DNA ENCODING DIPEPTIDE-SYNTHESI ING ENZYME (VARIANTS), BACTERIUM BELONGING TO THE GENUS ESCHERICHIA, AND METHODS

FOR PRODUCING DIPEPTIDES USING THEREOF

TECHNICAL FIELD

[0001]

The present invention relates to the biotechnology industry, and specifically to novel dipeptide-synthesizing enzymes and methods for producing dipeptides, in particula dipeptides having an acidic L-amino acid residue at the N- terminus . BACKGROUND ART

[0002]

Dipeptides are used in the fields of pharmaceuticals, foods, and various other fields. For example, the dipeptide Asp-Glu has been used for preparation of the diuretic and natriuretic pharmaceutical composition (FR2662359 Al) . A pharmaceutical composition, containing dipeptides having agonistic effects on NR1/NR2A and NR1/NR2B subtypes of NMDA receptor is known (JP 2009209131 A) . Taste properties of numerous dipeptides have been studied. For example, the dipeptide Asp-Val has sourness taste (Sogame S. and

Matsushita I., New Food Ind. , 1996, 38(12):44-49

(Japanese) ) . An excellent saltiness-strengthening agent is known obtained by using a dipeptide containing glutamic acid such as Glu-Ala, Glu-Asp, Glu-Glu, Glu-Ile, Asp-Glu, His-Glu, Trp-Glu, etc. (WO 2009113563 Al) .

[0003]

A variety of methods of producing dipeptides are known, including extraction from protein hydrolysates, chemical synthesis from protected and/or activated amino acids, and enzymatic synthesis using 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 cloning vehicle encoding peptide

comprised by the repeating amino acid sequence (Asp-Phe)n has been reported to be useful for production of benzylated and methylated derivatives of dipeptide Asp-Phe -(European Patent Application No. 0036258) .

[0004]

The synthesis of dipeptides using chemical and/or chemical-enzymatic approaches requires introduction and removal of protecting groups for functional groups of amino acids to be joined, and isolation of desired product from racemic mixture. The process is thus considered to be disadvantageous from the point of view cost, efficiency, and necessity to discard the concomitant chemicals such as organic solvents, salts, and the like.

[0005]

Several approaches for enzymatic synthesis of

dipeptides and derivatives thereof have been reported, which include a method using reverse reaction of proline iminopeptidase having ability to produce a peptide from an L-amino acid ester and an L-amino acid (Russian Patent No. 2279440), a method using non-ribosomal peptide synthetase

(NRPS) (U.S. Patent Nos . 5,795,738 and 5,652,116; Doekel S. and Marahiel M.A., Chem. Biol., 2000, 7:373-384; Dieckmann R. et al., FEBS Lett., 2001, 498:42-45), a method using aminoacyl-tRNA-synthetase (Japanese Patent Publication Nos.: 58-146539 (1983), 58-209992 (1983), and 59-106298 (1984)), and a method using a mutant protein having the peptide-synthesizing activity (Russian Patent Application 2007127719) . [0006]

The enzymes belonging to the ATP-dependent

carboxylate-amine/thiol a-ligase superfamily have been widely used for production of dipeptides having an - peptide bond between two L-amino acids. For example, by using the homology search function of SubtiList

(http://genolist.pasteur.fr/SubtiList/), which is a

database of the genomic DNA of Bacillus subtilis 168, and the amino acid sequence of D-Ala-D-Ala ligase gene derived from Bacillus subtilis 168, the ywfE gene has been found, which encodes the enzyme capable of synthesizing dipeptides having at the N-terminus the L-amino acid such as, in particular, L-Ala, L-Gly, L-Met, L-Ser, and L-Thr (Tabata K. et al., J. Bacterid., 2005, 187 (15) : 5195-5202; U.S. Patent Nos. 7,514,243 and 7,939,302). Despite the YwfE protein (bacilysin synthetase, enzyme classification number (EC) 6.3.2.28) has extremely broad substrate specificity, the enzyme does not accept highly charged 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. Bacterid . , 2005,

187 (15) : 5195-5202) . Also, a protein encoded by the

rhizocticin synthetase gene and having dipeptide- synthesizing activity has been described, which utilizes L- amino acids, Gly, and β-Ala as substrates (U.S. Patent No. 7,939,294). As confirmed by the liberated phosphoric acid (Pi) as well as TOFMS and NMR analyses, the enzyme places L-Arg and L-Lys on the N-terminus of dipeptide. The Hidden Markov Model (HMM) -based profile analysis revealed five L- amino acids a-ligases (Lais) originating from Treponema denticola ATCC 35405, Photorhabdus luminescence subsp.

laumondii TT01, Streptococcus mutants UA159, Streptococcus pneumoniae TIGR4, and Actinobacillus pleuropneumoniae serovar 1 str. 4074, capable of forming from L-amino acids various peptidyl compounds as proved by the release of phosphoric acid (Senoo A. et al., Biosci. Biotechnol.

Biochem., 2010, 74 (2) : 415-418) . No dipeptide formation was confirmed in combination of L-Glu or L-Asp with other L- amino acids. A mutant protein having the peptide- synthesizing activity has been confirmed by HPLC using standard samples to form dipeptides bearing L-Met at the N- terminus (Russian Patent Application 2007127719) . The in silico screening performed with the help of the NCBI's

BLAST service (http://www.ncbi.nlm.nih.gov/BLAST/) and based on the amino acid sequence of Lai from B. subtilis (BsLal) has revealed a protein RSpl486a from Ralstonia solanacearum, which is capable of forming dipeptide bond as confirmed by the release of phosphoric acid (Kino K. et al., Biochem. Biophys . Res. Comm., 2008, 371:536-540; European Patent Application No.1870454). The structural analysis using NMR technique confirmed formation of dipeptides having L-Ser, L-Met, L-Gln, L-Phe, L-His, L-Ala, and L-Cys at the N-terminus. Despite the inorganic phosphate release has been confirmed in the mixture containing RSpl486a and

L-Asp with L-Phe, L-His, L-Met, L-Cys or L-Ala; or RSpl486a and L-Glu with L-Phe, L-His, L-Met, L-Cys, L-Ser, or L-Ala, the structural analysis of reaction products has not been performed. No additional phosphoric acid release above background level has been observed in reaction mixture containing RSpl486a and L-Asp or L-Glu. A newly discovered L-amino acid ligase RizB from B. subtilis NBRC3134 has been found to synthesize various heteropeptides and homo- oligomers of branched-chain amino acids consisting of 2 to 5 amino acid residues (Kino K. , Yakugaku Zasshi, 2010,

130 (11) : 1463-1469) . For example, formation of dimer, trimer, and tetramer of L-Val has been proven by LC-ESI-MS analysis in the mixture containing RizB, L-Val, and L-Glu or L-Asp. No heteropeptides have been revealed.

DISCLOSURE OF INVENTION PROBLEM TO BE SOLVED BY THE INVENTION

[0007]

To date, no data has been reported demonstrating synthesis of dipeptides having an acidic L-amino acid residue such as L-Glu or L-Asp residue at the N-terminus and any other L-amino acid or a derivative thereof at the C-terminus using an L-amino acid -ligase (Lai) .

MEANS FOR SOLVING PROBLEM

[0008]

An aspect of the present invention is to provide a DNA encoding L-amino acid a-ligase (Lai) capable of

synthesizing dipeptide(s) having an acidic L-amino acid such as L-Asp or L-Glu at the N-terminus and any other L- amino acid or a derivative thereof at the C-terminus, in a reaction mixture which contains a high-energy molecule such as adenosine 5' -triphosphate (ATP), or a salt thereof.

[0009]

Another aspect of the present invention is to provide a bacterium of the genus Escherichia, exemplary belonging to the species Escherichia coli, which has been modified to contain the DNA encoding Lai as described herein.

[0010]

Another aspect of the present invention is to provide methods for producing dipeptides having an acidic L-amino acid such as L-Asp or L-Glu at the N-terminus and any other L-amino acid or a derivative thereof at the C-terminus in a reaction mixture, which contains a high-energy molecule such as adenosine 5 ' -triphosphate, or a salt thereof, using the Lai enzyme . as described herein or a bacterium of the genus Escherichia, which has been modified to contain the DNA encoding the Lai enzyme as described herein.

[0011]

These aims were achieved by the finding novel

bacterial L-amino acid -ligases (Lais) catalyzing

formation of dipeptides having an acidic L-amino acid such as L-Asp or L-Glu at the N-terminus.

[0012]

An aspect of the present invention is to provide a DNA encoding a protein having dipeptide-synthesizing activity, wherein the DNA is selected from the group consisting of:

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

_. (B) a DNA hybridizing under stringent conditions with the nucleotide sequence complementary to the sequence shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15 and 17, wherein the stringent conditions comprise washing one time or more in a solution containing a salt concentration of 1*SSC, 0.1% SDS or O.lxSSC, 0.1% SDS at 60°C or 65°C;

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

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

(E) a DNA encoding a protein having homology, defined in I LoglO (E-value) | -values, of 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 to the amino acids sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16 and 18, and dipeptide-synthesizing activity according to the amino acid sequence of SEQ ID NOs : 2, 4, 6, 8, 10, 12, 14, 16 and 18.

[0013] .

