoding a peptidase is attenuated is reduced compared to an unmodified bacterium, for example, a wild-type strain of a bacterium belonging to the Enterobacteriaceae family or more precisely the genus Escherichia, such as E. coli K-12 strain.

The term “peptidase encoded by a gene with weakened expression” may also mean that the modified bacterium includes a modified gene that encodes a mutant protein having reduced activity compared to the wild-type protein, or a site functionally linked to the gene, including sequences that control gene expression such as promoters, enhancers, attenuators, ribosome binding sites (RBS), Shine-Dalgarno (SD) sequences, etc., modified to reduce expression of g a peptide encoding peptidase and other examples (see, for example, WO95 / 34672; Carrier, T. A. and Keasling, J. D., Biotechnol. Prog., 1999, 15: 58-64).

The expression of the gene encoding the peptidase can be attenuated by replacing the sequence that controls the expression of the gene, such as a promoter on the chromosomal DNA, with a weaker one. The strength of the promoter is determined by the frequency of acts of initiation of RNA synthesis. Examples of methods for evaluating promoter strength are described in Goldstein et al., Prokaryotic promoters in biotechnology, Biotechnol. Annu. Rev. 1995, 1: 105-128, etc. In addition, it is also possible to introduce a nucleotide substitution into the promoter region of the target gene in such a way as to weaken the strength of the promoter, as described in International Patent Publication WO00 / 1835. In addition, it is known that the replacement of several nucleotides in the region between the SD sequence and the start codon in the ribosome-binding region (RBS), in particular, in the sequence located above and immediately after the start codon, significantly affects the translation efficiency of mRNA. A similar modification of RBS can be combined with a decrease in the level of transcription of the gene encoding the peptidase.

The expression of a gene encoding a peptidase can also be attenuated by inserting a transposon or IS factor into the coding region of the gene (U.S. Patent No. 5,175,107) or using conventional methods, such as mutagenesis, by treating microorganisms such as UV radiation or nitrosoguanidine ( N-methyl-N′-nitro-N-nitrosoguanidine). In addition, the introduction of a site-directed mutation by replacing a gene using homologous recombination, as described above, can also be carried out using a plasmid that is unable to replicate in the host strain.

The term “enzymatic activity is reduced” may mean that the enzymatic activity of the peptidase in the modified strain is lower than in the unmodified strain, for example, a wild-type strain of a bacterium belonging to the Enterobacteriaceae family or more specifically to the genus Escherichia. For example, the enzymatic activity of a peptidase encoded by a gene can be reduced by inactivating the gene.

The term "peptidase activity is reduced" can also mean that the activity of the peptidase in the peptide cleavage reaction is reduced compared to the wild-type peptidase encoded by wild-type genes such as peRA, peRB, pepD, pepE, pepP, pepQ, pepN, pepT, iadA, iaaA (ybiK), dapE, etc.

In a modified bacterium, peptidase activity may be reduced by at least at least 10% or more, at least 30% or more, at least 50% or more, at least 70% or more, at least 90%, or more than peptidase encoded by the wild-type gene in an unmodified bacterium belonging to the Enterobacteriaceae family or more precisely to the genus Escherichia.

The term "peptidase activity" or "proteolytic activity" may mean the activity of an enzyme that catalyzes the reaction of intramolecular cleavage of the peptide bond (R. Beynon (ed.) And JS Bond (ed.), "Proteolytic Enzymes: A Practical Approach", 2 ^nd ed. , Oxford University Press, USA (2001)).

The peptide cleavage activity of a microorganism can be measured by placing the peptide as a substrate and microorganism cells in a medium to carry out a peptide degradation reaction, followed by determining the amount of remaining peptide by a known method, for example, HPLC or as described in Kristjansson MM, Activity measurements of proteinases using synthetic substrates ( UNIT C2.1) or Akpinar O. and Penner MH, Peptidase activity assays using protein substrates (UNIT C2.2) in Current Protocols in Food Analytical Chemistry (UNIT C2, Proteolytic Enzymes), John Wiley 85 Sons, Inc. (2002).

The enzymatic activity of the peptidase can also be reduced by introducing a mutation into the gene on the chromosome so that the intracellular activity of the peptidase is reduced compared to the unmodified strain. Such a mutation in the gene (s) may be the replacement of one or several bases, leading to the replacement of the amino acid residue in the protein encoded by the gene (s) ("missense" mutation), the introduction of a stop codon ("nonsense" mutation), deletion of one or several bases, leading to a reading frame shift, insertion of a gene reporting antibiotic resistance, partial or complete removal of genes in the genome (Qiu Z. and Goodman MF, J. Biol. Chem. 1997, 272: 8611-8617; Kwon DH et al., J. Antimicrob. Chemother. 2000, 46: 793-796).

The term "inactivated gene encoding a peptidase" may mean that the modified gene encodes a completely inactive or non-functional peptidase. It is also possible that the modified portion of the DNA is not capable of naturally expressing the gene due to deletion of part of the gene or the whole gene, shift of the reading frame of the gene, insertion of the missense / nonsense mutation (s) or modification of the adjacent portion of the gene, inclusion of sequences that control gene expression, such as a promoter (s), enhancer (s), attenuator (s), ribosome binding site (s) (RBS, ribosome binding site), etc. Gene inactivation can also be carried out by conventional methods, such as mutagenesis, by treating microorganisms, for example, UV irradiation or nitrosoguanidine (N-methyl-N′-nitro-N-nitrosoguanidine), site-directed mutagenesis, disruption of the gene structure using homologous recombination and / or mutagenesis by insertion deletion (Yu D. et al., Proc. Natl. Acad. Sci. USA, 2000, 97 (12): 5978-83; Datsenko KA and Wanner BL, Proc. Natl. Acad Sci. USA 2000, 97 (12): 6640-45), also referred to as “λRed-dependent integration”.

The term "bacterium modified in such a way that it contains recombinant DNA" may mean that the bacterium is modified in the usual way so that it contains exogenous DNA. Conventional methods include, for example, transformation, transfection, infection, conjugation and mobilization. Transformation, transfection, infection, conjugation, or mobilization of a bacterium by a DNA molecule encoding a protein can impart to a bacterium the ability of a bacterium to synthesize a protein encoded by a DNA molecule. Methods of transformation, transfection, infection, conjugation and mobilization include any of the previously known methods. For example, a method is known for efficiently transforming and transfecting DNA by treating Escherichia coli K-12 recipient cells with calcium chloride to increase cell permeability for DNA (Mandel M. and Higa A., Calcium-dependent bacteriophage DNA infection, J. Mol. Biol. 1970, 53: 159-162). Methods of specialized and / or general transduction are described (Morse ML et al., Transduction in Escherichia coli K-12, Genetics, 1956, 41 (1): 142-156; Miller JH, Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold Spring Harbor La. Press, 1972). Other methods can be used for random and / or directional integration of DNA into the host genome, for example, “Mu-dependent integration / amplification” (Akhverdyan et al., Appl. Microbiol. Biotechnol., 2011, 91: 857-871), “ Red / ET-dependent "or" λRed / ET-dependent integration "(Datsenko KA and Wanner BL, Proc. Natl. Acad. Sci. USA 2000, 97 (12): 6640-45; Zhang Y., et al., Nature Genet., 1998, 20: 123-128). Moreover, for multiple insertions of desired genes in addition to Mu-dependent replicative transposition (Akhverdyan et al., Appl. Microbiol. Biotechnol., 2011, 91: 857-871) and chemically induced chromosome evolution based on recA-dependent homologous recombination leading to the amplification of the desired genes (Tuo KEJ et al., Nature Biotechnol., 2009, 27: 760-765), other approaches based on various combinations of transposition, site-specific and / or homologous Red / ET-dependent can be used. recombination, and / or P1-dependent total transduction (see, for example, Minaeva et al., BMC Biot echnology, 2008, 8:63; Koma D. et al., Appl. Microbiol. Biotechnol., 2012, 93 (2): 815-829).

The bacterium described in the present invention can be further modified so that the expression level of a gene encoding an L-amino acid-α-ligase (Lal) or one or more genes encoding one or more proteins involved in phenylalanine biosynthesis is increased. Examples of such a protein include pheA, aroG4, and aroL encoding chorizmmutase-prefenate dehydratase (CM-PD), 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase (DAHP synthetase), and shikimatin kinase (SK), respectively (see, for example, Japan Patent No. 3225597). Hereinafter, the term “Lal” can be replaced with a protein involved in phenylalanine biosynthesis to interpret the term “gene encoding proteins involved in phenylalanine biosynthesis” or the like. Thus, such replacement terms may be referred to in order to clarify the present invention.

The term "increased expression of the gene encoding Lal" may mean that the number of protein molecules encoded by the Lal-coding gene, in terms of one cell is increased or the activity in terms of the protein molecule (can be called as specific activity), encoded by this gene, is increased compared to an unmodified strain, such as a wild-type strain or a parent strain. An example of an unmodified comparison strain can be the wild-type strains of a microorganism of the Enterobacteriaceae family, such as E. coli strain MG1655 (ATCC 47076), strain W3110 (ATCC 27325), Pantoea ananatis strain AJ13335 (PERM BP-6614), etc.

The term "increased expression of the gene encoding the Lal" may also mean that the expression level of the Lal encoding gene in the modified strain is higher than the expression level in an unmodified strain, for example a wild-type strain or a parent strain.

Methods that can be used to increase expression of the Lal coding gene include, but are not limited to, examples, increasing the number of copies of the Lal coding gene in the bacterial genome on the chromosome and / or on an autonomously replicating plasmid and / or introducing the Lal coding gene into the vector such that it becomes able to increase the number of copies and / or the expression level of the Lal coding gene in Escherichia bacteria in accordance with genetic engineering methods known to those skilled in the art.

Examples of such vectors include, but are not limited to, vectors with a wide range of hosts (broad-host-range vectors), for example, pCMHO, pRK310, pVK101, pBBR122, pBHR1 and the like. Multiple copies of the Lal coding gene can be introduced into the chromosomal DNA of a bacterium through, for example, homologous recombination, Mu-dependent integration, or similar methods. Homologous recombination can be carried out using a multiple copy sequence in chromosomal DNA. Multiple sequences in chromosomal DNA include, but are not limited to, repeating portions of DNA or reverse repeats at the ends of a moving genetic element. It is also possible to introduce the Lal coding gene into a transposable transposon to introduce multiple copies of the Lal coding gene into the chromosomal DNA. When using Mu-dependent integration, more than 3 copies can be inserted into chromosomal DNA in one act (Akhverdyan V.Z. et al., Biotechnol. (Russian), 2007, 3: 3-20).

An increase in the expression level of the Lal-coding gene can also be achieved by modifying the regulatory region adjacent to the Lal-coding gene, or by introducing native and / or modified foreign regulatory regions. Examples of regulatory regions or sequences include promoters, enhancers, attenuators, transcription termination and anti-termination signals, ribosome binding sites (RBS), and other expression control elements (e.g., sites to which repressors or inducers bind, and / or binding sites of transcriptional and translational regulatory proteins, for example, as part of mRNA). Such regulatory sequences are described, for example, in Sambrook J., Fritsch EF and Maniatis T., Molecular Cloning: A Laboratory Manual, 2 ^nd ed., Cold Spring Harbor Laboratory Press (1989). Modifications of sites that control the expression of gene (s) can be combined with an increase in the number of copies of the modified gene (s) in the bacterial genome using known methods (see, for example, Akhverdyan VZ et al., Appl. Microbiol. Biotechnol., 2011, 91: 857-871; Tyo KEJ et al., Nature Biotechnol., 2009, 27: 760-765).

Strong promoters may be examples of promoters that enhance the expression of the Lal coding gene. For example, the lac promoter, trp promoter, trc promoter, tac promoter, P _R or P _L λ-phage promoters are known as strong promoters. Strong promoters providing a high level of gene expression in bacteria belonging to the Enterobacteriaceae family can be used. Also, the influence of the promoter can be enhanced, for example, by introducing a mutation in the promoter region of the Lal coding gene to obtain a promoter with a stronger function, thus leading to an increase in the level of transcription of the Lal coding gene located under the promoter. In addition, it is known that the replacement of several nucleotides in the region between the ribosome binding site and the start codon, especially in the sequence immediately before the start codon, significantly affects the translational ability of mRNA. For example, a 20-fold variation in the level of gene expression was found depending on the nature of the three nucleotides preceding the start codon (Gold L. et al., Annu. Rev. Microbiol., 1981, 35: 365-403; Hui A. et al. , EMBOJ., 1984, 3: 623-629).

Strengthening the heterologous expression of the Lal coding gene in the cell of the host microorganism can also be accomplished by replacing rare and / or poorly used codons with synonymous medium or frequently used codons, while the term “use of the codon” can be defined as the number of times (frequency) of translation codon in an organism cell per unit time or as the average frequency of codon occurrence in sequenced protein-coding reading frames (Zhang SP et al., Gene, 1991, 105 (1): 61-72). The use of codons in the body can be found in the Codon Usage Database, which is an extended web version of the CUTG (Codon Usage Tabulated from GenBank) (http://www.kazusa.or.jp/codon/, Nakamura Y. et al., Codon usage tabulated from the international DNA sequence databases: status for the year 2000, Nucl. Acids Res., 2000, 28 (1): 292). In E. coli, such mutations may include, but are not limited to, replacing the rare Arg codons AGA, AGG, CGG, CGA with CGT or CGC; rare Ile codon ATA on the ATC or ATT; the rare Leu codon STA on CTG, STS, CTT, TTA or TTG; rare Pro-codon CCC to CCG or CCA; the rare Ser codon TCG on TST, TCA, TCC, AGC or AGT; rare Gly codons GGA, GGG on GGT or GGC; etc. Replacing less-used codons with synonymous often-used codons is preferred. Replacing rare and / or poorly used codons with synonymous medium or frequently used codons can be combined with co-expression of genes encoding rare m-RNAs that recognize rare codons.