It is an aspect of the present invention to provide a recombinant DNA for expression of the DNA as described above containing the DNA ^■ as described above.

[0014]

It is an aspect of the present invention to provide a dipeptide-producing bacterium belonging to the genus

Escherichia modified to contain the recombinant DNA as described above.

[0015]

It is a further aspect of the present invention to provide the bacterium as described above, wherein the bacterium belongs to the species Escherichia coli.

[0016]

It is a further aspect of the present invention to provide the bacterium as described above, wherein the bacterium is modified to have attenuated or inactivated one or more genes encoding proteins having peptidase activity.

[0017]

It is a further aspect of the present invention to provide the bacterium as described above, wherein the genes encoding proteins having peptidase activity are selected from the group consisting of pepA, pepB, pepD, pepE, pepP, pepQ, pepN, pepT, iadA, iaaA(ybiK) , and dapE.

[0018]

It is an aspect of the present invention to provide a protein having the dipeptide-synthesizing activity, wherein the protein is selected from the group consisting of:

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

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

(H) a protein having homology, defined in |Logl0(E- value) I -values, of 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 to the amino acids sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16 and 18, and dipeptide-synthesizing activity according to the amino acid sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14 and 16.

[0019]

It is an aspect of the present invention to provide a method for producing the protein as described above

comprising:

(a) cultivating the bacterium as described above in a culture medium to produce the protein;

(b) accumulating the protein in the bacterium or culture medium, or both; and, if necessary,

(c) collecting the protein from the bacterium or the culture medium.

[0020]

It is an aspect of the present invention to provide a method for producing a dipeptide or a salt thereof

comprising the steps of:

(a) reacting L-amino acids or L-amino acid derivatives, or salts thereof under appropriate conditions in the presence of the protein as described above;

(b) accumulating the dipeptide or a salt thereof in an appropriate solvent; and, if necessary,

(c) collecting the dipeptide or a salt thereof from the appropriate solvent. [0021]

It is an aspect of the present invention to provide a method for producing a dipeptide or a salt thereof

comprising the steps of:

(a) cultivating the bacterium as described above in a culture medium;

(b) accumulating the dipeptide in the bacterium or culture medium, or both; and, if necessary,

(c) collecting the dipeptide from the bacterium or the culture medium.

[0022]

It is a further aspect of the present invention to provide the method as described above, wherein the L-amino acids or derivatives thereof 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 a lower alkyl ester of L-phenylalanine.

[0023]

It is a further aspect of the present invention to provide the method as described above, wherein the

dipeptide is represented by the formula:

Rl - R2

wherein Rl is an acidic L-amino acid residue or a derivative of acidic L-amino acid residue, and R2 is an L- amino acid residue or a derivative of L-amino acid residue, wherein the L-amino acid residue is 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 a lower alkyl ester of L-phenylalanine residue.

[0024]

It is a further aspect of the present invention to provide the method as described above, wherein Rl is the L- aspartic acid or L-glutamic acid residue, and R2 is the L- glutamic acid, L-isoleucine, L-phenylalanine, L-tryptophan L-valine or a lower alkyl ester of L-phenylalanine residue.

[0025]

It is a further aspect of the present invention to provide the method as described above, wherein Rl is L- aspartic acid residue, and R2 is L-phenylalanine or a lower alkyl ester of L-phenylalanine residue.

[0026]

It is a further aspect of the present invention to provide the method as described above, wherein the lower alkyl ester of L-phenylalanine is methyl, ethyl or propyl ester of the L-phenylalanine.

[0027]

The present invention is described in details below.

BRIEF DESCRIPTION OF DRAWINGS

[0028]

FIG. 1 shows the scheme for the ligation reaction catalyzed by L-amino acid ligases (Lais). R _A and R _B are side-chain groups which may be of the same or different kinds. ATP means adenosine 5' -triphosphate, ADP means adenosine 5' -diphosphate, and Pi means inorganic phosphate, phosphoric acid, or a salt thereof.

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

FIG. 3 shows the activity of BBR47_51900 in ligation of canonical L-amino acids of two different kinds, wherein one kind is L-Glu. Cnt: control (L-Asp) .

FIG. 4 shows the activity of BBR47_51900 in ligation of canonical L-amino acids of two different kinds, wherein one kind is L-Asp. Cnt: control (L-Asp).

FIG. 5 shows the activity of . Staur_4851 in ligation of canonical L-amino acids of two different kinds, wherein one kind is L-Asp.

FIG. 6 shows the activity of Staur_4851 in ligation of

L-Asp and L-Phe determined by TLC analysis. 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 pg) ; 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 pg) ;

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 pg) ; 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 pg) ; 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 pg) ; 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 pg) .

FIG. 7 shows the activity of BBR47_51900 in ligation of L-Asp and L-Phe determined by LC-QTOF/MS/MS analysis.

SP: sample; ST: standard (aAsp-Phe and βΑΞρ-Phe) .

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

FIG . 9 shows the activity of BBR47_51900 in ligation of L-Glu and L-Val determined by LC-QTOF/MS/MS analysis.

SP: sample; ST: standard (aGlu-Val and yGlu-Val) .

FIG. 10 shows the alignment of BBR47_51900 and

Staur_4851 (ClustalW, outputted in PIR format) .

FIG. 11 shows the output data obtained by the

HMMsearch program using the alignment of BBR47_51900 and

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

FIG. 13 shows the alignment of BBR47_51900, Staur_4851, DES and BCE (Clustal , outputted in PIR format) .

FIG. 14 shows the output data obtained by the

HMMsearch program using the alignment of BBR47_51900,

Staur_485l, DES and BCE (first 65 hits are presented) .

FIG. 15 shows the distribution diagram of |Logl0(E- value) I -values obtained by the HMMsearch program using the alignment of BBR47_51900, Staur_4851, DES and BCE (see Figure 13) . The following hits are marked with solid arrows: 1 - BBR47_51900, 2 - DES, 3 - BCE, 4 - Staur_4851, 5 - BMY, 8 - BTH, 18 - BUR, 33 - AME, 62 - SFL.

FIG. 16-1 shows the aligned BBR47_51900, Staur_4851 and DES in the alignment of BBR47_51900, Staur_4851, DES, BCE and BMY (ClustalW, outputted in PIR format) .

FIG. 16-2 shows the aligned BCE and BMY in the

alignment of BBR47_51900, Staur_4851, DES, BCE and BMY (ClustalW, outputted in PIR format) .

FIG. 17 shows the output data obtained by the

HMMsearch program using the alignment of BBR47_51900,

Staur_4851, DES, BCE and BMY (first 65 hits are presented) .

FIG. 18-1 shows the aligned Staur_4851, BBR47_51900 and BCE in the alignment of BBR47_51900, Staur_4851, DES, BCE, BMY and BTH (ClustalW, outputted in PIR format).

FIG. 18-2 shows the aligned DES, BMY and BTH in the alignment of BBR47_51900, Staur_4851, DES, BCE, BMY and BTH (ClustalW, outputted in PIR format) .

FIG. 19 shows the output data obtained by the HMMsearch program using the alignment of BBR47_51900,

Staur_4851, DES, BCE, BMY and BTH (first 73 hits are presented) .

FIG. 20-1 shows the aligned Staur_4851 and BBR47_51900 in the alignment of BBR47_51900, Staur_4851, DES, BCE, BMY, BTH and BUR (ClustalW, outputted in PIR format).

FIG. 20-2 shows the aligned BCE and DES in the

alignment of BBR47_51900, Staur_4851, DES, BCE, BMY, BTH and BUR (ClustalW, outputted in PIR format) .

FIG. 20-3 shows the aligned BMY and BTH in the

alignment of BBR47_51900, Staur_4851, DES, BCE, BMY, BTH and BUR (ClustalW, outputted in PIR format) .

FIG. 20-4 shows the aligned BUR in the alignment of BBR47_51900, Staur_4851, DES, BCE, BMY, BTH and BUR

(ClustalW, outputted in PIR format) .

FIG. 21 shows the output data obtained by the

HMMsearch program using the alignment of BBR47_51900,

Staur_4851, DES, BCE, BMY, BTH and BUR (first 104 hits are presented) .

FIG. 22-1 shows the aligned Staur_4851 and BBR47_51900 in the alignment of BBR47_51900, Staur_4851, DES, BCE, BMY, BTH, BUR and AME (ClustalW, outputted in PIR format) .

FIG. 22-2 shows the aligned BCE and DES in the

alignment of BBR47_51900, Staur_4851, DES, BCE, BMY, BTH, BUR and AME (ClustalW, outputted in PIR format) .

FIG. 22-3 shows the aligned BMY and BTH in the

alignment of BBR47_51900, Staur_4851, DES, BCE, BMY, BTH, BUR and AME (ClustalW, outputted in PIR format) .

FIG. 22-4 shows the aligned BUR and AME in the

alignment of BBR47_51900, Staur_4851, DES, BCE, BMY, BTH, BUR and AME (ClustalW, outputted in PIR format) .

FIG. 23 shows the output data obtained by the

HMMsearch program using the alignment of BBR47_51900, Staur_4851, DES, BCE, BMY, BTH, BUR and AME (first 65 hits are presented) .

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

FIG. 24-2 shows the aligned BGE and DES in the

alignment of BBR47_51900, Staur_4851, DES, BCE, BMY, BTH, BUR, AME and SFL (ClustalW, outputted in PIR format) .