The number of copies, the presence or absence of the gene and / or operon genes can be measured, for example, by restriction of chromosomal DNA followed by Southern blotting using a probe selected based on the gene sequence, in situ fluorescence hybridization (fluorescence in situ hybridization , FISH) and similar methods. The level of expression of the gene and / or operon genes can be determined using various known methods, including Northern blotting, quantitative RT-PCR (RT-PCR), etc. Additionally, the level of gene expression can be determined by measuring the amount of transcribed mRNA using well-known methods, including, for example, Northern blotting, quantitative RT-PCR (RT-PCR), etc. The amount of proteins encoded by the gene can be determined by known methods, including SDS-PAGE (SDS-PAGE) followed by Western blotting (Western blotting), or mass spectrometric analysis of protein samples, etc.

Methods for preparing plasmid DNA, cleavage, ligation, and transformation of DNA, selection of oligonucleotides as primers, etc. can be carried out by conventional methods known to the person skilled in the art. These methods are described, for example, in Sambrook J., Fritsch EF and Maniatis T., “Molecular Cloning: A Laboratory Manual, 2 ^nd ed.”, Cold Spring Harbor Laboratory Press (1989). Molecular cloning and expression of heterologous genes are described in Bernard R. Glick, Jack J. Pasternak and Cheryl L. Patten, Molecular Biotechnology: principles and applications of recombinant DNA, 4 ^th ed., Washington, DC: ASM Press (2009) ; Evans Jr., TC and Xu M.-Q., Heterologous gene expression in E. coli, 1 ^st ed., Humana Press (2011).

The term “operably linked to a gene” may mean that one or more regulatory sequences are linked to the nucleotide sequence of a nucleic acid molecule or a given gene in such a way that expression is possible (eg, enhanced, increased, constitutive, basal, attenuated, reduced or repressive expression) a nucleotide sequence, preferably expressing a gene product encoded by a nucleotide sequence.

The bacterium described in the present invention can be obtained by communicating the desired properties of a bacterium that is initially capable of producing a dipeptide. Similarly, a bacterium can be obtained by communicating the ability to produce a dipeptide of a bacterium that already has the desired properties.

In addition to the properties mentioned above, a bacterium can also have other specific properties, without going beyond the scope of this invention, such as: need various nutrients, have sensitivity, resistance and dependence on antibiotics.

3. Methods for producing dipeptides

The methods of the present invention can be methods of producing a dipeptide, more specifically a dipeptide having an acidic L-amino acid at the N-terminus, using L-amino acid-α-ligase (Lal) or a bacterium belonging to the Enterobacteriaceae family, modified to contain the specified Lal, and thus referred to as an enzymatic method and a bacterial fermentation method, respectively.

The term "amino acid" may mean a common amino acid known to a person skilled in the art, a derivative of an amino acid or its salt. For example, an amino acid may be an α-amino acid and a β-amino acid having a C ^α or C ^β chiral carbon atom, respectively, to which an amino group, a carboxyl group and a side chain group are attached. An example of a β-amino acid may be βAla. α-amino acids can be proteinogenic and non-proteinogenic amino acids. Proteinogenic amino acids can be L-amino acids, such as L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, glycine, L-glutamic acid, L-glutamine, 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, or their salts having a C ^α chiral carbon atom . Amino acids can be used in a protected and unprotected form. The protected form of amino acids, in contrast to the unprotected form, may mean the presence of one or more substituents associated with an amino group, a carboxy group and / or a side chain group. Amino acids having substituent (s) may be assigned to amino acid derivatives. Examples of amino acid derivatives can be lower alkyl esters of amino acids, such as the lower ester of L-phenylalanine. Lower esters include methyl ether, ethyl ether, propyl ether, and the like.

The term “amino acid” is equivalent to the term “Lal-catalyzed reaction substrate” or simply “substrate”.

The term “acidic L-amino acid” can mean aspartic acid (Asp) or glutamic acid (Glu) of the L-form or their salts.

The term "dipeptide" may mean an organic molecule or its salt, consisting of two amino acid residues, or residues of two derivatives of amino acids, or a combination thereof, as described above. For example, the term “dipeptide” may mean a dipeptide formed by two proteinogenic L-amino acid residues such that an acid L-amino acid residue such as L-Asp or L-Glu is located at the N-terminus of the dipeptide and the other L-amino acid residue the same or another species is located at the C-end of the dipeptide. It is also accepted that amino acid derivatives, for example a lower alkyl ester of an L-amino acid, such as methyl ester of L-Phe, can be located at the C-terminus of the dipeptide.

The dipeptide described herein is not limited to a dipeptide having acidic amino acid residues at the N-terminus. The dipeptide can be represented by the formula R1-R2, where R1 and R2 can be amino acid residues or their derivatives located at the N- and C-ends of the corresponding dipeptides and connected via a peptide bond. Examples of R1 and R2 may be L-amino acids such as L-Ala, L-Arg, L-Asp, L-Asn, L-Cys, Gly, L-Glu, L-Gln, L-His, L-Ile, L-Leu, L-Lys, L-Met, L-Phe, L-Pro, L-Ser, L-Thr, L-Trp, L-Tyr and L-Val, or derivatives thereof, such as L-PheOMe, or their salts. R1 and R2 may be the same or different.

The term “peptide bond” can mean a covalent chemical bond —C (O) NH— formed between two molecules when the carboxyl part of one molecule, the carboxy component, reacts with the amine part, the amine component, whereby a molecule forms. For example, amino acids can form peptide bonds by interacting with each other and forming a water molecule.

Any carboxy component may be used provided that it is capable of forming a peptide upon condensation with the amine component of another substrate. Examples of the carboxy component include L-amino acid esters, D-amino acid esters, L-amino acid amides and D-amino acid amides, and organic acid esters that do not have an unprotected amino group. Examples of amino acid esters include not only amino acid esters corresponding to natural amino acids, but also amino acid esters corresponding to unnatural amino acids or their derivatives. Also, examples of amino acid esters include α-amino acid esters, as well as β-, γ-, and ω-amino acid esters and the like having various amino linking sites. Typical examples of amino acid esters include methyl esters, ethyl esters, isopropyl esters, n-butyl esters, isobutyl esters, tert-butyl esters, phenyl amino acid esters. Also, the carboxy moiety of the carboxy moiety may be represented by a carboxyl group of COOH or its derivative COR, where R may be a halide group, such as, for example, chlorine.

Any amine component can be used provided that it is capable of forming a peptide upon condensation with the carboxyl component of another substrate. Examples of the amine component include L-amino acids, C-protected L-amino acids, D-amino acids, C-protected D-amino acids and amines. Also, examples of amino acids include not only naturally occurring amino acids, but also non-naturally occurring amino acids or their derivatives. They include α-amino acids, as well as β-, γ- or ω-amino acids and the like having sites linking carboxy groups.

3.1 Enzymatic method

The enzymatic method may include at least one step allowing the L-amino acid-α-ligase or Lal-containing substance to be contacted with one or more amino acids (s) of the same species or of various species, or derivatives thereof, or salts thereof, under acceptable conditions, to obtain a reaction product in accordance with the activity of Lal as described above.

A method capable of exposing a Lal or Lal-containing substance according to the present invention can be mixing with each other a Lal or Lal-containing substance, a molecule with a carboxyl part and a molecule with an amine part. More specifically, a method of adding a Lal or Lal-containing substance to a solution containing carboxyl and amine components can be used, allowing a reaction between the components to form a dipeptide. Also, in the case of using a bacterium that produces Lal, this method may include growing a bacterium that produces Lal, production and accumulation of Lal in bacteria or culture fluid, and then adding a molecule with a carboxyl component and a molecule with an amine component to the medium. The resulting dipeptide can be isolated by known methods and purified if necessary.

The term "Lal-containing substance" can mean any substance, provided that it contains Lal. Examples of the Lal-containing substance include a culture of bacteria that produce Lal, bacterial cells isolated from the culture, and a product obtained from the processing of bacterial cells (can also be used as "processed bacterial cell product"). A bacterial culture can mean what happens when the bacteria are grown and, more precisely, a mixture of bacterial cells, the medium used to grow the bacteria, the substance produced by the bacteria grown, etc. Bacterial cells can be washed and used in the form of washed cells. The treated bacterial cell product includes degradation products, lysis or lyophilized bacterial cells and the like, as well as a crude enzyme obtained by treating bacterial cells, etc., as well as a purified enzyme obtained by purification of a crude enzyme, and the like. . A partially purified enzyme obtained by various purification methods can be used as a purified enzyme; or immobilized enzymes can be used that have been immobilized by the method of covalent binding, adsorption, capture and the like. Due to the fact that some bacteria are partially destroyed during cultivation, depending on the microorganism used, extracellular fluid (supernatant) can also be used as an enzyme-containing substance.

Wild-type strains can be used as bacteria that contain Lal, or recombinant strains expressing Lal can be used as described above. Bacteria are not limited to intact bacterial cells, but acetone-treated bacterial cells, lyophilized bacterial cells, or bacterial cells otherwise treated may also be used. Can also be used immobilized bacterial cells or the product of immobilized treated bacterial cells obtained by immobilization of bacterial cells, or the product of treated bacterial cells obtained by covalent binding, adsorption, capture or other methods, as well as immobilized treated bacterial cells.

When using cell cultures, grown bacterial cells, washed bacterial cells, or the washed bacterial cell product that was obtained by disrupting or lysing bacterial cells, it often happens that the resulting peptides are cleaved by the enzymes contained in them that are not involved in the formation of the peptide. In this situation, it may be preferable in some cases to add a metalloprotease inhibitor such as ethylenediaminetetraacetic acid (EDTA). The amount added may be in the range of 0.1 mM to 300 mM, or more preferably in the range of 1 mM to 100 mM.

An example of the enzymatic method of the present invention is a method in which the transformed cells described herein are grown in culture fluid to accumulate a peptide-forming enzyme (Lal) in the culture fluid and / or transformed cells. Since the peptide-forming enzyme can be easily obtained in large quantities using a transformant, dipeptides can also be obtained quickly and in large quantities.

The amount of Lal or Lal-containing substance used may be sufficient if it is the amount at which the expected effect (effective amount) is exerted, and this effective amount can be easily determined by a person familiar with the art in a simple preliminary experiment. In the case of using Lal, for example, the amount that can be used is from 0.1 g / l to 10 g / l (see Example 3), while in the case of using washed bacterial cells, the amount used can be higher. which is determined by the amount of Lal in the bacterial cell.

The term “acceptable conditions” may mean conditions under which a Lal-catalyzed reaction can occur; those. the reaction product, for example a dipeptide, can be formed from a carboxy component and an amine component. The term "acceptable conditions" may include, without limitation, the terms "enzyme", "amino acid", "substrate", "acceptable solvent", "macroergic molecule", "acceptable temperature conditions", etc.

The term “macroergic molecule” can mean any organic or inorganic molecule necessary for the Lal-catalyzed reaction to take place under acceptable conditions. Traditionally, cofactors can be an example of macroergic molecules. More precisely, an example of a macroergic molecule may be adenosine 5′-triphosphate (ATP) or its salt. Salts of sodium, potassium, ammonium and the like may be used in any combination thereof.

The term "acceptable solvent" may mean any solvent in which the Lal-catalyzed reaction proceeds so that a reaction product, such as a dipeptide, can be formed. Organic and aqueous solvents or mixtures thereof in various ratios may be examples of an acceptable solvent. A suitable solvent may contain the Lal enzyme according to the present invention; cofactor such as ATP and the like; cations such as sodium, potassium, ammonium, calcium and magnesium ions and the like; anions such as sulfate, chloride, phosphate ions and the like; other inorganic and / or organic molecules necessary for the activity of the L-amino acid-α-ligase. Tris (hydroxymethyl) aminomethane (Tris), N-tris (hydroxymethyl) methylglycine (tricin) or N, N-bis (2-hydroxyethyl) glycine (bicin) or the like described in Carmody W.R. J. Chem. Educ., 1961, 38 (11): 559-560, can be added to the reaction mixture as buffering agents. The acidity of the reaction mixture (pH) may be between 6.5 and 10.5, or between 7.0 and 10.0, or between 7.5 and 9.5, or between 8 and 9. A suitable solvent may be in acceptable temperature conditions.

The term "acceptable temperature conditions" may mean the temperature conditions under which the Lal-catalyzed reaction can proceed so that a reaction product, such as a dipeptide, can be formed. Acceptable temperature conditions can be between 0 and 60 ° C, or 20 and 40 ° C, or 25 and 37 ° C, or 28 and 35 ° C.

The concentration of the carboxy component and the amine component used as starting compounds may be from 1 mM to 10 M and preferably from 50 mM to 2 M, respectively; however, there are cases in which it is preferable to add the amine component in an equimolar amount or excess with respect to the carboxy component. In cases where a high concentration of substrates inhibits the reaction, they can be added sequentially during the reaction until the concentration of these substrates begins to cause inhibition.