FIG. 24-3 shows the aligned BMY and BTH in the

alignment of BBR47_51900, Staur_4851, DES, BCE, BMY, BTH, BUR, AME and SFL (ClustalW, outputted in PIR format) .

FIG. 24-4 shows the aligned BUR and AME in the

alignment of BBR47_51900, Staur_4851, DES, BCE, BMY, BTH, BUR, AME and SFL (ClustalW, outputted in PIR format) .

FIG. 24-5 shows the aligned SFL in the alignment of

BBR47_51900, Staur_4851, DES, BCE, BMY, BTH, BUR, AME and SFL (ClustalW, outputted in PIR format) .

FIG. 25 shows the output data obtained by the

HMMsearch program using the alignment of BBR47_51900,

Staur_4851, DES, BCE, BMY, BTH, BUR, AME and SFL (first 38 hits are presented) .

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

FIG. 27 shows the TLC-analysis of the specific

aspartic peptide-hydrolyzing ( DP3-hydrolyzing) activity in the E. coli 4-5Δ strains. A solution (1 μL) of the 5- fluorotryptophan (standard) was used for calibration: (1) 3 mM, (2) 2 mM, (3) 1 mM, and (4) 0.5 mM. An aliquot (1 L) of reaction mixture .containing Mn ²⁺ (5, 6) or Zn ²⁺ (7, 8) was loaded onto TLC-plate. Abbreviations: 5FT - 5- fluorotryptophan, DP3 - L-Asp-L-5-fluorotryptophane

dipeptide, Asp - L-Aspartate. FIG. 28 shows the scheme for investigation of the DP3 toxicity due to the specific aspartic peptide-hydrolyzing activity in the E. coli 1-5Δ strains. The E. coli strain is grown in the presence of DP3 dipeptide. Being accepted by the peptidase plus strain (E. coli P ⁺ ) , DP3 is hydrolyzed resulting in formation of L-aspartate and 5- fluorotryptophane (5FT) . The 5FT is toxic for cell thus resulting in growth arrest. The DP3 dipeptide is stable and does not affect cell growth in a peptidase-deficient strain or in the -strain with low peptidase activity {E. coli P ^" ) .

BEST MODE(S) FOR CARRYING OUT THE INVENTION

[0029]

1. Enzyme

The phrase "an enzyme" can mean an L-amino acid - ligase (Lai) having activity of joining amino acids in a high-energy molecule-dependent manner to form the peptide bond between amino acid residues.

[0030]

The enzyme of the present invention can be an L-amino acid a-ligase selected from the group consisting of

BBR47_51900 (a hypothetical protein), Staur_4851

(argininosuccinate lyase 2-like protein) , DES (pyridoxal- phosphate dependent enzyme) , BUR (putative lyase) , BCE

(argininosuccinate lyase domain protein) , BTH (hypothetical protein YBT020_25570 ) , AME (conserved hypothetical protein), SFL (protein of unknown function DUF201), and BMY

(argininosuccinate lyase domain protein) , which is not limited to the aforementioned proteins.

[0031]

The nucleotide sequence of the gene (NCBI Reference Sequence: YP_002774671.1; nucleotide positions: 5464162 to 5465418, complement; Gene ID: 7721040) from Brevibacillus brevis NBRC 100599 (NCBI Taxonomy ID: 358681) and the amino acid sequence of BBR47_51900 encoded by the gene are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

The nucleotide sequence of the gene (NCBI Reference Sequence: AD072629.1; nucleotide positions: 5973963 to 5975216, complement; Gene ID: 9878344) from Stigmatella aurantiaca DW4/3-1 (NCBI Taxonomy ID: 378806) and the amino acid sequence of Staur_4851 encoded by the gene are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

The nucleotide sequence of the gene (GenBank accession

No. EGK06810.1, GI : 332967701) from Desmospora sp. 8437 (NCBI Taxonomy ID: 997346) and the amino acid sequence of DES encoded by the gene are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

The nucleotide sequence of the gene (GenBank accession

No. EBA51208.1, GI : 134251129) from Burkholderia

pseudomallei 305 (NCBI Taxonomy ID: 425067) and the amino acid sequence of BUR encoded by the gene are shown in SEQ ID NO: 7 and SEQ ID NO:. 8, respectively.

The nucleotide sequence of the gene (GenBank accession

No. EEK72190.1, GI : 228615090) from Bacillus cereus AH621 (NCBI Taxonomy ID: 526972) and the amino acid sequence of BCE encoded by the gene are shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively.

The nucleotide sequence of the gene (GenBank accession

No. ADY24341.1, GI : 324329081) from Bacillus thuringiensis subsp. finitimus (strain YBT-020) (NCBI Taxonomy ID:

930170) and the amino acid sequence of BTH encoded by the gene are shown in SEQ ID NO: 11 and SEQ ID NO: 12,

respectively.

The nucleotide sequence of the gene (GenBank accession No. ABR48216.1, GI: 149949688) from Alkaliphilus

metalliredigens QYMF (NCBI Taxonomy ID: 293826) and the amino acid sequence of AME encoded by the gene are shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively.

The nucleotide sequence of the gene (GenBank accession No. ADW01942.1, GI : 320007092) from Streptomyces

flavogriseus ATCC 33331 (NCBI Taxonomy ID: 591167) and the amino acid sequence of SFL encoded by the gene are shown in SEQ ID NO: 15 and SEQ ID NO: 16, respectively.

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

[0032]

Since there may be some differences in DNA sequences between the genera or species and strains of said genera, the genes encoding BBR47_51900, Staur_4851, DES, BUR, BCE, BTH, AME, SFL and BMY are not limited to the genes shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, or 17 but may include genes which are variant nucleotide sequences of or homologous to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, or 17, and which encode variants of the BBR47_51900, Staur_4851, DES, BUR, BCE, BTH, AME, SFL and BMY proteins. Moreover, the genes encoding BBR47_51900, Staur_4851, DES, BUR, BCE, BTH, AME, SFL and BMY can be variant nucleotide sequences.

[0033]

The phrase "a variant nucleotide sequence" can mean a nucleotide sequence which encodes "a variant protein".

The phrase "a variant nucleotide sequence" can mean a nucleotide sequence which encodes "a variant protein" using any synonymous amino acid codons according to the standard genetic code table (see, for example, Lewin B., Genes VIII, 2004, Pearson Education, Inc., Upper Saddle River, NJ

07458) . [0034]

The phrase "a variant nucleotide sequence" can also mean a nucleotide sequence which hybridizes under stringent conditions with the nucleotide sequence complementary to the sequence shown in SEQ ID NOs : 1, 3, 5, 7, 9, 11, 13, 15, or 17, or a probe which can be prepared from the nucleotide sequence under stringent conditions provided that it

encodes functional L-amino acid a-ligase. "Stringent

conditions" include those under which a specific hybrid, for example, a hybrid having similarity of 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% is formed, and a non-specific hybrid, for example, a hybrid having homology lower than the above is not formed. For example, stringent conditions can be exemplified by washing one time or more, or in another example, two or three times, at a salt concentration of 1*SSC (standard sodium citrate or standard sodium chloride) and 0.1% SDS (sodium dodecyl sulfate), or in another example, O.lxSSC and 0.1% SDS, at 60°C or 65°C. Duration of washing depends on the type of membrane used for blotting and, as a rule, should be what is recommended by the manufacturer. For example, the recommended duration of washing for the

Amersham Hybond™-N+ positively charged nylon membrane (GE Healthcare) under stringent conditions is 15 minutes. The washing step can be performed 2 to 3 times. As the probe, a part of the sequence complementary to the sequences shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, or 17 may also be used. Such a probe can be produced by PCR using

oligonucleotides as primers prepared on the basis of the sequences shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, or 17, and a DNA fragment containing the nucleotide

sequence as a template. The length of the probe is recommended to be >50 bp; it can be suitably selected depending on the hybridization conditions, and is usually 100 bp to 1 kbp. For example, when a DNA fragment having a length of about 300 bp is used as the probe, the washing conditions after hybridization can be exemplified by 2>SSC and 0.1% SDS at 50°C, 60°C or 65°C. Alternatively, the stringent condition may be hybridization in 6xSCC at about 45°C followed by one or two or more washings in 0.2xSCC and 0.1% SDS at 50 to 65°C.

[0035]

As the genes encoding the BBR47_51900, Staur_4851, DES, BUR, BCE, BTH, AME, SFL and BMY proteins have already been elucidated (see above) , the variant nucleotide sequences encoding variant proteins of the BBR47_51900, Staur_4851, DES, BUR, BCE, BTH, AME, SFL and BMY proteins can be obtained by PCR (polymerase chain reaction; refer to White T.J. et al., Trends Genet., 1989, 5:185-189) utilizing primers prepared based on the nucleotide sequence of the genes encoding BBR47_51900, Staur_4851, DES, BUR, BCE, BTH, AME, SFL and BMY. Genes encoding the BBR47_51900,

Staur_4851, DES, BUR, BCE, BTH, AME, SFL and BMY proteins or their variant proteins of other microorganisms can be obtained in a similar manner.