After the dipeptide is obtained and accumulated in an acceptable solvent in the required amount, solid particles such as cells, cell debris and denatured proteins can be removed from the culture fluid by centrifugation or membrane filtration, and then the target dipeptide can be recovered from an acceptable solvent using any combination of known methods, such as concentration, ion exchange chromatography, high performance liquid chromatography (HPLC), crystallization, and the like.

Isolation and purification of the enzyme

L-Amino acid-α-ligase can be isolated from a bacterium belonging to the genus Escherichia, for example, E. coli. The method of accumulating, isolating and purifying Lal from a bacterium may be a conventional method known to one skilled in the art.

The bacterium is grown in culture fluid, as described here, to produce Lal. Bacterial cell extract can be prepared from cells by disrupting cells using physical methods, such as ultrasonic disruption, or an enzymatic method using enzymes that destroy cells, and removing the insoluble fraction by centrifugation, etc. The peptide-forming proteins can be further purified by fractionation of the bacterial cell extract solution obtained by the above method with a combination of conventional protein purification methods such as anion exchange chromatography, cation exchange chromatography or gel filtration.

Examples of anion exchange chromatography carriers may be Q-Sepharose HP or DEAE agarose (GE Healthcare) and the like. The enzyme can be extracted from the unbound fraction under neutral pH conditions (7-8) when the cell extract containing enzymes is passed through a column filled with a carrier. Different eluents may be used depending on the carrier. For example, if MonoS HR (GE Healthcare) is used in cation exchange chromatography, the cell extract containing the enzyme is passed through a column filled with a carrier, and the column is washed with a high salt concentration buffer solution to elute the enzyme. At the same time, the salt concentration may increase sequentially, or a concentration gradient may be applied. As a physiological saline solution, NaCl from 0 to 0.5 M can be used. Examples of gel filtration media may be Superdex 200 HR or Sephadex 200 (GE Healthcare).

In the aforementioned purification method, each fraction containing the enzyme can be tested for Lal activity in accordance with the method described in the Examples described below.

3.2. The method of bacterial fermentation

The method in accordance with the present invention may also be a method for producing a dipeptide, more specifically, a dipeptide having an acidic L-amino acid at the N-terminus, by growing the bacteria of the present invention in a culture fluid for the purpose of production, excretion and accumulation of the dipeptide in the culture fluid , and isolation of the dipeptide from the culture fluid.

The cultivation of bacteria according to the present invention, the accumulation and purification of the dipeptide from the environment, etc. can be carried out in a manner similar to traditional fermentation methods, characterized in that the dipeptide or amino acid is produced using a microorganism. The nutrient medium for producing the dipeptide may be a typical medium that contains a carbon source, a nitrogen source, inorganic ions and other necessary organic components. Sugars such as glucose, lactose, galactose, fructose, arabinose, maltose, xylose, trehalose, ribose and starch hydrolysates can be used as a carbon source; alcohols such as glycerin, mannitol and sorbitol; organic acids such as gluconic acid, fumaric acid, citric acid, malic acid and succinic acid and the like. Inorganic ammonium salts such as ammonium sulfate, ammonium chloride, ammonium phosphate can be used as a nitrogen source; organic nutrients such as soybean hydrolysates; ammonia gas; aqueous ammonia and the like Vitamins, such as vitamin B1, essential substances, for example, organic nutrients, such as nucleic acids, such as adenine and RNA or yeast extract and the like, may be present in appropriate amounts or in the form of traces. In addition to the above, small amounts of calcium phosphate, magnesium sulfate, iron ions, manganese ions and the like can be added if necessary.

In order to increase the dipeptide-producing ability of the bacteria of the present invention, the culture medium may be further saturated with amino acids, amino acid derivatives, cofactors and other (bio) chemicals. For example, to increase the ability of a bacterium to produce an Asp-Phe dipeptide, additional amounts of L-Phe and L-Asp may be added to the culture medium.

The cultivation is preferably carried out under aerobic conditions from 16 to 72 hours, the temperature of the cultivation, in the range of which the cultivation can be controlled from 15 to 45 ° C, or in the range from 28 to 37 ° C. The acidity of the medium (pH) is maintained in the range of 5-8, or in the range of 6.5-7.2 using inorganic or organic acidic or alkaline substances such as gaseous ammonia.

After growth, solid particles such as cell debris can be removed from the liquid medium by centrifugation or membrane filtration, and then the target dipeptide can be isolated from the enzymatic mixture by a combination of known methods such as concentration, ion exchange chromatography, high performance liquid chromatography (HPLC), crystallization and etc.

Examples

The present invention will be described in more detail below with reference to the following non-limiting Examples.

Example 1

Cloning BBR47_51900 from Brevibacillus brevis NBRC 100599 and Staur_4851 from Stigmatella aurantiaca DW4 / 3-1.

The primary structure of genes encoding the hypothetical proteins BBR47_51900 and Staur_4851 has been optimized for expression in E. coli. Genes encoding BBR47_51900 from Brevibacillus brevis NBRC 100599 and Staur_4851 from Stigmatella aurantiaca DW4 / 3-1 were synthesized using the SlonoGene ™ gene synthesis service (http://www.sloning.com/) and delivered as a set of plasmids pSlo.X, containing a synthesized XbaI-EcoRI fragment that includes target genes having optimized sequences. XbaI-EcoRI fragments containing genes with optimized sequences encoding the BBR47_51900 and Staur_4851 proteins are shown in SEQ ID NOs: 19 and 20, respectively.

For the construction of plasmids pET-HT-BBR and pET-HT-STA, the corresponding XbaI-EcoRI fragments of plasmids pSlo.X were removed by digestion with XbaI and EcoRI and then ligated with the vector pET15 (b +) (Novagen, USA), digested the same restriction enzymes.

Example 2

Expression and purification of His6-labeled BBR47_51900 and Staur_4851.

Plasmids pET-HT-BBR and pET-HT-STA were introduced into strain BL21 (DE3) (Novagen, USA) by Ca ²⁺ -dependent transformation for the construction of strains BL21 (DE3) [pET-HT-BBR] and BL21 (DE3) [pET-HT-STA]. Electrotransformation was performed using a Bio-Rad electroporator (USA) (No. 165-2098, version 2-89) in accordance with the manufacturer's instructions.

The cells of each of the strains BL21 (DE3) [pET-HT-BBR] and BL21 (DE3) [pET-HT-STA] were grown in LB medium (also referred to as lysogenic medium, as described in Sambrook, J. and Russell, DW ( 2001) Molecular Cloning:.. A Laboratory Manual ( 3 ^{rd ed) Cold Spring Harbor Laboratory Press} ) at 37 ° C and 150 rev / min, up to OD ₅₄₀ ~ 1. Isopropyl-β-D-thio-galactosyl (IPTG) was added at a final concentration of 1 mM, and the cell culture was incubated for 2 hours at 37 ° C and 150 rpm. Induced cells were removed from 1 L of fermentation medium, resuspended in 60-80 ml of NT-I buffer (20 mM NaH ₂ PO ₄ , 0.5 M NaCl, 20 mM imidazole, pH was maintained in the region of 7.4 with KOH solution) and digested at 2000 Psi (~ 140 bar) using French-press (Thermo Spectronic). Debris was removed by centrifugation in two stages: at 4 ° C and 13000 rpm./min, followed by filtration through a 0.45 μm filter (CHROMAFIL Xtra CA-45/25, MACHEREY-NAGEL GmbH). The crude protein solution was loaded onto a HiTrap Chelating column (GE Healthcare), filled with a chromatographic sorbent (IMAC) with immobilized metal affinity, per ml of total volume and calibrated with HT-I buffer. IMAC was used in accordance with the manufacturer's recommendations. Active fractions were collected, combined and desalted using a PD10 column (GE Healthcare) calibrated with SB buffer (20 mM Tris-HCl, 120 mM NaCl, 1 mM β-mercaptoethanol, 15% glycerol, pH 7.5). The collected protein was divided into aliquots of 200 μl and stored at -70 ° C. Protein concentration was determined using BIO-RAD PROTEIN ASSAY (BIO-RAD, USA).

Example 3

Preliminary analysis of substrate specificity BBR47_51900 and Staur_4851.

Substrate specificity BBR47_51900 and Staur_4851 were studied in a reaction medium containing an enzyme and the same or two different canonical L-amino acids, such as L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, glycine, L-glutamic acid, L-glutamine, 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 or their salts.

The composition of the reaction mixture with a total volume of 50 μl was as shown below, unless otherwise indicated:

BBR47_51900 or Staur_4851 4 mcg Tris-HCl pH 8.0 50 mm The first L-amino acid 10 mm The second L-amino acid 10 mm Adenosine 5′-triphosphate (ATP) 10 mm MgSO ₄ × 7H ₂ O 10 mm H ₂ O up to 50 μl

The reaction was carried out at 32 ° C for 15 hours. Portions of the reaction mixture with a volume of 1-2 μl were studied using thin layer chromatography (TLC) using 2-propanol: acetone: 250 mM ammonia: H ₂ O as 100: 100: 12: 28 as the mobile phase. A solution (0.3%, w / v) of ninhydrin in acetone was used as a coloring reagent. The determination was carried out at 540 nm. TLC plates were developed after treatment with reaction mixtures containing:

1) BBR47_51900 and L-Glu / L-Glu, or L-Glu / L-Asp, or L-Glu / L-Val, or L-Glu / L-Ile, or L-Asp / L-Ile, or L -Asp / L-Val (Figures 2-4);

2) Staur_4851 and L-Asp / L-Phe or L-Asp / L-Trp or L-Asp / L-Thr) (Figures 5 and 6).

The results indicate that BBR47_51900 and Staur_4851 catalyze the ligation of acidic L-amino acids, such as L-Glu and L-Asp, with other L-amino acids.

Example 4

HPLC determination of dipeptides synthesized using BBR47_51900 and Staur_4851.

The dipeptides obtained using BBR47_51900 and Staur_4851 were determined using the HPLC method in a reaction mixture with a total volume of 400 μl, which contained:

BBR47_51900 or Staur_4851 160 mcg Tris-HCl pH 9.0 50 mm L-Asp or L-Glu 10 mm L-Phe or L-PheOMe, L-Val, L-Trp 10 mm Adenosine 5′-triphosphate (ATP) 10 mm MgSO ₄ × 7H ₂ O 10 mm

where Me means a methyl group.

Reactions were carried out at 32 ° C for 15 hours. Then, 0.5 ml of the reaction mixture was filtered through Amicon Ultra-0.5 ml, 3K Centrifugal Filters (Millipore, # UFC500396) and analyzed by HPLC.

The analysis conditions were as follows:

Equipment: HITACHI L-2000 series.

Column: Inertsil ODS-3 4.6 × 250 mm, 5 mm (GL Sciences Inc.).

Temperature: 40 ° C.

Buffers:

A (for a mixture of L-Asp / L-Val and L-Glu / L-Val): 0.1 M KH ₂ PO ₄ (pH 2.2) + 5 mM sodium octanesulfonate: CH ₃ CN as 4: 1 (vol. ./about.),

B (for L-Asp / L-Phe mixture): 0.1 M KH ₂ PO ₄ (pH 2.2) + 5 mM sodium octanesulfonate: CH ₃ CN as 7: 3 (v / v),

C (for a mixture of L-Asp / L-PheOMe, L-Asp / L-Trp and L-Val / L-Val): 0.1 M KH ₂ PO ₄ (pH 2.2) + 5 mM sodium octanesulfonate: CH ₃ CN as 3: 2 (v / v),

D (for L-Phe / L-Phe mixture): 0.1 M KH ₂ PO ₄ (pH 2.2) + 5 mM sodium octanesulfonate: CH ₃ CN as 1: 1 (v / v).

Gradient Profile: Isocratic.

Flow rate: 1.5 ml / min.

Injection volume: 10 μl.

Scope: UV 210 nm.

Chemical reagents that were used for the HPLC method:

L-Asp (L-Aspartic Acid, Sodium Salt): Nacalai Tesque, Inc. # 03504-75

L-Phe (L-Phenylalanine): Nacalai Tesque, Inc. # 26901-35

L-Val (L-Valine): Ajinomoto Co., Inc. # 317LG13

L-Trp (L-tryptophan): Ajinomoto Co., Inc. # 0000002205

L-PheOMe (L-phenylalanine methyl ester hydrochloride): Tokyo Chemical Industry Co., Ltd. # P1278

ATP: Oriental yeast Co., Ltd. # 45142000

MgSO ₄ × 7H ₂ O: Junsei Chemical Co., Ltd. # 83580-0301

αAsp-Phe (H-Asp-Phe-OH): Bachem G-1620

βAsp-Phe (H-Asp (Phe-OH) -OH): Bachem G-4750

αAsp-Asp (H-Asp-Asp-OH): Bachem G-1565

Phe-Asp (H-Phe-Asp-OH): Bachem G-2870

Phe-Phe (H-Phe-Phe-OH): Bachem G-2925

αAsp-Val (H-Asp-Val-OH): Bachem G-1635

Val-Asp (H-Val-Asp-OH): Bachem G-3510

Val-Val (H-Val-Val-OH): Bachem G-3595

αGlu-Val (H-Glu-Val-OH): Bachem G-2010

γGlu-Val (H-Glu (Val-OH) -OH): Bachem G-2015

Val-Glu (H-Val-Glu-OH): Bachem G-3520

αGlu-Glu (H-Glu-Glu-OH): Bachem G-1915

αAspartame (H-Asp-Phe-OMe): Bachem G-1545

βAspartame: (H-Asp (Phe-OMe) -OH): Bachem G-3725

Asp-Trp (H-Asp-Trp-OH): Bachem G-3705.

Solutions of αGlu-Val, γGlu-Val, αAsp-Val (10 mM each) and αAsp-Phe, βAsp-Phe (5 mM each) were prepared for analysis by HPLC. The concentration of the obtained dipeptide was determined using appropriate calibration curves. For the LC-QTOF / MS / MS method, each solution with a standard sample was dissolved 50-fold or 100-fold (Example 5).

The results of the HPLC method for the reaction mixture are presented in Table 1. As can be seen from Table 1, BBR47_51900 and Staur_4851 catalyze the formation of a dipeptide having acidic L-amino acids, such as L-Asp and L-Glu, at the N-terminus.

Example 5

Determination by LC-QTOF / MS / MS of the dipeptides obtained using BBR47_51900.

Samples of the reaction mixtures and standard solutions obtained as described in Example 4 were investigated by LC-QTOF / MS / MS.

The analysis conditions were as follows:

Equipment: LC (Agilentl200SL), MS (Micromass Q-TOF Premier)

LC conditions:

Column: Develosil C30 2.0 × 250 mm, 3 μm (Nomura Chemical).

Temperature: 20 ° C.

Buffers:

A: H ₂ O (0.025% formic acid),

B: CH ₃ CN (0.025% formic acid).

Gradient Profile:

Time (min) AT (%) 0 0 twenty 22.5 20.1 one hundred 25 one hundred

Flow rate: 0.3 ml / min.

Injection volume: 2-5 μl.

MS conditions:

Capillary voltage: 3.0 kV.

Needle voltage: 20 V.

Collision voltage: 4 V (MS / MS 12B).

Temperature source: 80 ° C.

Evaporation temperature: 120 ° C.

Needle gas flow rate: 50 l / h.

Desolvation gas flow rate: 700 l / h.

The results of the analysis of the reaction mixture by LC-QTOF / MS / MS are shown in Figures 7-9. As can be seen from Figures 7-9, BBR47_51900 catalyzes the formation of αAsp-Phe, αAsp-Val, and αGlu-Val dipeptides.

Example 6

Search for enzymes that are isofunctional to BBR47_51900 and Staur_4851.

The HMMER method was used to search the database for homologous protein sequences and to align protein sequences. The method is based on a probabilistic model, referred to as the Hidden Markov model profile (HMM profile) (Finn RD et al., HMMER web server: interactive sequence similarity searching, Nucleic Acids Res., 2011, 39 (Web Server issue) : W29-37).

Compared to BLAST, FASTA computer algorithms and other sequence alignment and database search programs based on outdated methodology, the HMMER method has achieved significantly more accurate and more reliable determination of very different homologies, such as isofunctional proteins, due to the efficiency of mathematical models lying at the heart of the method. Earlier, efficiency was achieved with significant computational costs, but in the new HMMER3 project, HMMER became much faster, like BLAST (Finn RD et al., HMMER web server: interactive sequence similarity searching, Nucleic Acids Res., 2011, 39 (Web Server issue ): W29-37).

In order to find enzymes that are isofunctional BBR47_51900 and Staur_4851, BBR47_51900 and Staur_4851 (Figure 10) were aligned using the HMMsearch search program from the HMMER3 software suite that allows one or more profiles to be searched against the protein sequence database (http: // hmmer .janelia.org /). A HMM profile was created based on sequence alignment BBR47_51900 and Staur_4851 (Model 1), which was used for homology searches. A list of the nearest isofunctional proteins (hits) found is shown in Figure 11. Analysis of the distribution chart of values | Log (E-value) | showed that the values | Log (E-value) | distributed over five groups of proteins, and the indicated values between the members of the groups were lower than the same values between the groups (Figure 12). The first group included BBR47_51900 and Staur_4851, the second group included DES and ALL, the third group included BMY, the fourth group included BTN and the fifth group included BUR. The remaining proteins are a set of ungrouped homologues having stochastic values | Log (E-value) |. The AME and SFL proteins were selected from ungrouped homologs as a negative control.

Provided that the proteins from the first (BBR47_51900, Staur_4851) and second (DES and ALL) groups are isofunctional, a new HMM profile can be obtained (Model 2) based on alignment of BBR47_51900, Staur_4851, DES and ALL (Figure 13); it can be used to search for isofunctional proteins using the HMMsearch program as described above. Thus, a new list of isofunctional proteins can be obtained (Figure 14), which is described by a | Log ₁₀ (E-value) | distribution chart that differs from the initial diagram (Figure 15). A new list of isofunctional proteins may include three groups, such as the first group, including BBR47_51900, Staur_4851, DES and ALL; the second group, including BMY and VTN; the third group, including BUR, characterized in that AME is closer to the first group (position number has changed from No. 47 (Figure 12) to No. 33 (Figure 15)) and SFL is located further from the first group (position number has changed from No. 49 ( Figure 12) at No. 62 (Figure 15)).

Provided that the proteins from the first group (BBR47_51900, Staur_4851, DES and ALL) and the second group (such as BMY) are isofunctional, the new NMM profile (Model 3), based on the alignment of BBR47_51900, Staur_4851, DES, ALL and BMY (Figure 16) can be obtained; it can be used to search for isofunctional proteins using the HMMsearch program, as described above. Thus, a new list of isofunctional proteins can be obtained (FIG. 17). A new list of isofunctional proteins may include three groups, such as the first group, including BBR47_51900, Staur_4851, DES, ALL and BMY; the second group, including VTN; the third group, including BUR, characterized in that AME is present at position No. 37 and SFL is present at position No. 65 (Figure 17).

Provided that the proteins from the first group (BBR47_51900, Staur_4851, DES, ALL and BMY) and the second group (BTN) are isofunctional, a new HMM profile can be obtained (Model 4) based on alignment of BBR47_51900, Staur_4851, DES, ALL, BMY and BTN (Figure 18), which can be used to search for isofunctional proteins, using the HMMsearch program as described above. Thus, a new list of isofunctional proteins can be obtained (Figure 19). A new list of isofunctional proteins may include three groups, such as the first group, which includes BBR47_51900, Staur_4851, DES, ALL, BMY and BTN; the second group, which includes BUR; the third group, which includes AME, characterized in that the SFL is in position No. 73 (Figure 19).

Provided that the proteins from the first group (BBR47_51900, Staur_4851, DES, ALL, BMY and BTN) and the second group (BUR) are isofunctional, a new HMM profile can be obtained (Model 5) based on alignment of BBR47_51900, Staur_4851, DES, ALL, BMY, BTN and BUR (Figure 20), which can be used to search for isofunctional proteins using the HMMsearch program as described above. Thus, a new list of isofunctional proteins can be obtained (Figure 21). A new list of isofunctional proteins may include three groups, such as the first group including BUR; the second group, including BBR47_51900, Staur_4851, DES, ALL, BMY and VTN; the third group, including AME, characterized in that the SFL is in position No. 104 (Figure 21).

Provided that the proteins from the first group (BUR), the second group (BBR47_51900, Staur_4851, DES, ALL, BMY and BTN) and the third group (AME) are isofunctional, a new HMM profile can be obtained (Model 6), based on alignment BBR47_51900, Staur_4851, DES, ALL, BMY, BTN, BUR and AME (Figure 22), which can be used to search for isofunctional proteins using the HMMsearch program as described above. Thus, a new list of isofunctional proteins can be obtained (Figure 23). A new list of isofunctional proteins may include two groups, such as the first group including BUR; the second group, including BBR47_51900, Staur_4851, DES, ALL, BMY, VTN and AME, characterized in that the SFL is in position No. 65 (Figure 23).

Provided that the proteins from the first group (BUR) and the second group (BBR47_51900, Staur_4851, DES, ALL, BMY, BTN and AME) and the SFL protein are isofunctional, a new HMM profile can be obtained (Model 7) based on alignment of BBR47_51900 , Staur_4851, DES, ALL, BMY, BTN, BUR, AME and SFL (Figure 24), which can be used to search for isofunctional proteins using the HMMsearch program as described above. Thus, a new list of isofunctional proteins can be obtained (Figure 25). A new list of isofunctional proteins may include three groups, such as the first group including BUR; the second group, including BBR47_51900, Staur_4851, DES, ALL, BMY, VTN and AME; and a third group, including SFL, which is in position No. 38 (Figure 25).

In a similar way, new lists of isofunctional L-amino acid-α-ligases capable of synthesizing a dipeptide having an acidic L-amino acid such as L-Asp or L-Glu at the N-terminus and any other L-amino acid or its derivative at C-end. If the proteins BBR47_51900 and Staur_4851 are used to build the HMM profile (Model 1), then the value | Log ₁₀ (E-value) | ≥233 can be used to search for new isofunctional Lals; if the BBR47_51900, Staur_4851, DES, and ALL proteins are used to build the NMM profile (Model 2), then the value | Log ₁₀ (E-value) | ≥196 can be used to search for new isofunctional Lals; if the BBR47_51900, Staur_4851, DES, ALL and BMY proteins are used to build the NMM profile (Model 3), then the value | Log ₁₀ (E-value) | ≥182 can be used to search for new isofunctional Lals; if the BBR47_51900, Staur_4851, DES, ALL, BMY and BTN proteins are used to construct the NMM profile (Model 4), then the value | Log ₁₀ (E-value) | ≥175 can be used to search for new isofunctional Lals; if the BBR47_51900, Staur_4851, DES, ALL, BMY, BTN and BUR proteins are used to build the NMM profile (Model 5), then the value | Log ₁₀ (E-value) | ≥162 can be used to search for new isofunctional Lals; if the BBR47_51900, Staur_4851, DES, ALL, BMY, BTN, BUR and AME proteins are used to construct the NMM profile (Model 6), then the value | Log ₁₀ (E-value) | ≥142 can be used to search for new isofunctional Lals; and if the BBR47_51900, Staur_4851, DES, ALL, BMY, BTN, BUR, AME and SFL proteins are used to construct the NMM profile (Model 7), then the value | Log ₁₀ (E-value) | ≥128 can be used to search for new isofunctional Lals (Figure 26), where E-value is a parameter of the HMMsearch program (Finn RD et al., HMMER web server: interactive sequence similarity searching, Nucleic Acids Res., 2011, 39 (Web Server issue): W29- 37).

Example 7

Cloning, expression and purification of DES, BUR, ALL, BTN, AME, SFL and BMY enzymes.

The primary structure of the gene encoding the DES, BUR, ALL, BTN, AME, SFL, and BMY proteins was optimized for expression in E. coli using the "Back translation" function of Gene Designer (Villalobos A. et al., Gene Designer: a synthetic biology tool for constructing artificial DNA segments, BMC Bioinformatics, 2006, 7: 285). All constructs were synthesized using the SlonoGene ™ gene synthesis service (http://www.sloning.com/) and delivered as pSlo.X plasmids containing the synthesized XbaI-EcoRI fragment that includes the target gene having optimized sequences. XbaI-EcoRI fragments containing genes with optimized sequences encoding proteins DES, BUR, ALL, BTH, AME, SFL and BMY are presented in SEQ ID NOs: 21, 22, 23, 24, 25, 26 and 27, respectively.

Proteins DES, BUR, ALL, BTN, AME, SFL and BMY can be expressed in E. coli, purified, and their activity can be tested as described for BBR47_51900 and Staur_4851 in Examples 1-5.

Example 8

The design of peptidase-deficient strains of E. coli 1-6Δ.

8.1. The design of peptidase-deficient strains of E. coli 1-6Δ.

The iadA gene was deleted in E. coli strain BW25113 (KEIO collection, strain No. ME9062) having the Δpep-B mutation (KEIO collection, strain No. JW2507; The E. coli Genetic Stock Center, Yale University, New Haven, USA, accession number CGSC9995 ) (E. coli 1Δ strain). For this purpose, a DNA fragment carrying the λattL-cat-λattR cassette was obtained by PCR (polymerase chain reaction) using primers P1 (SEQ ID NO: 28) and P2 (SEQ ID NO: 29) and plasmid pMW118-λattR-cat- λattL as a matrix (Katashkina Zh.I. et al., Mol. Biol. (Mosk.), 2005, 39 (5): 823-831). The obtained DNA fragment was introduced into the E. coli strain BW25113 (ΔpepB) / pKD46 by the method of electrotransformation using the Bio-Rad electroporator (USA) (No. 165-2098, version 2-89) in accordance with the manufacturer's instructions. The recombinant plasmid pKD46 (Datsenko KA and Wanner BL, Proc. Natl. Acad. Sci. USA, 2000, 97: 6640-6645) with a temperature-sensitive replicon was used as a donor of the phage λ genes responsible for the λRed-dependent recombinant the system. Plasmid pKD46 can be integrated into E. coli BW25113 (ΔpepB) to obtain the E. coli strain BW25113 (ΔpepB) / pKD46 by the described method (Datsenko KA and Wanner BL, Proc. Natl. Acad. Sci. USA, 2000, 97: 6640- 6645). In addition, E. coli strain BW25113 (ΔpepB) containing the recombinant plasmid pKD46 can be obtained from E. coli Genetic Stock Center, Yale University, New Haven, USA, accession number CGSC7739.