[0036]

The phrase "a variant protein" can mean a protein which has one or several changes in the sequence compared with SEQ ID NOs : 2, 4, 6, 8, 10, 12, 14, 16, and 18, whether they are substitutions, deletions, insertions, and/or additions of amino acid residues, but still

maintains an activity similar to that of the BBR47_51900, Staur_4851, DES, BUR, BCE, BTH, AME, SFL and BMY proteins, respectively, or the three-dimensional structure of the BBR47_51900, Staur_4851, DES, BUR, BCE, BTH, AME, SFL and BMY proteins is not significantly changed relative to the wild-type or non-modified proteins. The number of changes in the variant protein depends on the position or the type of amino acid residues in the three dimensional structure of the protein. It can be, but is not strictly limited to, 1 to 45, or 1 to 30, or 1 to 15, or 1 to 10, or 1 to 5, in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16 and 18.

[0037]

The exemplary substitution, deletion, insertion, and/or addition of one or several amino acid residues can be a conservative mutation (s) so that the activity and features of the variant protein are maintained, and are similar to those of the BBR47_51900, Staur_4851, DES, BUR, BCE, BTH, AME, SFL and BMY proteins. The representative conservative mutation is a conservative substitution. The conservative substitution can.be a substitution, wherein substitution takes place mutually among Phe, Trp and Tyr, if the substitution site is an aromatic amino acid; among Ala, Leu, lie and Val, if the substitution site is a hydrophobic amino acid; between Glu, Asp, Gin, Asn, Ser, His and Thr, if the substitution site is a hydrophilic amino acid; between Gin and Asn, if the substitution site is a polar amino acid; among 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 hydroxyl group. Examples of conservative substitutions include substitution of Ser or Thr for Ala, substitution of Gin, His or Lys for Arg, substitution of Glu, Gin, Lys, His or Asp for Asn, substitution Asn, Glu o. Gin for Asp, substitution of Ser or Ala for Cys,

substitution Asn, Glu, Lys, His, Asp or Arg for Gin, substitution Asn, Gin, Lys or Asp for Glu, substitution of Pro for Gly, substitution Asn, Lys, Gin, Arg or Tyr for His, substitution of Leu, Met, Val or Phe for lie, substitution of lie, Met, Val or Phe for Leu, substitution Asn, Glu, Gin, His or Arg for Lys, substitution of lie, Leu, Val or Phe for Met, substitution of Trp, Tyr, Met, lie or Leu for Phe, substitution of Thr or Ala for Ser, substitution of Ser or Ala for Thr, substitution of Phe or Tyr for Trp,

substitution of His, Phe or Trp for Tyr, and substitution of Met, lie or Leu for Val. These changes in the variant protein can occur in regions of the protein which are not critical for the function of the protein. This is because some amino acids have high homology to one another so that the three dimensional structure or activity is not affected by such a change. Therefore, the protein variants encoded by the genes shown in SEQ ID NOs : 1, 3, 5, 7, 9, 11, 13, 15, or 17 may have a similarity or identity of not less than 40%, not less than 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 entire amino acid sequences shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16 and 18 as long as the functionality of the BBR47_51900, Staur_4851, DES, BUR, BCE, BTH, AME, SFL and BMY proteins, respectively, is maintained. Alternatively, the protein variants encoded by the genes shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, or 17 may have a homology, which can be defined using the | LoglO (E-value ) | -values calculated by the HMMsearch program when the profile hidden Markov model

(profile HMM) based on the aforementioned program is

originated (Finn R.D. 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, of 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, with respect to the entire amino acid sequences shown in SEQ ID NOs : 2, 4, 6, 8, 10, 12, 14, 16 and 18 as long as the functionality of the BBR47_51900,

Staur_4851, DES, BUR, BCE, BTH, AME, SFL and BMY proteins, respectively, is maintained.

[0038]

The exemplary substitution, deletion, insertion, and/or addition of one or several amino acid residues can also be a non-conservative mutation (s) provided that the mutation (s) is/are compensated for by one or more secondary mutations in the different position (s) of amino acids sequence so that the activity and features of the variant protein are maintained, and are similar to those of the

BBR47_51900, Staur_4851, DES, BUR, BCE, BTH, AME, SFL and BMY proteins.

[0039]

To evaluate the degree of protein or DNA homology, several calculation methods can be used, such as BLAST search, FASTA search and Clustal method. The BLAST (Basic Local Alignment Search Tool, www.ncbi.nlm.nih.gov/BLAST/) search is the heuristic search algorithm employed by the programs blastp, blastn, blastx, megablast, tblastn, and tblastx; these programs ascribe significance to their findings using the statistical methods of 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 computer program BLAST calculates three parameters: score, identity and similarity. The FASTA search method is described by Pearson W.R. ("Rapid and sensitive sequence comparison with FASTP and FASTA", Methods Enzymol., 1990, 183:63-98). The

ClustalW method is described by 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).

[0040]

The phrase "activity of an L-amino acids -ligase (Lai)" can mean the activity of an enzyme catalyzing reaction of joining amino acids in a high-energy molecule- dependent manner to form the peptide bond between amino acid residues. Dipeptide, tripeptide or peptide of linear or branched structure consisting of more than three amino acids residues, or derivatives thereof may be the product of the reaction catalyzed by Lai. The reaction scheme for the Lal-catalyzed reaction may be described as shown in Figure 1 without limiting to the kind of amino acids or derivatives thereof and reaction conditions used in the following non-limiting Examples. The activity of Lai can be measured, for example, by the assay described in the

Example 3 or Tabata K. et al., J. Bacterid., 2005,

187 (15) : 5195-5202. The phrase "activity of L-amino acids a- ligase (Lai)" can be equivalent, in particular, to the phrase "dipeptide-synthesizing activity".

[0041]

Furthermore, when an amino acid sequence that contains a substitution, deletion, insertion, and/or addition of one or several amino acid residues in the amino acid sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16 and 18., it can retain activity of L-amino acids a-ligase by 10% or more, by 30% or more, by 50% or more, by 70% or more, and by 90% or more of a protein having the amino acid sequence of SEQ ID NOs: 2, 4/ 6, 8, 10, 12, 14, 16 and 18.

[0042]

The phrase "an isofunctional protein" can mean the protein having the activity of an L-amino acids a-ligase (Lai) as described above. Exemplary, the isofunctional protein can synthesize dipeptide having an acidic L-amino acid residue such as L-Glu or L-Asp residue at the N- terminus and any other L-amino acid or a derivative thereof at the C-terminus.

[0043]

2. Bacterium

The phrase "a dipeptide-producing bacterium" can mean a bacterium of the family Enterobacteriaceae such as a bacterium belonging to the genus Escherichia, which has an ability to produce and cause accumulation of a dipeptide in a culture medium when the bacterium is cultured in the medium. The dipeptide-producing ability can mean the ability of the bacterium to produce a dipeptide in a medium or the bacterial cells and cause accumulation of the dipeptide to such an extent that the dipeptide can be collected from the medium or the bacterial cells when the bacterium is cultured in the medium.

[0044]

The bacterium may inherently have the dipeptide- producing ability or may be modified to have a dipeptide- producing ability by using mutation methods or DNA

recombination techniques.

[0045]

The bacteria belonging to the family

Enterobacteriaceae can be from the genera Enterobacter, Erwinia, Escherichia, Klebsiella, Morganella, Pantoea, Photorhabdus, Providencia, Salmonella, Yersinia, and so forth, and can have the ability to produce a dipeptide.

Specifically, those classified into the family

Enterobacteriaceae according to the taxonomy used in the NCBI (National ^' Center for Biotechnology Information)

database

(www. ncbi . nlm. nih. gov/Taxonomy/Browser/wwwtax. cgi?id=543) can be used. Examples of strains from the family

Enterobacteriaceae which can be modified include a

bacterium of the genus Escherichia , Enterobacter or Pantoea.

[0046]

Strains of Escherichia bacterium which can be modified to obtain Escherichia bacteria in accordance with the presently disclosed subject matter are not particularly limited, and specifically, those described in the work of Neidhardt et al. can be used (Bachmann, B.J., Derivations and genotypes of some mutant derivatives of E. coli K-12, p. 2460-2488. In F.C. Neidhardt et al. (ed. ) , E. coli and

Salmonella: cellular and molecular biology, 2 ^nd ed. ASM

Press, Washington, D.C., 1996). The species E. coli is a particular example. Specific examples of E. coli include E. coli W3110 (ATCC 27325), E. coli MG1655 (ATCC 47076), and so forth, which are derived from the prototype wild-type strain, K-12 strain. These strains are available from, for example, the American Type Culture Collection (P.O. Box 1549, Manassas, VA 20108, United States of America) . That is, registration numbers are given to each of the strains, and the strains can be ordered by using these registration numbers (refer to www.atcc.org). The registration numbers of the strains are listed in the catalogue of the American Type Culture Collection.

[0047]

Examples of the Enterobacter bacteria include

Enterobacter agglomerans, Enterobacter aerogenes, and so forth. Examples, of the Pantoea bacteria include Pantoea ananatis, and so forth. Some strains of Enterobacter agglomerans were recently reclassified into Pantoea

agglomerans, Pantoea ananatis or Pantoea stewartii on the basis of nucleotide sequence analysis of 16S rRNA, etc.. A bacterium belonging to any of the genus Enterobacter or Pantoea may be used so long as it is a bacterium classified into the family Enterobacteriaceae . When a Pantoea ananatis strain is bred by genetic engineering techniques, Pantoea ananatis AJ13355 strain (FERM BP-6614), AJ13356 strain (FERM BP-6615), AJ13601 strain (FERM BP-7207) and

derivatives thereof can be used. These strains were

identified as Enterobacter agglomerans when they were isolated, and deposited as Enterobacter agglomerans .

However, they were recently re-classified as Pantoea ananatis on the basis of nucleotide sequencing of 16S rRNA and so forth as described above.

[0048]

The dipeptide-producing bacterium as described herein can be modified to have attenuated or inactivated one or more genes of one or more kinds encoding protein (s) having peptidase, or proteolytic activity so that the activity of peptidase (s) is decreased. For example, one or more

proteases encoding genes such as pepA (KEGG, Kyoto

Encyclopedia of Genes and Genomes, entry No. b4260), pepB

(KEGG, entry No. b2523) , pepD (KEGG, entry No. b0237), pepE (KEGG, entry No. b4021), pepP (KEGG, entry No. b2908), pepQ (KEGG, entry No. b3847), pepN (KEGG, entry No. b0932), pepT (GenBank accession No. AAC74211), iadA (KEGG, entry No. b4328), iaaA(ybiK) (KEGG, entry No. b0828), dapE (KEGG, entry No. b2472), and so forth can be attenuated and/or inactivated.

[0049] The dipeptide-producing bacterium as described herein can be also modified to have attenuated or inactivated one or more genes of one or more kinds encoding protein (s) having dipeptide permease (dpp) activity so that the activity of peptide permease (s) is decreased. For example, one or more dipeptide permeases encoding genes such as dppA (KEGG, entry No. b3544), dppB (KEGG, entry No. b3543), dppC (KEGG, entry No. b3542), dppD (KEGG, entry No . b3541), dppF (KEGG, entry No. b3540), and so forth can be attenuated and/or inactivated. Deletion of the entire dpp gene operon (dppA, dppB, dppC, dppD and dppF) may be also preferred in the dipeptide-producing bacterium.

[0050]

The dipeptide-producing bacterium as described herein can be also modified to have attenuated or inactivated one or more genes of one or more kinds encoding protein (s) involved in biosynthesis of aromatic amino acids so that the activity of the protein (s) is decreased. For example, one or more proteins encoding genes such as tyrR (KEGG, entry No. bl323), tryA (KEGG, entry No. b2600), and so forth can be attenuated and/or inactivated.

[0051]

The phrase "an attenuated gene encoding peptidase" or "an attenuated gene encoding protein" is equivalent to the phrase "a peptidase encoding gene with attenuated

expression" or "a protein encoding gene with attenuated expression", respectively. Hereinafter, the term

"peptidase" may be replaced with "protein" as recited above (e.g., protein(s) having dipeptide permease (dpp) activity, or protein (s) involved in biosynthesis of aromatic amino acids) for interpreting the phrase "an attenuated gene encoding protein" or the like. Therefore, such replaced phases may be recited as elements for specifying the present invention.

[0052]

The phrase "a peptidase encoding gene with attenuated expression" can mean that an amount of a peptidase in the modified bacterium, in which expression of the peptidase encoding gene is attenuated, is reduced as compared with a non-modified bacterium, for example, a wild-type strain of the bacterium belonging to the family Enterobacteriaceae, or more specifically, genus Escherichia such as the E. coli K-12 strain.

[0053]

The phrase "a peptidase encoding gene with attenuated expression" can also mean that the modified bacterium includes a modified gene, which encodes a mutant protein having decreased activity as compared with the wild-type protein, or a region operably linked to the gene, including sequences controlling gene expression such as promoters, enhancers, attenuators, ribosome-binding sites (RBS) ,

Shine-Dalgarno (SD) sequences, etc., is modified resulting in a decrease in the expression level of the peptidase encoding gene, and other examples (see, for example,

095/34672; Carrier T.A. and Keasling J.D., Biotechnol. Prog. , 1999, 15: 58-64) .

[0054]

Expression of the peptidase encoding gene can be attenuated by replacing an expression control sequence of the gene, such as a promoter on the chromosomal DNA, with a weaker one. The strength of a promoter is defined by the frequency of initiation acts of RNA synthesis. Examples of methods for evaluating the strength of promoters and strong promoters are described in Goldstein et al. (Prokaryotic promoters in biotechnology, Biotechnol. Annu. Rev., 1995, 1:105-128), and so forth. Furthermore, it is also possible to introduce nucleotide substitution for several

nucleotides in a promoter region of a target gene and thereby modify the promoter to be weakened as disclosed in International Patent Publication WO00/18935. Furthermore, it is known that substitution of several nucleotides in the spacer between the SD sequence and the start codon in the RBS, in particular, a sequence immediately upstream from the start codon, greatly affects the translation efficiency of mRNA. This modification of the RBS may be combined with decreasing transcription of a peptidase encoding gene.

[0055]

Expression of the peptidase encoding gene can also be attenuated by insertion of a transposon or an IS factor into the coding region of the gene" ^' (U.S. Patent No.

5,175,107) or by conventional methods, such as mutagenesis with ultraviolet irradiation (UV) irradiation or

nitrosoguanidine (N-methyl-N' -nitro-N-nitrosoguanidine) .

Furthermore, the incorporation of a site-specific mutation by gene substitution using homologous recombination such as set forth above can also be conducted with a plasmid which is unable to replicate in the host.

[0056]

The phrase "enzymatic activity is decreased" can mean that the enzymatic activity of a peptidase is lower than that in a non-modified strain, for example, a wild-type strain of the bacterium belonging to the family

Enterobacteriaceae, or more specifically, genus Escherichia. Exemplary, the enzymatic activity of the peptidase encoding gene can be abolished by the gene inactivation .

' [0057]

The phrase "the activity of peptidase is decreased" can also mean that the peptide degrading activity is

decreased compared with a wild-type peptidase encoded by the wild-type gene such as pepA, pepB, pepD, pepE, pepP, pepQ, pepN, pepT, iadA, iaaA(ybiK) , dapE, and so forth.

[0058]

In the modified bacterium, the activity of peptidase can be decreased by at least 10% or more, by at least 30% or more, by at least 50% or more, by at least 70% or more, by at least 90% or more as compared with a peptidase

encoded by a wild-type gene in a non-modified bacterium belonging to the family Enterobacteriaceae, more

specifically to the genus Escherichia .

[0059]

The phrase "peptidase activity" or "proteolytic

activity" can mean the activity of an enzyme catalyzing reaction of intramolecular digestion of the peptide bond (R. Beynon (ed.) and J.S. Bond (ed.), "Proteolytic Enzymes: A Practical Approach", 2 ^nd ed., Oxford University Press, USA (2001) ) .

[0060]

The peptide degrading activity of a microorganism can be measured by allowing a peptide as a substrate and

microorganism cells to be present in a medium, thereby performing peptide degrading reaction, and then determining the amount of the remaining peptide by a known method, for example, HPLC analysis, or as described in Kristjansson M.M., Activity measurements of proteinases using synthetic substrates (UNIT C2.1) or Akpinar 0. and Penner M.H.,

Peptidase activity assays using protein substrates (UNIT C2.2) in Current Protocols in Food Analytical Chemistry (UNIT C2, Proteolytic Enzymes), John Wiley & Sons, Inc.

(2002) .

[0061]

The enzymatic activity of a peptidase can be decreased by introducing a mutation into the chromosome so that intracellular activity of the peptidase is decreased as compared with a non-modified strain. Such a mutation on the gene(s) or upstream the genes in the operon structure can be the replacement of one base or more to cause an amino. acid substitution in the protein encoded by the gene(s)

(missense mutation) , introduction of a stop codon (nonsense mutation) , deletion of one or two bases to cause a frame shift, insertion of a drug-resistance gene and/or

transcription termination signal, deletion of a part of the gene(s) or deletion of the entire gene(s) (Qiu Z. and

Goodman M.F., J. Biol. Chem. , 1997, 272:8611-8617; Kwon D.H. et a-1., J. Antimicrob. Chemother., 2000, 46:793-796).

[0062]

The phrase "an inactivated gene encoding peptidase" can mean that the modified gene encodes a completely

inactive or non-functional peptidase. It is also possible that the modified DNA region is unable to naturally express the gene due to deletion of a part of or the entire gene, shifting of the reading frame of the gene, introduction of missense/nonsense mutation (s), or modification of an

adjacent region of the gene, including sequences

controlling gene expression, such as promoter (s),

enhancer (s), attenuator ( s ) , ribosome-binding site(s), etc. Inactivation of the gene can also be performed by

conventional methods such as a mutagenesis treatment using UV irradiation or nitrosoguanidine (N-methyl-N' -nitro-N- nitrosoguanidine ) , site-directed mutagenesis, gene

disruption using homologous recombination, or/and

insertion-deletion mutagenesis (Yu D. et al., Proc. Natl. Acad. Sci. USA, 2000, 97 ( 12 ): 5978-83 ; Datsenko K.A. and

Wanner B.L., Proc. Natl. Acad. Sci. USA, 2000, 97(12): 6640- 45) , also called "Red-driven integration" or "ARed-mediated integration" . [0063]

The phrase "a bacterium modified to contain the

recombinant DNA" can mean the bacterium modified to contain an exogenous DNA by, for example, conventional methods such as, for example, transformation, transfection, infection, conjugation, and mobilization. Transformation, transfection, infection, conjugation or mobilization of a bacterium with the DNA encoding a protein can impart the bacterium an ability to synthesize the protein encoded by the DNA.