The transformant E. coli BW25113 (ΔpepB, iadA :: λattR-cat-λattL) is resistant to chloramphenicol and instead of iadA it contains a chloramphenicol resistance marker (Cm ^R- marker) encoded by the cat gene on the chromosome. The Cm ^R marker was excised as described in (Katashkina Zh.I. et al., Mol. Biol. (Mosk.), 2005, 39 (5): 823-831) for the construction of E. coli strain BW25113 (ΔpepB, iadA :: λattB) (E. coli strain 2Δ).

The repE gene was removed in E. coli strain BW25113 (ΔrepB, iadA :: λattB). A DNA fragment carrying the λattL-cat-λattR cassette was obtained by PCR using primers P3 (SEQ ID NO: 30) and P4 (SEQ ID NO: 31) and plasmid pMW118-λattR-cat-λattL as a template. The resulting DNA fragment was introduced into the E. coli strain BW25113 (ΔpepB, iadA :: λattB) / pKD46 by the method of electrotransformation, as described above. Marker Cm ^{R was} excised from E. coli BW25113 transformant (ΔrepB, iadA :: λattB, pepE :: λattR-cat-λattL) for the construction of E. coli strain BW25113 (ΔrepB, iadA :: λattB, peE :: λattB) (strain E .coli 3Δ).

The ybiK gene was deleted in E. coli strain BW25113 (ΔrepB, iadA :: λattB, pepE :: λattB). A DNA fragment carrying the λattL-cat-λattR cassette was obtained by PCR using primers P5 (SEQ ID NO: 32) and P6 (SEQ ID NO: 33) and plasmid pMW118-λattR-cat-λattL as a template. The resulting DNA fragment was introduced into the E. coli strain BW25113 (ΔrepB, iadA :: λattB, pepE :: λattB) / pKD46 by the method of electrotransformation, as described above. The Cm ^R marker was excised from the E. coli transform BW25113 (ΔpepB, iadA :: λattB, pepE :: λattB, ybiK :: λattR-cat-λattL) for the construction of the E. coli strain BW25113 (Δrerp, iadA :: λattB, pepE :: λattB, ybiK :: λattB) (E. coli strain 4Δ).

The dapE gene was deleted in the E. coli strain BW25113 (ΔrepB, iadA :: λattB, pepE :: λattB, ybiK :: λattB). A DNA fragment carrying the λattL-cat-λattR cassette was obtained by PCR using primers P7 (SEQ ID NO: 34) and P8 (SEQ ID NO: 35) and plasmid pMW118-λattR-cat-λattL as a template. The obtained DNA fragment was introduced into the E. coli strain BW25113 (ΔrepB, iadA :: λattB, pepE :: λattB, ybiK :: λattB) / pKD46 by the method of electrotransformation, as described above. Marker Cm ^{R was} excised from the E. coli transform BW25113 (ΔpepB, iadA :: λattB, pepE :: λattB, ybiK :: λattB, dapE :: λattR-cat-λattL) for the construction of the E. coli strain BW25113 (ΔpepB, iadA :: λattB, pepE :: λattB, ybiK :: λattB, dapE :: λattB) (E. coli strain 5Δ). Thus, E. coli strains having one to five deleted genes (E. coli 1-5Δ strains) encoding peptidases were obtained.

8.2 Analysis of specific aspartate-peptide-hydrolyzing activity in E. coli 5Δ strain.

To analyze the specific aspartate-peptide-hydrolyzing activity, the artificial DP3 dipeptide (L-Asp-L-5-fluorotryptophan) was synthesized (branch of the Institute of Bioorganic Chemistry, RAS, Pushchino, Russian Federation). Peptide-hydrolyzing activity was investigated in vitro and in vivo.

For in vitro studies, cells of E. coli BW25113 and E. coli 5Δ strains were grown in LB and M9-salts + Glucose media (0.2%, w / v) (Sambrook, J. and Russell, DW (2001) Molecular Cloning: A Laboratory Manual (3 ^{rd ed.). Cold Spring Harbor} Laboratory Press) at 37 ° C until cell density OD _595nm ~ 1. The grown cells were planted by centrifugation (4 ° C, 10,000 rpm), resuspended in buffer E (50 mM Tris-HCl pH 8.0, 20 mM NaCl), destroyed by ultrasound, then centrifuged (14000 g, 4 ° C, 20 min ) to remove cell debris. The concentration of the crude protein can be determined by the Bradford method (Bradford MM, Anal. Biochem., 1976, 72: 248-254) using bovine serum albumin as a standard.

Crude proteins were used to study DP3 hydrolyzing activity.

The reaction mixture contained:

Tris-HCl pH 8.0 50 mm NaCl 20 mm DP3 dipeptide 5 mm ZnSO ₄ or MnCl ₂ 1 mm Crude protein 24 mcg H ₂ O up to 10 μl

The reaction mixtures were incubated at 37 ° C for various times. DP3-hydrolyzing activity was measured by quantitative TLC, measuring the amount of released 5-fluorotryptophan (Figure 27, Table 4). A mixture of 2-propanol: acetone: H ₂ O as 25: 25: 4 was used as the mobile phase. A solution (0.3%, w / v) of ninhydrin in acetone was used as a coloring reagent. The results showed that aspartate-peptide-hydrolyzing activity can be determined in strain 5Δ, which suggests the presence of unknown peptidases having DP3-hydrolyzing activity in E. coli 5Δ strain.

For in vivo studies, the toxicity of the DP3 dipeptide was studied for engineered peptidase-deficient E. coli 1-5Δ strains (Figure 28). Cells of E. coli BW25113 and E. coli 1-5Δ strains were grown in M9-salts medium containing D-glucose or glycerin (0.4%, w / v) to a density of OD _555nm ~ 2. Cellular biomass was dissolved and about 106 cells were placed on agar with M9-salts, D-glucose or glycerin (0.4%, w / v) and DP3-dipeptide. The plates were incubated at 37 ° C. for 48 hours (for D-glucose) or 72 hours (for glycerol) (Figure 29, Table 5). Visual analysis showed that a deletion of five known asparagine-peptide-hydrolyzing enzymes encoded by the peB, iadA, peE, ybiK and dapE genes reduces the toxicity of DP3 in E. coli strains 1-5Δ (from 6 μM for BW25113 to 30 μM for strain 5Δ grown in M9-salts + D-glucose). An increase in the concentration of DP3 to 50 μM led to growth retardation of strains of E. coli 1-5Δ, which suggests residual intracellular DP3-peptidase activity.

8.3. Determination of residual peptidases in E. coli 5Δ strain having specific aspartate-peptide-hydrolyzing activity.

To determine the residual intracellular peptidases having specific aspartate-peptide-hydrolyzing activity, the following procedure was used. Cells of E. coli strain 5Δ were grown at 37 ° C. overnight in 4 L of M9-salts containing D-glucose (0.4%, w / v). The grown cells were planted by centrifugation (4 ° C, 10,000 rpm) and resuspended in 100 ml of buffer F (20 mm Tris-HCl pH 7.5, 20 mm NaCl).

Cleaning was carried out in the following way:

Step 1. Cells were digested twice by passing through French-press (Thermo Spectronic), then centrifuged (14000 g, 4 ° C, 20 min) to remove cell debris. The resulting crude proteins were loaded onto a DEAE FF 16/10 column (20 ml) (GE Healthcare) calibrated with F buffer (20 mM Tris-HCl pH 7.5, 20 mM NaCl). Elution was carried out at a flow rate of 1 ml / min using a linear NaCl gradient (from 20 to 600 mm in 20 column volumes) in buffer F. Fractions (10 ml each) were selected and analyzed as described in Example 8.2. Fractions 16-21 were found to be active.

Step 2. Proteins from the collected fractions 16-21 were precipitated with saturated solution (60%) (NH ₄ ) ₂ SO ₄ , placed in 2 ml of buffer F and loaded onto a standard column Superdex 200 HR 10 / 30A (GE Healthcare) calibrated with buffer G (20 mM Tris-HCl pH 7). Isocratic elution was carried out at a flow rate of 0.5 ml / min using buffer G. Fractions with a volume of 0.5 ml (0.5 ml each) were collected and analyzed as described in Example 8.2. Active fractions were detected (12-13).

Step 3. Proteins from the selected fractions 12-13 (Step 2) were loaded onto a Sourse15Q column (1.6 ml) (GE Healthcare) calibrated with buffer G (20 mM Tris-HCl pH 7). Elution was performed at a flow rate of 0.5 ml / min using a linear NaCl gradient (from 0 to 400 mm in 20 column volumes) in buffer G. Fractions (0.5 ml each) were selected and analyzed as described in Example 8.2. Active fractions were detected (15-17). Summarized cleaning data are shown in Table 6.

Step 4. To identify peptidases having specific aspartate-peptide-hydrolyzing activity, proteins of several fractions (15-17) were subjected to denaturing gel electrophoresis (DDS / PAGE) (Laemmli UK, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 1970, 227: 680-685). The elution profile of activity was compared with the protein elution profile. Only one protein was found for which the activity and elution profiles were identical.

The purified protein was extracted from DDS gel and digested with trypsin (Govorun V.M. et al., Biochemistry (Mosc.), 2003, 68 (1): 42-49).

The resulting mixture was analyzed using the MALDI-TOF mass spectrometry method as described in (Govorun V.M. et al., Biochemistry (Mosc.), 2003, 68 (1): 42-49). The mass spectrum data of the isolated protein coincided with the data obtained for E. coli aminopeptidase A / I (PEPA). Thus, the sixth peptidase was found in E. coli 5Δ strain.

8.4. Obtaining peptidase-deficient strain of E. coli 6Δ.

The peR gene was deleted in E. coli strain BW25113 (ΔpepB, iadA :: λattB, pepE :: λattB, ybiK :: λattB, dapE :: λattB), as described in Example 8.1. A DNA fragment carrying the λattL-cat-λattR cassette was obtained by PCR using primers P9 (SEQ ID NO: 36) and P10 (SEQ ID NO: 37) and plasmid pMW118-λattR-cat-λattL as a template. The obtained DNA fragment was introduced into the E. coli strain BW25113 (ΔpepB, iadA :: λattB, pepE :: λattB, ybiK :: λattB, dapE :: λattB) / pKD46 by the method of electrical transformation as described above to obtain E. coli strain BW25113 (ΔpepB , iadA :: λattB, pepE :: λattB, ybiK :: λattB, dapE :: λattB, pepA :: λattL-cat-λattR) (E. coli strain 6Δ).

8.5. Analysis of specific peptide-hydrolyzing activity in E. coli 6Δ strain.

Specific aspartate-peptide-hydrolyzing activity was analyzed in vitro as described in Example 8.2. The results obtained (Table 4) showed that aspartate-peptide-hydrolyzing activity can be determined in strain 6Δ and which turned out to be lower than in strains 4-5Δ.

Example 9

Enzymatic production of dipeptides having an acidic L-amino acid, such as L-Asp or L-Glu, at the N-terminus using modified E. coli 5Δ and 6Δ strains having Lal activity.

Dipeptides containing an acidic L-amino acid, such as L-Asp or L-Glu, at the N-terminus, are obtained using bacteria of the Enterobacteriaceae family, more specifically, bacteria belonging to the genus Escherichia, having the ability to produce dipeptides in a medium containing or lacking, for example, but not limited to, essential amino acids. The dipeptide-producing bacterium is an E. coli strain 5Δ or 6Δ as described above, with reduced peptidase activity, which was further modified to have L-amino acid-α-ligase activity. The gene (s) encoding (e) Lal (s) is selected (s) from the group consisting of BBR47_51900, Staur_4851, DES, BUR, ALL, BTH, AME, SFL and BMY, which are introduced into the E. coli chromosome or introduced into a bacterial cell on a plasmid having a gene encoding Lal. Gene (s) encoding (e) Lal (s), placed (s) under the promoter.

Modified E. coli 5Δ or 6Δ strains containing the gene (s) encoding (e) Lal (s) and control 5Δ or 6Δ strains are each grown at 28-37 ° C for 18-72 hours in Luria-Bertani medium ( also called lysogenic medium as described in Sambrook, J. and Russell, DW (2001) Molecular Cloning: a Laboratory Manual (3 ^{rd ed.) Cold Spring Harbor Laboratory} Press).. The E. coli strain 5Δ or 6Δ containing the gene (s) encoding (e) Lal (s) is added to 2 ml of culture medium in 20 × 200 mm tubes and grown at 28-37 ° C for 18-72 hours on rotary shaker at 250 rpm.

The composition of the enzymatic medium (g / l):

Glucose 5-40 NaCl 0.8 (NH ₄ ) ₂ SO ₄ 22 K ₂ HPO ₄ 2.0 MgSO ₄ × 7H ₂ O 0.8 MnSO ₄ × 5H ₂ O 0.02 FeSO ₄ × 7H ₂ O 0.02 Thiamine hydrochloride 0.002 Yeast extract 1.0-2.0 CaCO ₃ thirty L-phe 0-100 (mm) L-asp 0-100 (mm)

The fermentation medium is sterilized at 116 ° C for 30 minutes, glucose and CaCO _{3 are} sterilized separately under the following conditions: glucose at 110 ° C for 30 minutes, CaCO ₃ at 116 ° C for 30 minutes. The pH is adjusted at 5-8 with a KOH solution.

After growing, the accumulated dipeptide is measured using thin layer chromatography (TLC). TLC plates (10 × 20 cm) coated with a layer of silica gel (0.11 mm) contain a non-fluorescent indicator (Sorbpolymer, Krasnodar, Russian Federation). TLC plates were placed in a mobile phase containing 2-propanol: acetone: 250 mM ammonia: H20 as 100: 100: 12: 28. A solution of ninhydrin in acetone (0.3%, w / v) is used as a coloring reagent. The determination is performed at 540 nm.