Methods of transformation, transfection, infection,

conjugation and mobilization include any known methods that have been reported. For example, a method of treating recipient cells with calcium chloride so as to increase permeability of the cells of Escherichia coli K-12 to DNA has been reported for efficient DNA transformation and transfection (Mandel M. and Higa A. , Calcium-dependent bacteriophage DNA infection, J. Mol. Biol., 1970, 53:159- 162). Methods of specialized and/or generalized

transduction are described (Morse M.L. et al., Transduction in Escherichia coli K-12, Genetics, 1956, 41 (1 ): 142-156;

Miller J.H., Experiments in Molecular Genetics. Cold Spring Harbor, N.Y.: Cold Spring Harbor La. Press, 1972). Other methods for random and/or targeted integration of DNA into the host genome can be applied, for example, "Mu-driven integration/amplification" (Akhverdyan et al., Appl .

Microbiol. Biotechnol. , 2011, 91:857-871), "Red/ET-driven integration" or "ARed/ET-mediated integration" (Datsenko K. A. and Wanner B.L., 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-driven replicative transposition (Akhverdyan et al., Appl. Microbiol. Biotechnol., 2011, 91:857-871) and chemically inducible chromosomal evolution based on recA-dependent homologous recombination resulted in an amplification of desired genes (Tyo K.E.J, et al., Nature Biotechnol., 2009, 27 : 760-765) , another methods can be used, which utilize different combinations of

transposition, site-specific and/or homologous Red/ET- mediated recombinations, and/or Pl-mediated generalized transduction (see, for example, Minaeva et al., BMC

Biotechnology, 2008, 8:63; Koma D. et al., Appl . Microbiol. Biotechnol. , 2012, 93 (2 ) : 815-829 ) .

[0064]

The bacterium of the present invention can be modified further in such a way that expression level of a gene encoding L-amino acid -ligase (Lai) or one or more genes encoding one or more proteins involved in biosynthesis of phenylalanine are enhanced. Examples of such a protein include pheA, aroG4 and aroL encoding chorismate mutase- prephenate dehydratase (CM-PD) , 3-deoxy-D- arabinoheptulosonate-7-phosphate synthetase (DAHP

synthetase) and shikimate kinase (SK) , respectively (see, e.g., Japanese Patent No. 3225597). Hereinafter, the term "Lai" may be replaced with the protein involved in

biosynthesis of phenylalanine, for interpreting the phrase "a gene encoding proteins involved in biosynthesis of phenylalanine" or the like. Therefore, such replaced phases may be recited as elements for specifying the present invention.

[0065]

The phrase "enhanced expression of a gene encoding Lai" can mean that the number of the molecules encoded by the Lal-encoding gene per cell is increased, or the

activity per molecule (may be referred to as a specific activity) of the protein encoded by these gene improved, as compared with a non-modified strain such as a wild-type or a parent strain. Examples of a non-modified strain serving as a reference for the above comparison include a wild-type strain of a microorganism belonging to the family

Enterobacteriaceae such as the E. coli G1655 strain (ATCC 47076), 3110 strain (ATCC 27325), Pantoea ananatis AJ13335 strain (FERM BP-6614), and so forth.

[0066]

The phrase "enhanced expression of a gene encoding Lai" can also mean that the expression level of the Lal- encoding gene is higher than that level in a non-modified strain, for example, a wild-type or parent strain.

[0067]

Methods which can be used to enhance expression of the Lal-encoding gene include, but are not limited to,

increasing the Lal-encoding gene copy number in bacterial genome (in the chromosome and/or in the autonomously replicated plasmid) and/or introducing the Lal-encoding gene into a vector that is able to increase the copy number and/or the expression level of the Lal-encoding gene in a bacterium of the genus Escherichia according to genetic engineering methods known to the one skilled in the art.

[0068]

Examples of the vectors include, but are not limited to broad-host-range vectors such as pCMHO, pRK310, pVKlOl, pBBRl22, pBHRl, and the like. Multiple copies of the Lal- encoding gene can also be introduced into the chromosomal DNA of a bacterium by, for example, homologous

recombination, Mu-driven integration, or the like.

Homologous recombination can be carried out using a

sequence multiple copies in the chromosomal DNA. Sequences with multiple copies in the chromosomal DNA include, but are not limited to repetitive DNA or inverted repeats present at the end of a transposable element. In addition, it is possible to incorporate the Lal-encoding gene into a transposon and allow it to be transferred to introduce multiple copies of the Lal-encoding gene into the

chromosomal DNA. By using Mu-driven integration, more than 3 copies of the gene can be introduced into the chromosomal DNA during a single act (Akhverdyan V.Z. et al., Biotechnol. (Russian), 2007, 3:3-20).

[0069]

Enhancing of the Lal-encoding gene expression can also be -achieved by increasing the expression level of the Lal- encoding gene by modification of adjacent regulatory

regions of the Lal-encoding gene or introducing native and/or modified foreign regulatory regions. Regulatory regions or sequences can be exemplified by promoters, enhancers, attenuators and transcription termination

signals, anti-termination signals, ribosome-binding sites (RBS) and other expression control elements (e.g., regions to which repressors or inducers bind and/or binding sites for transcriptional and translational regulatory proteins, for example, in the transcribed mRNA) . Such regulatory sequences are described, for example, in Sambrook J. ,

Fritsch E.F. and Maniatis T., "Molecular Cloning: A

Laboratory Manual", 2 ^nd ed., Cold Spring Harbor Laboratory Press (1989). Modifications of regions controlling gene(s) expression can be combined with increasing the copy number of the modified gene(s) in bacterial genome using the known methods (see, for example, Akhverdyan V.Z. et al . , Appl .

Microbiol. Biotechnol. , 2011, 91:857-871; Tyo K.E.J, et al., Nature Biotechnol. , 2009, 27:760-765).

[0070]

The exemplary promoters enhancing the Lal-encoding gene expression can be the potent promoters. For example, the lac promoter, the trp promoter, the trc promoter, the tac promoter, the P _R or the P _L promoters of lambda phage are all known to be potent promoters. Potent promoters providing a high level of gene expression in a bacterium belonging to the family Enterobacteriaceae can be used.

Alternatively, the effect of a promoter can be enhanced by, for example, introducing a mutation into the promoter region of the Lal-encoding gene to obtain a stronger

promoter function, thus resulting in the increased

transcription level of the Lal-encoding gene located

downstream of the promoter. Furthermore, it is known that substitution of several nucleotides in the ribosome binding site (RBS) , especially the sequences immediately upstream of the start codon, profoundly affect the mRNA

translatability. For example, a 20-fold range in the

expression levels 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., EMBO J. , 1984: 3, 623-629).

[0071]

Enhancing of the Lal-encoding gene heterologous

expression in host microorganisms can also be achieved by substituting rare and/or low-usage codons for synonymous middle- or high-usage codons, where codon usage can be defined as the number of times (frequency) a codon is translated per unit time in the cell of an organism or an average codon frequency of the sequenced protein-coding reading frames of an organism (Zhang S.P. et al . , Gene, 1991, 105 (1) : 61-72) . The codon usage per organism 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 can include, without limiting, the substitution of rare Arg codons AGA, AGG, CGG, CGA for CGT or CGC; rare lie codon ATA for ATC or ATT; rare Leu codon CTA for CTG, CTC, CTT, TTA or TTG; rare Pro codon CCC for CCG or CCA; rare Ser codon TCG for TCT, TCA, TCC, AGC or AGT; rare Gly codons GGA, GGG for GGT or GGC; and so forth. The substitution of low-usage codons for synonymous high-usage codons can be preferable. The substituting rare and/or low-usage codons for synonymous middle- or high-usage codons may be combined with co-expression of the genes which encode rare tRNAs recognizing rare codons.

[0072]

The copy number, presence or absence of the gene and/or operon genes can be measured, for example, by restricting the chromosomal DNA followed by Southern blotting using a probe based on the gene sequence,

fluorescence in situ hybridization (FISH), and the like. The level of the gene and/or operon gene's expression can be measured by various known methods including Northern blotting, quantitative RT-PCR, and the like. In addition, the level of gene expression can be determined by measuring the amount of mRNA transcribed from the gene using various well-known methods, including Northern blotting,

quantitative RT-PCR, and the like. The amount of the protein coded by the gene can be measured by known methods including SDS-PAGE followed by immunoblotting assay

(Western blotting analysis) , or mass spectrometry analysis of the protein samples, and the like.

[0073]

Methods for preparation of plasmid DNA, digestion, ligation and transformation of DNA, selection of an

oligonucleotide as a primer, and the like may be ordinary methods well-known to the one skilled in the art. These methods are described, for instance, in Sambrook J. ,

Fritsch E.F. and Maniatis T., "Molecular Cloning: A

Laboratory Manual", 2 ^nd ed. , Cold Spring Harbor Laboratory Press (1989) or Green M.R. and Sambrook J.R., "Molecular Cloning: A Laboratory Manual", 4 ^th ed., Cold Spring Harbor Laboratory Press (2012). Methods for molecular cloning and heterologous gene expression 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, D.C: ASM Press (2009); Evans Jr., T.C. and Xu M.-Q., "Heterologous gene expression in E.

coli" , 1 ^st ed., Humana Press (2011).