Example 10

Enzymatic production of Asp-Phe using BBR47_51900 and Staur_4851 using an ATP regeneration system

In order to prevent enzyme inhibition at high ATP concentrations, the yield of Asp-Phe was studied in a reaction mixture containing BBR47_51900 and Staur_4851, as well as phosphoenolpyruvate and pyruvate kinase for ATP regeneration. The composition of the reaction mixture was as follows:

BBR47_51900 or Staur_4851 0.15 units Tris-HCl pH 9.0 50 mm L-AspNa 100 mm L-phe 100 mm ATP 10 mm Phosphoenolpyruvate 100 mm MgSO ₄ × 7H ₂ O 10 mm Pyruvate kinase 25 units H ₂ O up to 1 ml

Reactions were carried out at 37 ° C for 1-48 hours. 100 μl were taken from 1 ml of the reaction mixture at each reaction time. Each reaction mixture to which 10 μl of 1 M EDTA (pH 9.0) was added in order to stop the reaction was investigated by HPLC. The conditions are shown in Example 4. It was shown that BBR47_51900 and Staur_4851 produce Asp-Phe under the conditions of the ATP regeneration system (Table 9).

Example 11

Analysis of specific Asp-Phe-hydrolyzing activity in E. coli 7Δ strain

11.1. Construction of E. coli 7Δ strains deficient in Asp-Phe-hydrolyzing peptidases

The pepD gene was removed in E. coli strain JM109. A DNA fragment carrying the ΔattL-cat-ΔattR cassette was obtained by PCR using primers P11 (SEQ ID NO: 38) and P12 (SEQ ID NO: 39) and plasmid pMW118-ΔattL-cat-ΔattR as a template. The resulting DNA fragment was introduced into the E. coli strain JM109 / pKD46, as described above. The Cm ^R marker was excised from the E. coli JM109 transform (pepD :: ΔattR-cat-ΔattL) for the construction of the E. coli JM109 strain (pepD :: ΔattB).

The peE gene was removed in the E. coli strain JM109 (pepD :: ΔattB). A DNA fragment carrying the ΔattL-cat-ΔattR cassette was obtained by PCR using primers P13 (SEQ ID NO: 40) and P14 (SEQ ID NO: 41) and plasmid pMW118-ΔattL-cat-ΔattR as a template. The resulting DNA fragment was introduced into the E. coli strain JM109 (pepB :: ΔattB) / pKD46, as described above. The Cm ^R marker was excised from the E. coli JM109 transform (pepD :: ΔattB, pepE :: ΔattR-cat-ΔattL) for the construction of the E. coli JM109 strain (pepD :: ΔattB, pepE :: ΔattB).

The iadA gene was deleted in the E. coli strain JM109 (pepD :: ΔattB, pepE :: ΔattB). A DNA fragment carrying the ΔattL-cat-ΔattR cassette was obtained by PCR using primers P15 (SEQ ID NO: 42) and P16 (SEQ ID NO: 43) and plasmid pMW118-ΔattL-cat-ΔattR as a template. The resulting DNA fragment was introduced into the E. coli strain JM109 (pepD :: ΔattB, pepE :: ΔattB) / pKD46, as described above. The Cm ^R marker was excised from the E. coli JM109 transform (pepD :: ΔattB, pepE :: ΔattB, iadA :: ΔattR-cat-ΔattL) for the construction of the E. coli strain JM109 (pepD :: ΔattB, pepE :: ΔattB, iadA :: ΔattB).

The peR gene was deleted in the E. coli strain JM109 (pepD :: ΔattB, pepE :: ΔattB, iadA :: ΔattB). A DNA fragment carrying the ΔattL-cat-ΔattR cassette was obtained by PCR using primers P17 (SEQ ID NO: 44) and P18 (SEQ ID NO: 45) and plasmid pMW118-ΔattL-cat-ΔattR as a template. The resulting DNA fragment was introduced into the E. coli strain JM109 (pepD :: ΔattB, pepE :: ΔattB, iadA :: ΔattB) / pKD46, as described above. The Cm ^R marker was excised from the E. coli JM109 transform (pepD :: ΔattB, pepE :: ΔattB, iadA :: ΔattB, pepA :: ΔattR-cat-ΔattL) for the construction of the E. coli strain JM109 (pepD :: ΔattB, pepE :: ΔattB, iadA :: ΔattB, pepA :: ΔattB).

The reB gene was deleted in the E. coli strain JM109 [pepD :: ΔattB, pepE :: ΔattB, iadA :: ΔattB, pepA :: ΔattB). A DNA fragment carrying the ΔattL-cat-ΔattR cassette was obtained by PCR using primers P19 (SEQ ID NO: 46) and P20 (SEQ ID NO: 47) and plasmid pMW118-ΔattL-cat-ΔattR as a template. The resulting DNA fragment was introduced into the E. coli strain JM109 (pepD :: ΔattB, pepE :: ΔattB, iadA :: ΔattB, pepA :: ΔattB) / pKD46, as described above. The Cm ^R marker was excised from the E. coli transform JM109 (pepD :: ΔattB, pepE :: ΔattB, iadA :: ΔattB, pepA :: ΔattB, pepB :: ΔattR-cat-ΔattL) for the construction of E. coli strain JM109 (pepD :: ΔattB, pepE :: ΔattB, iadA :: ΔattB, pepA :: ΔattB, pepB :: ΔattB).

The iaaA gene was deleted in the E. coli strain JM109 (pepD :: ΔattB, pepE :: ΔattB, iadA :: ΔattB, pepA :: ΔattB, pepB :: ΔattB). A DNA fragment carrying the ΔattL-cat-ΔattR cassette was obtained by PCR using primers P21 (SEQ ID NO: 48) and P22 (SEQ ID NO: 49) and plasmid pMW118-ΔattL-cat-ΔattR as a template. The resulting DNA fragment was introduced into the E. coli strain JM109 (pepD :: ΔattB, pepE :: ΔattB, iadA :: ΔattB, pepA :: ΔattB, pepB :: ΔattB) / pKD46, as described above. The Cm ^R marker was excised from the E. coli transformant JM109 {pepD :: ΔattB, pepE :: ΔattB, iadA :: ΔattB, pepA :: ΔattB, pepB :: ΔattB, iaaA :: ΔattR-cat-ΔattL) for the construction of strain E .coli JM109 (pepD :: ΔattB, pepE :: ΔattB, iadA :: ΔattB, pepA :: ΔattB, pepB :: ΔattB, iaaA :: ΔattB).

The operon of the dpp genes (dppA, dppB, dppC, dppD, dppF] was deleted in the E. coli strain JM109 [pepD :: ΔattB, pepE :: ΔattB, iadA :: ΔattB, pepA :: ΔattB, pepB :: ΔattB, iaaA :: ΔattB). A DNA fragment carrying the ΔattL-cat-ΔattR cassette was obtained by PCR using primers P23 (SEQ ID NO: 50) and P24 (SEQ ID NO: 51) and plasmid pMW118-ΔattL-cat-ΔattR as a template. The final DNA fragment was introduced into the E. coli strain JM109 (pepD :: ΔattB, pepE :: ΔattB, iadA :: ΔattB, pepA :: ΔattB, pepB :: ΔattB, iaaA :: ΔattB) / pKD46, as described above. The Cm ^R marker was excised from the E. coli transform JM109 (pepD :: ΔattB, pepE :: ΔattB, iadA :: ΔattB, pepA :: ΔattB, pepB :: ΔattB, iaaA :: ΔattB, dpp :: ΔattR-cat-ΔattL ) for the construction of E. coli strain JM109 (pepD :: ΔattB, pepE :: ΔattB, iadA ::: ΔattB, pepA :: ΔattB, pepB :: ΔattB, iaaA :: ΔattB, dpp :: ΔattB).

11.2. Analysis of specific Asp-Phe-hydrolyzing activity in E. coli 7Δ strain

Cells of E. coli JM109 and strain E. coli 7Δ were grown in medium with LB agar at 37 ° C for 16 hours. The grown cells were inoculated in 20 ml of MS medium and grown at 37 ° C to an optical density of OD _{610 nm} ~ 20. Then, Asp-Phe (final concentration of 2 mM) was added to the medium and the cells were grown further for 32 hours. The final culture at each time of cultivation was centrifuged to obtain a culture supernatant. The Asp-Phe residue in the culture supernatant was analyzed by HPLC. The results are presented in Table 10. The hydrolysing activity against Asp-Phe in the culture of E. coli 7Δ strain was much less compared to that in E. coli JM109 strain.

The composition of the MS environment (g / l):

Glucose twenty (NH ₄ ) ₂ SO ₄ 8 KH ₃ PO ₄ 0.5 FeSO ₄ × 7H ₂ O 0.005 MnSO ₄ × 7H ₂ O 0.005 Yeast extract one L-tyr 0.05 MgSO ₄ × 7H ₂ O 0.5 CaCO ₃ thirty

The fermentation medium was sterilized at 121 ° C for 20 minutes, except that glucose, MgSO ₄ × 7H ₂ O and CaCO _{3 were} sterilized separately under the following conditions: glucose and MgSO ₄ × 7H ₂ O at 121 ° C for 20 min, CaCO ₃ - at 180 ° C for 2 hours. The pH was maintained to 7 with a KOH solution. Specific Asp-Phe-hydrolyzing activity was lower in E. coli 7Δ strain when compared with that in E. coli strain JM109 (Table 10).

Example 12

Determination of the ability to produce Asp-Phe strain E. coli 7Δ, sverkhekspressiya Lal gene

The primary structure of genes encoding BBR47_51900 and Staur_4851 was optimized for expression in E. coli. Genes encoding BBR47_51900 from Brevibacillus brevis NBRC 100599 and Staur_4851 from Stigmatella aurantiaca DW4 / 3-1 were synthesized using GenScript and obtained as a set of plasmids pUC57 (pUC57-cBBR and pUC57-cSTA). The nucleotide sequences of the obtained cBBR and cSTA are represented by SEQ ID NO: 68 and SEQ ID NO: 69, respectively.

12.1. Construction of pSF12-cBBR and pSF12-cSTA

A DNA fragment carrying BBR47_51900 was obtained by PCR using primers P25 (SEQ ID NO: 52) and P2b (SEQ ID NO: 53) and plasmid pUC57-cBBR as a template. PCR was performed using the following steps: 98 ° C, 30 seconds; (98 ° C, 15 seconds; 58 ° C, 10 seconds; 72 ° C, 1 minute) × 30 cycles; 72 ° C, 5 minutes in 50 μl of the reaction mixture, including 0.04 μg of plasmid DNA, 0.2 μmol / L of each primer, 1.0 Unit. high-precision DNA polymerase (Phusion High-Fidelity DNA Polymerase, New England Labs), 10 μl of 5 × Phusion HF buffer and 0.2 mmol / L of each dNTPs. Purification of the obtained DNA fragment was performed using the MinElute PCR Purification Kit (Qiagen).

The DNA fragment carrying Staur_4851 was obtained by PCR using primers P27 (SEQ ID NO: 54) and P28 (SEQ ID NO: 55) and plasmid pUC57-cSTA as a template. PCR conditions and purification method are described above.

The solutions thus obtained were treated with restriction enzymes Nde I and Pst I to digest the obtained DNA, and then each of the DNA fragments (1.3 kb) was purified using the MinElute Reaction Cleanup Kit (Qiagen).

The pSF12-ggt vector was constructed from the pUC18 vector and contains the rpoH promoter and the ggt gene encoding gamma-glutamyl transpeptidase from E. coli strain W3110 (WO2013051685 A1). The pSF12-ggt vector was digested with Nde I and Pst I. DNA fragments were separated by agarose gel electrophoresis. DNA fragment (3.0 kb) was isolated using QIAquick Gel Extraction Kit (Qiagen).

A DNA fragment (1.3 kb) containing the BBR47_51900 or Staur_4851 gene and a DNA fragment (3.0 kb) obtained above were subjected to a ligation reaction using TaKaRa Ligation Kit Ver. 2.1 ( TaKaRa) at 16 ° C for 30 minutes. Competent E. coli JM109 cells (TaKaRa) were transformed by heat shock using a ligation reaction mixture, placed on LB agar medium containing 100 μg / ml ampicillin and grown at 30 ° C overnight. The plasmid was isolated from the colony of the transformant grown in the medium, in accordance with the known method. Thus, pSF12-cBBR and p8F12-cSTA were obtained. The sequence of DNA vectors was confirmed using 3130 Genetic Analyzer (Applied Biosystems).

12.2. Enzymatic Asp-Phe production using modified E. coli 7Δ strains having Lal activity

The dipeptide producing bacterium is the E. coli 7Δ strain described above, deficient in peptidase and dipeptide-permease activity, further modified to have L-amino acid-α-ligase activity. The dipeptide producing strains contain a gene encoding BBR47_51900 or Staur_4851 introduced into the bacterial cell on the plasmid pSF12-cBBR or pSF12-cSTA, respectively. Each of the genes encoding Lals is placed under the rpoH promoter. Modified E. coli 7Δ strains containing the gene encoding Lal and the control 7Δ strain were grown at 25 ° C for 24 hours in LB agar medium (containing 100 μg / ml ampicillin for modified E. coli 7Δ strains containing the gene, encoding Lal). E. coli 7Δ strains containing the gene encoding Lal and the control strain 7Δ were inoculated in 20 ml of MS medium containing 100 mM L-Asp and 100 mM L-Phe in a 500 ml Sakaguchi flask and grown at 25 ° C for 32 hours on a reciprocating shaker at 120 rpm The resulting culture was centrifuged to obtain a culture supernatant. The Asp-Phe culture accumulated in the supernatant was analyzed by HPLC. The results are presented in Table 11. As can be seen from Table 11, Asp-Phe was not obtained using strain 7Δ, while Asp-Phe was produced by strain 7Δ containing the gene encoding Lal.