[0074]

The phrase "operably linked to a gene" can mean that the regulatory sequence (s) is linked to the nucleotide sequence of the nucleic acid molecule or gene of interest in a manner which allows for expression (e.g., enhanced, increased, constitutive, basal, attenuated, decreased or repressed expression) of the nucleotide sequence,

preferably expression of a gene product encoded by the nucleotide sequence.

[0075]

The bacterium as described herein can be obtained by imparting the required properties to a bacterium inherently having the ability to produce a dipeptide. Alternatively, the bacterium can be obtained by imparting the ability to produce a dipeptide to a bacterium which already has the required properties.

[0076]

The bacterium can have, in addition to the properties already mentioned, other specific properties such as

various nutrient requirements, drug resistance, drug sensitivity, and drug dependence, without departing from the scope of the present invention.

[0077]

3. Methods for producing dipeptides

The methods of the present invention can be the methods for producing a dipeptide, more specifically a dipeptide having an acidic L-amino acid at the N-terminus, using an L-amino acid a-ligase (Lai) or a bacterium

belonging to the family Enterobacteriaceae modified to contain said Lai, therefore, referred to as a -enzymatic method and a fermentative method, respectively.

[0078]

The phrase "an amino acid" can mean an ordinal amino acid known to the one skilled in the art, a derivative of amino acid, or salts thereof. The exemplary amino acids can be a-amino acids and β-amino acids having C ^a or C ^p chiral carbon atom respectively, to which the amino group, carboxy group, and side-chain group are attached. The β-amino acids can be exemplified by pAla. The a-amino acids can be exemplified by proteinogenic and non-proteinogenic amino acids. Proteinogenic amino acids can be exemplified by 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 salts thereof, having C" chiral carbon atom. The amino acids can be used in unprotected or protected form. The protected form of amino acid can mean, contrary to the unprotected form, an amino acid having one or more substituents

attached to amino group, carboxy group, and/or side-chain group. The amino acid having substituents ( s) attached can be referred to as an amino acid derivative. The amino acid derivative can be exemplified by a lower alkyl ester of amino acid such as a lower alkyl ester of L-phenylalanine . As lower alkyl ester, methyl ester, ethyl ester, and propyl ester, or the like can be mentioned.

[0079]

The phrase "an amino acid" can be equivalent to the phrase "a substrate of the Lal-catalyzed reaction" or "a substrate" for the reasons of simplicity.

[0080]

The phrase "an acidic L-amino acid" can mean the aspartic acid (Asp) and glutamic acid (Glu) of L-form, or salts thereof.

[0081]

The phrase "a dipeptide" can mean an organic molecule or a salt thereof consisting of two amino acid residues or derivatives of two amino acid residues, or a combination thereof, joined via peptide bond. The dipeptide can be consisted of amino acids or derivatives thereof, which are specified above. For example, the phrase "a dipeptide" can mean a dipeptide formed by two proteinogenic L-amino acid residues in such a way that an acidic L-amino acid residue such as L-Asp or L-Glu is located at the N-terminus of the dipeptide and another L-amino acid residue of the same kind or different kind is located at the C-terminus of the dipeptide. It is also accepted that a derivative of an amino acid, for example, a lower alkyl ester of an L-amino acid such as the methyl ester of L-Phe can be located at the C-terminus of the dipeptide.

[0082]

The dipeptide as described herein is. not limited to the dipeptide having an acidic amino acid residue at the N- terminus. The dipeptide can be represented by the formula R1-R2, where Rl and R2 can mean amino acid residues or derivatives thereof located at the N- and C-terminus of the dipeptide respectively and joined via peptide bond. Rl and R2 can be exemplified by 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 salts thereof. Rl and R2 may be of the same kind or different kinds.

[0083]

The phrase "peptide bond" can. mean a covalent chemical bond -C(0)NH- formed between two molecules when the carboxy part of one molecule, referred to as a carboxy component, reacts with the amino part of another molecule, referred to as an amino component, causing the release of a molecule. For example, amino acids can form the peptide bond upon joining with the release of a molecule of water.

[0084]

Any carboxy component may be used as far as it can form a peptide by condensation with the other substrate in the form of the amine component. Examples of carboxy

component include L-amino acid esters, D-amino acid esters, L-amino acid amides, and D-amino acid amides as well as organic acid esters not having unprotected an amino group. In addition, examples of amino acid esters include not only amino acid esters corresponding to naturally-occurring amino acids, but also amino acid esters corresponding to non-naturally-occurring amino acids or their derivatives. In addition, examples of amino acid esters include -amino acid esters as well as β-, γ-and ω-amino acid esters and the like having different amino group bonding sites.

Typical examples of amino acid esters include methyl esters, ethyl esters, n-propyl esters, iso-propyl esters, n-butyl esters, iso-butyl esters, and tert-butyl esters of amino acids. Also, the carboxy part of carboxy component can be exemplified by the carboxyl group COOH or a derivative thereof COR, where R can mean a substituted phenyl group or a halogenyl group such as chloro group.

[0085]

Any amine component may be used as far as it can form a peptide by condensation with the other substrate in the form of the carboxy component. Examples of the amine component include L-amino acids, C-protected L-amino acids, D-amino acids, C-protected D-amino acids, and amines. In addition, examples of the amines include not only

naturally-occurring amines, but also non-naturally- occurring amines or their derivatives. In addition,

examples of the amino acids include not only naturally- occurring amino acids, but also non-naturally-occurring amino acids or their derivatives. These include -amino acids as well as β-, γ- or ω-amino acids and the like having different carboxy group bonding sites.

[0086]

3.1. Enzymatic method

The enzymatic method can include at least the step of allowing the L-amino acids a-ligase or Lal-containing substance to contact with one or more amino acid(s) of the same kind or different kinds, or derivatives thereof, or salts thereof, under appropriate conditions to obtain the reaction product in accordance with the activity of Lai as described above.

[0087]

The method of allowing the Lai or Lal-containing substance used in the present invention to act on a carboxy component and an amino component may be mixing the Lai or Lal-containing substance, the molecule with carboxy part, and the molecule with amino part with each other. More specifically, a method of adding the Lai or Lal-containing substance to a solution containing carboxy and amino

components to form a dipeptide and allowing them to react may be used. Alternatively, in the case of using a

bacterium that produces the Lai, a method may be used that includes culturing the bacterium that forms the Lai,

producing, and accumulating the Lai in the bacterium or cultivation medium, and then adding the molecule with carboxy component and the molecule with amine component to the medium. The produced dipeptide can then be collected by established methods and purified as necessary.

[0088]

The phrase "Lal-containing substance" can mean any substance so far as it contains the Lai, and examples of specific forms thereof include a culture of bacteria that produce the Lai, bacterial cells isolated from the culture, and a product obtained by treating the bacterial cells

(also referred to as "treated bacterial cell product") . A culture of bacteria can mean what is obtained by culturing a bacterium, and more specifically, a mixture of bacterial cells, the medium used for culturing the bacterium, and substances produced by the cultured bacterium, and so forth. In addition, the bacterial cells may be washed and used in the form of washed bacterial cells. In addition, the

treated bacterial cell product includes the products of disrupted, lysed or freeze-dried bacterial cells, and the like, and also includes a crude enzyme recovered by

treating bacterial cells, and so forth, as well as a

purified enzyme obtained by purification of the crude enzyme, and so forth. A partially purified enzyme obtained by various types of purification methods may be used for the purified enzyme, or immobilized enzymes may be used that have been immobilized by a covalent bonding method, an adsorption method, an entrapment method, or the like. In addition, since some bacteria are partially lysed during culturing depending on the microbes used, the culture supernatant may also be used as the enzyme-containing substance in such cases.

[0089]

In addition, wild-type strains may be used as bacteria that contain the Lai, or gene recombinant strains that express the Lai may also be used as described above. The bacteria are not limited to intact bacterial cells, but rather acetone-treated bacterial cells, freeze-dried bacterial cells or other treated bacterial cells may also be used. Immobilized bacterial cells and an immobilized treated bacterial cell product obtained by immobilizing the bacterial cells or treated bacterial cell product by covalent bonding, adsorption, entrapment or other methods, as well as treated immobilized bacterial cells, may also be used.

[0090]

Furthermore, when using cultures, cultured bacterial cells, washed bacterial cells or a treated bacterial cell product that has been obtained by disrupting or lysing bacterial cells, it is often the case that an enzyme exists therein that decomposes the formed peptides without being involved in peptide formation. In this situation, it may be rather preferable in some cases to add a metal protease inhibitor like ethylene diamine tetraacetic acid (EDTA) . The addition amount can be within the range of 0.1 mM to 300 mM, and preferably within the range of 1 mM to 100 mM.

[0091]

An exemplary mode of the enzymatic method of the present invention is a method in which the transformed cells described herein are cultured in a medium, and a peptide-forming enzyme (Lai) is allowed to accumulate in the medium and/or transformed cells. Since the peptide- forming enzyme can be easily produced in large volumes by using a transformant , dipeptides can be produced in large amounts and rapidly.