Example 13

Determination of the ability to produce Asp-Phe strain E. coli 9Δ / pMGAL1 / pHSG-cLal

13.1. Construction of E. coli 7Δ strain deficient in both tyrR and tyrA genes.

First, for derepression of the synthesis of enzymes involved in the biosynthesis of aromatic amino acids, the tyrR gene in the E. coli strain JM109 (pepD :: ΔattB, pepE :: ΔattB, iadA :: ΔattB, pepA :: ΔattB, pepB :: ΔattB, iaaA: : ΔattB, dpp :: ΔattB). A DNA fragment carrying the ΔattL-cat-ΔattR cassette was obtained by PCR using primers P29 (SEQ ID NO: 56) and P30 (SEQ ID NO: 57) and plasmid pMW118-ΔattL-cat-ΔattR as a template. The resulting DNA fragment was introduced into E. coli strain JM109 (pepD :: ΔattB, pepE :: ΔattB, iadA :: ΔattB, pepA :: ΔattB, pepB :: ΔattB, iaaA :: ΔattB, dpp :: ΔattB) / pKD46, as described in Example 11.1. The Cm ^R marker was excised from the E. coli transform JM109 (pepD :: ΔattB, pepE :: ΔattB, iadA :: ΔattB, pepA :: ΔattB, pepB :: ΔattB, iaaA :: ΔattB, dpp :: ΔattB, tyrR :: ΔattR-cat-ΔattL) for the construction of E. coli strain JM109 (pepD :: ΔattB, pepE :: ΔattB, iadA :: ΔattB, pepA :: ΔattB, pepB :: ΔattB, iaaA :: ΔattB, dpp :: ΔattB, tyrR :: ΔattB).

Then, the tyrA gene in E. coli strain JM109 was removed (pepD :: ΔattB, pepE :: ΔattB, iadA :: ΔattB, pepA :: ΔattB, pepB :: ΔattB, iaaA :: ΔattB, dpp :: ΔattB, tyrR :: ΔattB), so that the prefenate, a common intermediate in the biosynthesis of Phe and Tyr, could not be utilized in the biosynthesis of Tyr. A DNA fragment carrying the ΔattL-cat-ΔattR cassette was obtained by PCR using primers P31 (SEQ ID NO: 58) and P32 (SEQ ID NO: 59) and plasmid pMW118-ΔattL-cat-ΔattR as a template. The final DNA fragment was introduced into the E. coli strain JM109 (pepD :: ΔattB, pepE :: ΔattB, iadA :: ΔattB, pepA :: ΔattB, pepB :: ΔattB, iaaA :: ΔattB, dpp :: ΔattB, tyrR :: ΔattB) / pKD46 as described above. The Cm ^R marker was excised from the E. coli transform JM109 (pepD :: ΔattB, pepE :: ΔattB, iadA :: ΔattB, pepA :: ΔattB, pepB :: ΔattB, iaaA :: ΔattB, dpp :: ΔattB, tyrR :: ΔattB, tyrA :: ΔattR-cat-ΔattL) for the construction of E. coli strain JM109 (pepD :: ΔattB, pepE :: ΔattB, iadA :: ΔattB, pepA :: ΔattB, pepB :: ΔattB, iaaA :: ΔattB, dpp :: ΔattB, tyrR :: ΔattB, tyrA :: ΔattB).

13.2. Construction of pHSG-cBBR and pHSG-cSTA

To construct the pHSG-cBBR plasmid, the corresponding EcoRI - SphI fragment containing the rpoH promoter and the BBR47_51900 gene was excised from the plasmid pSF12-cBBR by digestion with EcoRI and SphI and then ligated with the pHSG396 (TaKaRa) vector treated with the same restriction enzymes.

For the construction of plasmid pHSG-cSTA, a DNA fragment carrying Staur_4851 under the rpoH promoter was obtained by PCR using REM primers (SEQ ID NO: 60) and P34 (SEQ ID NO: 61) and plasmid pSF12-cSTA as a template. PCR was performed using the following program: 98 ° C, 30 seconds; (98 ° C, 15 seconds; 58 ° C, 10 seconds; 72 ° C, 1 minute) × 30 cycles; 72 ° C, 5 minutes in 50 μl of the reaction mixture, including 0.04 μg of the plasmid DNA, 0.2 μmol / L of each primer, 1.0 Unit high-precision DNA polymerase (Phusion High-Fidelity DNA Polymerase, New England Labs), 10 μl of 5 × Phusion HF buffer and 0.2 mmol / L of each dNTPs. Then the DNA fragment (1.5 kb) digested with BamHI and XhoI was ligated with the pHSG396 vector treated with the same restriction enzymes.

13.3. Transformation of pMGAL1 and pHSG-cLal vectors into E. coli 9Δ strain

The pMGAL1 vector was constructed from pMW19 (Wako), and contains three genes involved in Phe biosynthesis in E. coli: pheA, aroG4 and aroL encoding chorizmmutase-prefenate dehydratase (CM-PD), 3-deoxy-D-arabinoheptulosonate-7- phosphate synthetase (DAHP synthetase) and shikimate kinase (SK), respectively (JP3225597). Each of the native pheA and aroG4 genes was mutated to remove Phe feedback inhibition.

The vectors pMGAL1 and pHSG-cLal were simultaneously introduced into the E. coli 9Δ strain by electroporation using LB agar medium containing 100 μg / ml ampicillin and 25 μg / ml chloramphenicol. The strain thus obtained was named E. coli 9Δ / pMGALl / pHSG-cLal.

13.4. Enzymatic production of Asp-Phe using E. coli 9Δ / pMGAL1 / pHSG-cLal

The modified E. coli strain 9Δ / pMGAL1 / pHSG-cLal and the control strain 9Δ / pMGAL1 were each grown at 25 ° C for 24 hours in LB agar medium (containing 100 μg / ml ampicillin and 25 μg / ml chloramphenicol for E. coli 9Δ / pMGAL1 / pHSG-cLal). The E. coli strain 9Δ / pMGAL1 / pHSG-cLal and the control strain 9Δ / pMGAL1 were inoculated in 20 ml of MS medium containing 100 mM L-Asp in a 500 ml Sakaguchi flask and grown at 25 ° C for 72 hours on a reciprocating rocking chair at 120 rpm. The resulting culture was centrifuged to obtain a culture supernatant. Phe and Asp-Phe cultures accumulated in the supernatant were analyzed by HPLC. The results are shown in Table 12. As can be seen from Table 12, Asp-Phe was not obtained using strain 9Δ / pMGAL1, while Asp-Phe was produced by strain 9Δ / pMGAL1 containing the gene encoding Lal.

Example 14

DES substrate specificity analysis

14.1. Construction of the plasmid pELAC-MBP-DES-HT

14.1.1. Construction of the auxiliary plasmid pELAC

The DNA fragment was obtained by PCR using primers P35 (SEQ ID NO: 62), P36 (SEQ ID NO: 63) and DNA plasmid pUC18 (GenBank: L08752.1) as a template. The resulting DNA fragment was digested with BglII and XbaI and cloned into plasmid pET22 (b +) (Novagen, catalog No. 69744-3), cut with the same endonucleases. So the plasmid pELAC was constructed.

14.1.2. Construction of the auxiliary plasmid pELAC-MBP-HT

The malE gene (without signal peptide sequence) was obtained by PCR using primers P37 (SEQ ID NO: 64), P38 (SEQ ID NO: 65) and E. coli chromosomal DNA MG1655 as a template. The resulting DNA fragment was digested with XbaI and BamHI and cloned into the pELAC- / XbaI-BamHI vector. So the plasmid pELAC-MBP-HT was constructed.

14.1.3. Construction of the plasmid pELAC-MBP-DES-HT

To construct the plasmid pELAC-MBP-DES-HT, a DNA fragment containing the DES gene was obtained using primers P39 (SEQ ID NO: 66), P40 (SEQ ID NO: 67) and plasmid pUC57-DES (present patent application) as a matrix. The resulting DNA fragment was digested with BamHI and NotI and ligated with the vector pELAC-MBP-HT / BamHI-NotI. Thus, the plasmid pELAC-MBP-DES-HT was constructed.

14.2. Expression and purification of MBP-hybrid DES with His ₆ tag

E. coli 7Δ cells containing pELAC-MBP-DES-HT were grown in LB medium containing 100 μg / ml ampicillin in test tubes at 37 ° C until an OD of _{610 nm} ~ 2 was _reached . Then, 2 ml of the obtained culture was inoculated in 100 ml of LB-medium containing IPTG (final concentration 0.1 mmol / L) in a 500 ml Sakaguchi flask at 30 ° C for 8 hours. Induced cells were taken from 1.6 L of nutrient broth, resuspended in 200-240 ml of HT-II buffer (50 mM Tris-HCl pH 8.0, 0.3 M NaCl, 10 mM imidazole, 15% glycerol) and sonicated with using a sonicator (INSONATOR 201M, KUBOTA). Debris was removed by centrifugation at 4 ° C and 14,000 rpm for 15 minutes, then passed through a 0.45 mm filter (Milhpore). The untreated protein solution was loaded onto a HisTALON Superflow 5 ml cartridge (Clontech) using AKTA avant 25 (GE Healthcare) according to the manufacturer's recommendations. Fractions containing a MBP hybrid DES with a Hise tag were pooled and salted out using PD-10 columns (GE Healthcare) calibrated with SC buffer (50 mM Tris-HCl pH8.0, 0.3 M NaCl, 15% glycerol) .

14.3. Analysis of Asp-Phe-synthesizing activity of MBP-hybrid DES with His ₆ tag

The dipeptides synthesized using the MBP hybrid DES with His ₆ tag were determined using an HPLC analysis of a reaction mixture that contained:

DES 160 mcg Tris-HCl pH 9.0 50 mm L-asp 100 mm L-phe 100 mm Adenosine 5′-triphosphate (ATP) 10 mm MgSO ₄ × 7H ₂ O 10 mm H ₂ O up to 400 μl

Reactions were carried out at 37 ° C for 15 hours. Then, an HPLC analysis of the reaction mixture was carried out, to which 10 μl of 1 M EDTA, pH 9.0 was added to stop the enzymatic reaction. The reaction conditions were the same as described in Example 4. DES was shown to catalyze the formation of 0.30 mM Asp-Phe.

Additional example 1.

Table 2 (identity) and Table 3 (similarity) show the multiple alignments of the BBR47_51900 and Staur_4851c proteins with known L-amino acid-α-ligases (Lals). As can be seen from Tables 2 and 3, the proteins BBR47_51900 and Staur_4851 are identical by no more than 25% (Table 2) and are similar to no more than 43% (Table 3) with the known Lals.

Additional example 2.

Table 7 (identity) and Table 8 (similarity) show paired alignment values for the BBR47_51900, Staur_4851, DES, BUR, ALL, BTN, AME, SFL, and BMY proteins. As can be seen from Tables 7 and 8, the proteins BBR47_51900, Staur_4851, DES, BUR, ALL, BTN, AME, SFL and BMY are identical by no more than 25% (Table 7) and similar to no more than 44% (Table 8).

Although the invention has been described in detail with reference to the best methods of carrying out the invention, it will be apparent to those skilled in the art that various changes can be made and equivalent replacements made, and such changes and replacements are not outside the scope of the present invention. Each of the above documents has a link, and all cited documents are part of the description of the present invention.

Table 1 Dipeptides synthesized using BBR47_51900 and Staur_4851. Enzyme Dipeptides (mm) Asp-phe Asp-pheome Asp-trp Asp-val Glu-val BBR47_51900 1.22 0,03 0.13 3.9 1.48 Staur_4851 2.54 0.02 1.48 4.4 0.40

table 2 Multiple alignment (identity,%) of BBR47_51900 and Staur_4851 with famous Lals. Enzyme Lals with known substrate specificity one 2 3 four 5 6 7 8 9 10 eleven 12 13 fourteen fifteen 16 17 eighteen 19 BBR47_51900 one hundred 38 23 25 twenty 17 19 fifteen 12 13 fifteen fifteen fifteen 16 12 13 eighteen 10 13 Staur_4851 38 one hundred 25 23 17 16 21 17 13 13 13 16 16 17 fourteen fourteen fourteen 9 13 Identity can be defined as the percentage of identical amino acid residues among all positions without gaps between pairs. Names of proteins: 1 - BBR47_51900 (NCBI Link to sequence: YP_002774671.1); 2 - Staur_4851 (NCBI Link to sequence: ADO72629.1); 3 - TDE2209 (NCBI Link to sequence: NP_972809.1); 4 - BL00235 (NCBI Link to sequence: YP_081312.1); 5 - plu1218 (NCBI Link to sequence: NP_928530.1; 6 - YwfE (UniProtKB / Swiss-Prot: P39641.1); 7 - Rsp1486 (NCBI Link to sequence: NP_523045.1); 8 - NP_900476 (NCBI Link to sequence: NP_900476.1); 9 - Aple02000835 (NCBI Link to sequence: ZP_00134462.2); 10 - SMU1321c (NCBI Link to sequence: NP_721690.1); 11 - YP_816266 (NCBI Link to sequence: YP_816266.1); 12 - YP_001544794 (NCBI Link to sequence: YP_001544794.1); 13 - YP_077482 (NCBI Link to sequence: YP_077482.1); 14 - BAH56723 (GenBank: BAH56723.1); 15 - NP_358563 (NCBI Link to sequence: NP_358563.1); 16 - YP_910063 (NCBI Link to sequence: YP_910063.1); 17 - BAG72134 (GenBank: BAG72134.1); 18 - plul440 (NCBI Link to sequence: NP_928738.1); 19 - AAZ37741 (GenBank: AAZ37741.1).

Table 3 Multiple alignment (similarity,%) BBR47_51900 and Staur_4851 with the famous Lals. Enzyme Lals with known substrate specificity one 2 3 four 5 6 7 8 9 10 eleven 12 13 fourteen fifteen 16 17 eighteen 19 BBR47_51900 one hundred 57 42 43 37 33 39 33 thirty thirty 31 32 34 36 34 32 37 26 29th Staur_4851 57 one hundred 43 42 36 32 38 31 32 32 thirty 32 33 35 29th 34 34 twenty thirty Similarity can be defined as the percentage of identity and similarity of amino acid residues among all positions without gaps between pairs. Names of proteins: 1 - BBR47_51900 (NCBI Link to sequence: YP_002774671.1); 2 - Staur_4851 (NCBI Link to sequence: ADO72629.1); 3 - TDE2209 (NCBI Link to sequence: NP_972809.1); 4 - BL00235 (NCBI Link to sequence: YP_081312.1); 5 - plu1218 (NCBI Link to sequence: NP_928530.1); 6 - YwfE (UniProtKB / Swiss-Prot: P39641.1); 7 - Rsp1486 (NCBI Link to sequence: NP_523045.1); 8 - NP_900476 (NCBI Link to sequence: NP_900476.1); 9 - Aple02000835 (NCBI Link to sequence: ZP_00134462.2); 10 - SMU1321c (NCBI Link to sequence: NP_721690.1); 11 - YP_816266 (NCBI Link to sequence: YP_816266.1); 12 - YP_001544794 (NCBI Link to sequence: YP_001544794.1); 13 - YP_077482 (NCBI Link to sequence: YP_077482.1); 14 - BAN56723 (GenBank: BAH56723.1); 15 - NP_358563 (NCBI Link to sequence: NP_358563.1); 16 - YP_910063 (NCBI Link to sequence: YP_910063.1); 17 - BAG72134 (GenBank: BAG72134.1); 18 - plu1440 (NCBI Link to sequence: NP_928738.1); 19 - AAZ37741 (GenBank: AAZ37741.1)

Table 4 Specific aspartic peptide-hydrolyzing activity (A, in nmol / mg min) in E. coli 4-6Δ strains. Cofactors Strain WT * 4Δ 5Δ 6Δ - 110.48 6.98 7.41 6.07 Zn ²⁺ 123.03 6.38 5.63 4.69 Mn ²⁺ 282.34 13.26 14.14 6.86 Standard deviation: <5%. * - wild type Names: WT - BW25113; 4Δ - BW25113 (ΔpepB, iadA :: λattB, pepE :: λattB, ybiK :: λattB); 5Δ - BW25113 (ΔpepB, iadA :: λattB, pepE :: λattB, ybiK :: λattB, dapE :: λattB; 6Δ - BW25113 (ΔpepB, iadA :: λattB, pepE :: λattB, ybiK :: λattB, dapE :: λattB, pepA :: λattL-cat-λattR).

Table 5 The study of the toxicity of DP3 due to aspartate-peptide-hydrolyzing activity of E. coli 5Δ strain. DP3 conc. (mm) Strains Culture medium M9-salt / Glucose (0.4%, w / v) M9-salt / Glycerin (0.4%, w / v) 0 Wt ++ ++ 5Δ ++ ++ 2 Wt ++ ++ 5Δ ++ ++ four Wt + + 5Δ ++ ++ 6 Wt + - + - 5Δ ++ ++ 10 Wt - - 5Δ ++ + fifteen Wt - - 5Δ ++ + twenty Wt - - 5Δ ++ + - thirty Wt - - 5Δ + - - fifty Wt - - 5Δ - - Designations: ++: the level of growth is equal to the level observed in the wild-type strain at 0 mm DP3, +: the growth level is lower compared to the level observed in the wild-type strain at 0 mM DP3, + -: the level of growth that is observed, but it is very small compared to the level that is observed in the wild-type strain at 0 mM DP3, -: no growth observed

Table 6 Purification of peptidases having specific aspartic peptide-hydrolyzing (DP3-hydrolyzing) activity. Fraction V (ml) Conc. (mg / ml) Protein (mg) Activity (nmol / mg min) Total activity (nmol / min) Lysate 30,0 4,550 136.5 14.7 2006.6 Unrelated 30,0 1,080 32,4 1.4 45.4 DEAE (16-21) 60.0 1,100 66.0 22.4 1477.8 Superdex (12-13) 2.0 0.520 1,04 69.8 72.5 Source 15Q (15) 0.5 0,050 0,025 116.6 2.9 Source 15Q (16) 0.5 0,050 0,025 382.8 9.6 Source 15Q (17) 0.5 0,050 0,025 155.7 3.9

Table 7 Pair alignment (identity,%) Lals. Enzyme AME SFL BBR ALL DES Bmy VTN Bur STA AME one hundred 36 29th 29th 25 27 31 25 29th SFL one hundred 32 thirty thirty 28 26 28 36 BBR one hundred 55 57 46 40 35 36 ALL one hundred 61 47 38 thirty 33 DES one hundred 47 40 32 36 Bmy one hundred 41 thirty 34 VTN one hundred thirty 32 Bur one hundred 38 STA one hundred Identity can be defined as the percentage of identical amino acid residues among all positions without gaps between pairs. BBR means BBR47_51900 and STA means Staur_4851.

Table 8 Pair alignment (similarity,%) Lals. Enzyme AME SFL BBR Bce DES Bmy VTN Bur STA AME one hundred 58 51 52 51 fifty 51 45 fifty SFL one hundred 47 48 49 46 45 44 fifty BBR one hundred 74 76 65 64 54 55 ALL one hundred 79 65 64 49 54 DES one hundred 67 66 53 56 Bmy one hundred 61 48 53 VTN one hundred fifty 51 Bur one hundred 55 STA one hundred Similarity can be defined as the percentage of identical and similar amino acid residues among all positions without gaps between pairs. BBR means BBR47_51900, and STA means Staur_4851.

Table 9 Asp-Phe (mm) 1 hour 2 h 4 h 8 h 24 h 48 h BBR47_51900 7 13 24 40 68 74 Staur_5841 10 twenty 33 49 56 56

Table 10 0 h 2 h 4 h 6 h 8 h 24 h 32 h Jm109 one hundred 0 0 0 0 0 0 strain 7Δ one hundred 97 96 96 95 91 87

Table 11 Strain E. coli Asp-Phe (mm) 7Δ 0 7Δ / pSF12-cBBR 0.20 7Δ / pSF12-cSTA 0.24

Table 12 Strain E. coli Phe (mm) Asp-Phe (mm) 9Δ / pMGAL1 24.4 but. 9Δ / pMGAL1 / pHSG2-cBBR 21,4 0.003 9Δ / pMGAL1 / pHSG2-cSTA 25.5 0,0006 but. - undefined

Claims (16)

1. A dipeptide-producing bacterium belonging to the genus Escherichia and modified in such a way as to contain DNA encoding a protein having a dipeptide-synthesizing activity, and characterized in that said DNA is selected from the group consisting of:
(A) a DNA having the nucleotide sequence of SEQ ID NOs: 1, 3, and 5;
(B) DNA hybridizing under stringent conditions with a nucleotide sequence complementary to the sequence shown in SEQ ID NOs: 1, 3, and 5, characterized in that said stringent conditions include washing one or more times in a solution containing a salt concentration of 1 × SSC , 0.1% SDS or 0.1 × SSC, 0.1% SDS, at 60 ° C or 65 ° C;
(C) DNA encoding a protein having the amino acid sequence of SEQ ID NOs: 2, 4, and 6;
(D) DNA encoding a variant of a protein having the amino acid sequence of SEQ ID NOs: 2, 4 and 6, but which contains the substitution, deletion, insertion or addition of one or more amino acid residues, and having a dipeptide-synthesizing activity in accordance with the amino acid sequence of SEQ ID NOs: 2, 4, and 6;
(E) DNA encoding a protein having homology as defined by $$\left|Lo{g}_{10}\left(E-value\right)\right|$$

, not less than 128, not less than 142, not less than 162, not less than 175, not less than 182, not less than 196 or not less than 233 compared to the amino acid sequence of SEQ ID NOs: 2, 4 and 6, and having a dipeptide-synthesizing activity in accordance with the amino acid sequence of SEQ ID NOs: 2, 4, and 6. 2. The bacterium according to claim 1, characterized in that said bacterium belongs to the species Escherichia coli. 3. The bacterium according to any one of paragraphs. 1 or 2, characterized in that the bacterium is modified so as to have attenuated or inactivated one or more genes encoding proteins having peptidase activity. 4. The bacterium according to claim 3, characterized in that said genes encoding proteins having peptidase activity are selected from the group consisting of peRA, peB, pepD, peE, peR, pepQ, pepN, peR, iadA, iaaA (ybiK) and dapE. 5. A method of producing a dipeptide or its salt, including:
(a) the reaction of L-amino acids, or derivatives of L-amino acids, or their salts under acceptable conditions in the presence of a protein having a dipeptide-synthesizing activity, characterized in that the protein is selected from the group consisting of:
(F) a protein having the amino acid sequence of SEQ ID NOs: 2, 4, and 6;
(G) a variant of a protein having the amino acid sequence of SEQ ID NOs: 2, 4 and 6, but which contains the substitution, deletion, insertion or addition of one or more amino acid residues, and having a dipeptide-synthesizing activity in accordance with the amino acid sequence of SEQ ID NOs: 2, 4 and 6;
(H) a protein having homology defined in terms of $$\left|Lo{g}_{10}\left(E-value\right)\right|$$

, not less than 128, not less than 142, not less than 162, not less than 175, not less than 182, not less than 196 or not less than 233 compared to the amino acid sequence of SEQ ID NOs: 2, 4 and 6, and having a dipeptide-synthesizing activity in accordance with the amino acid sequence of SEQ ID NOs: 2, 4, and 6;
(b) the accumulation of the specified dipeptide or its salt in an acceptable solvent and, if necessary,
(c) isolating said dipeptide or salt thereof from an acceptable solvent. 6. The method according to p. 5, characterized in that the specified L-amino acid or its derivatives are selected from the group consisting of L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid , L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine , L-valine and lower alkyl ester of L-phenylalanine. 7. The method according to p. 5, characterized in that said dipeptide is represented by the formula
R1-R2,
characterized in that R1 is an acid L-amino acid residue or a derivative of an acid L-amino acid residue and R2 is an L-amino acid residue or a derivative of an L-amino acid residue, said L-amino acid residue selected from the group consisting of L-alanine residue, L arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine , L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine and lower alkyl ester of L-phenylalanine. 8. The method according to p. 7, characterized in that R1 is the remainder of L-aspartic acid or the remainder of L-glutamic acid and R2 is the remainder of L-glutamic acid, L-isoleucine, L-phenylalanine, L-tryptophan, L-valine or lower alkyl ester of L-phenylalanine. 9. The method according to p. 7, characterized in that R1 is the residue of L-aspartic acid and R2 is the residue of L-phenylalanine or lower alkyl ester of L-phenylalanine. 10. The method according to p. 6, characterized in that the lower alkyl ester of L-phenylalanine is methyl, ethyl or propyl ester of L-phenylalanine. 11. A method of producing a dipeptide or its salt, including:
(a) growing bacteria according to any one of paragraphs. 1-4 in a nutrient medium;
(b) the accumulation of the specified dipeptide in bacteria and / or culture fluid and, if necessary,
(c) isolating said dipeptide from a bacterium or culture fluid. 12. The method according to p. 11, characterized in that the specified L-amino acid or its derivatives are selected from the group consisting of L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid , L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine , L-valine and lower alkyl ester of L-phenylalanine. 13. The method according to p. 11, characterized in that the dipeptide is represented by the formula
R1-R2,
characterized in that R1 is an acid L-amino acid residue or a derivative of an acid L-amino acid residue and R2 is an L-amino acid residue or a derivative of an L-amino acid residue, said L-amino acid residue selected from the group consisting of L-alanine residue, L arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine , L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine and lower alkyl ester of L-phenylalanine. 14. The method according to p. 13, wherein R1 is a residue of L-aspartic acid or a residue of L-glutamic acid, and R2 is a residue of L-glutamic acid, L-isoleucine, L-phenylalanine, L-tryptophan, L-valine or lower alkyl ester of L-phenylalanine. 15. The method according to p. 13, wherein R1 is the residue of L-aspartic acid and R2 is the residue of L-phenylalanine or a lower alkyl ester of L-phenylalanine. 16. The method according to p. 12, characterized in that the lower alkyl ester of L-phenylalanine is methyl, ethyl or propyl ester of L-phenylalanine.

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