[0092]

The amount of Lai pr Lal-containing substance used may be enough if it is an amount at which the target effect is demonstrated (effective amount), and this effective amount can be easily determined through simple, preliminary experimentation by a person with ordinary skill in the art. In the case of using the Lai, for example, the amount used can be about 0.1 g/L to 10 g/L (see, for example, Example 3), while in the case of using washed bacterial cells, the amount used can be higher that depends on the amount of Lai in a bacterial cell.

[0093]

The phase "appropriate conditions" can mean the conditions under which the Lal-catalyzed reaction can proceed; i.e. a reaction product, for example, a dipeptide can be formed from a carboxy component and an amino

component. The phrase "appropriate conditions" can include without limiting the phrases "an enzyme", "an amino acid", "a substrate", "an appropriate solvent", "a high-energy , molecule", "appropriate temperature conditions", and so forth.

[0094]

The phrase "a high-energy molecule" can mean any organic or inorganic molecule required for the Lal- catalyzed reaction to proceed under appropriate conditions. Conventionally, cofactors may be exemplified as the high- energy molecule. More specifically, the high-energy

molecule can be exemplified by the adenosine 5' - triphosphate (ATP) or a. salt thereof. Sodium, potassium, ammonium salts, or the like in any combinations thereof can be used.

[0095]

The phrase "an appropriate solvent" can mean any solvent, in which the Lal-catalyzed reaction can proceed, that is a reaction product, for example, a dipeptide can be formed. Organic and aqueous solvents, or mixtures thereof in various proportions may be an appropriate solvent. An appropriate solvent may contain the Lai enzyme of the present invention; co ^' factors such as ATP, and the like; metal ions such as sodium, potassium, ammonium, calcium, magnesium ions, and the like; anions such as sulfate, chloride, phosphate ions, and the like; other inorganic and/or organic molecules required for the activity of L- amino acids a-ligase. Tris (hydroxymethyl ) aminomethane

(Tris), N-tris (hydroxymethyl ) methylglycine (Tricine) or N, N-bis (2-hydroxyethyl) glycine (Bicine) , or the like as described in Carmody W.R. J. Chem. Educ, 1961, 38(11) :559- 560 can be added into a reaction mixture as a buffering agent. The acidity (pH) of a ^' reaction mixture may be maintained 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. The appropriate solvent may be subjected to appropriate temperature

conditions .

[0096]

The phrase "appropriate temperature conditions" can mean temperature conditions in which the Lal-catalyzed reaction can proceed, that is a reaction product, for example, a dipeptide can be formed. The appropriate

temperature conditions may be between 0 and 60 °C, or 20 and 40°C, or between 25 and 37°C, or between 28 and 35°C.

[0097] The concentrations of the carboxy component and amine component serving as starting materials can be 1 mM to 10 M, and preferably 50 mM to 2 M, respectively; however, there are cases where it is preferable to add amine component in an amount equimolar or excess molar with respect to the carboxy component. In addition, in cases where high

concentrations of substrates inhibit the reaction, these can be added stepwise during the reaction after they are adjusted to concentrations that do not cause inhibition.

[0098]

After the dipeptide is produced and accumulated in an appropriate solvent in a required amount, solids such as cells, cell debris and denaturated proteins can be removed from a medium by centrifugation or membrane filtration, and then the target dipeptide can be recovered from the

appropriate solvent by any combination of conventional techniques such as concentration, ion-exchange

chromatography, high-performance liquid chromatography

(HPLC) , crystallization, and so forth.

[0099]

Collecting and purification of enzyme

The L-amino acids a-ligase can be purified from the bacterium belonging to the genus Escherichia , for example, the species E. coli. A method for accumulating, collecting, and purifying the Lai from the bacterium can be an ordinary method known to the one skilled in the art.

[0100]

The bacterium is grown in a culture medium as

described hereinafter to produce Lai. A bacterial cell extract can be prepared from the cells by disrupting the cells using a physical method such as ultrasonic disruption or an enzymatic method using a cell wall-dissolving enzyme and removing the insoluble fraction by centrifugation and so forth. The peptide-forming enzyme can then be purified by fractionating the bacterial cell extract solution

obtained in the above manner by combining ordinary protein purification methods such as anion exchange chromatography, cation exchange chromatography or gel filtration

chromatography.

[0101]

The examples of the carriers for use in anion exchange chromatography can be Q-Sepharose HP or DEAE

(diethylaminoethyl) agarose (GE Healthcare), and so forth. The enzyme can be recovered in the non-adsorbed fraction under conditions of neutral pH such as between 7 and 8 when the cell extract containing the enzyme is allowed to pass through a column packed with the carrier. Various eluents can be used depending on the carrier. For example, when the cation exchange chromatography is performed using the MonoS HR (GE Healthcare) , the cell extract containing the enzyme is allowed to pass through a column packed with the carrier. To elute the enzyme, the column can be washed with a buffer solution having a high salt concentration. At that time, the salt concentration may be sequentially increased or a concentration gradient may be applied. As a saline solution, NaCl of about 0 to about 0.5 M can be applied. If required, the enzyme can be purified by gel filtration chromatography. The examples of the carrier for use in gel filtration chromatography can be Superdex 200 HR or Sephadex 200 (GE Healthcare) .

[0102]

In the aforementioned purification procedure, the fraction containing the enzyme can be verified by assaying the Lai activity of each fraction according to the method indicated in the examples to be described later.

[0103] 3.2. Fermentative method

The method of the present invention can also be a method, for producing a dipeptide, more specifically a dipeptide having an acidic L-amino acid at the N-terminus, by cultivating the bacterium of the present invention in a culture medium to allow the dipeptide to be produced, excreted, and accumulated in the culture medium, and collecting the dipeptide from the culture medium.

The cultivation of a bacterium of the invention, collection, and the purification of a dipeptide from the medium and the like may be performed in a manner similar to conventional fermentation methods, wherein a dipeptide or an amino acid is produced using a microorganism. The culture medium for a dipeptide production may be a typical medium that contains a carbon source, a nitrogen source, inorganic ions, and other organic components as required. As the carbon source, saccharides such as glucose, lactose, galactose, fructose, arabinose, maltose, xylose, trehalose, ribose, and hydrolyzates of starches; alcohols such as glycerol, mannitol, and sorbitol; organic acids such as gluconic acid, fumaric acid, citric acid, malic acid, and succinic acid; and the like can be used. As the nitrogen source, inorganic ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate; organic nitrogen such as of soy bean hydrolyzates; ammonia gas; aqueous ammonia; and the like can be used. Vitamins such as vitamin Bl, required 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, even if trace, amounts. Other than these, small amounts of calcium phosphate, magnesium sulfate, iron ions, manganese ions, and the like may be added, if necessary.

[0104] To increase the dipeptide-producing ability of a bacterium of the present invention, the culture medium can be additionally supplemented with amino acids or amino acid derivatives, cofactors, and other (bio) chemicals . For example, to increase the ability of a bacterium to produce the Asp-Phe dipeptide, the culture medium may be

supplemented with additional quantities of L-Phe and L-Asp.

[0105]

Cultivation can be performed under aerobic conditions for 16 to 72 hours, the culture temperature during

cultivation can be controlled within 15 to 45°C, or within 28 to 37°C. The acidity (pH) can be adjusted between 5 and 8, or between 6.5 and 7.2 by using an inorganic or organic acidic or alkaline substance, as well as ammonia gas.

[0106]

After cultivation, solids such as cells and cell debris can be removed from the liquid medium by

centrifugation or membrane filtration, and then the target dipeptide can be recovered from the fermentation liquor by any combination of conventional techniques such as

concentration, ion-exchange chromatography, high- performance liquid chromatography (HPLC) , crystallization, and so forth. [0107]

Examples

The present invention is more precisely explained below with reference to the following non-limiting Examples.

[0108]

Example 1. Cloning of BBR47_51900 from Brevibacillus brevis NBRC 100599 and Staur_4851 from Stigmatella aurantiaca

DW4/3-1.

The primary structure of the genes encoding the hypothetical proteins BBR47_51900 and Staur_4851 was optimized for expression in E. coli The genes encoding BBR47_51900 from Brevibacillus brevis NBRC 100599 and

Staur_4851 from Stigmatella aurantiaca DW4/3-1 were

synthesized by the SlonoGene™ gene synthesis service

(http://www.sloning.com/) and delivered as a set of pSlo.X plasmids harboring the synthesized Xbal-EcoRI fragment which included the target genes having optimized sequences. The Xbal-EcoRI fragments harboring genes with optimized sequences encoding the BBR47_51900 and Staur_4851 proteins are shown in SEQ ID NOs: 19 and 20, respectively.

[0109]

To construct the pET-HT-BBR and pET-HT-STA plasmids, the corresponding Xbal-EcoRI fragments of the pSlo.X plasmids were excised by digestion with Xbal and EcoRI and then ligated with the pET15(b+) vector (Novagen, USA) digested by the same restrictases .

[0110]

Example 2. Expression and purification of His6-tagged

BBR47_51900 and Staur_4851.

Plasmids pET-HT-BBR and pET-HT-STA were introduced into BL21 (DE3). strain (Novagen, USA) by Ca ²⁺ -dependent transformation to construct the BL21 (DE3) [pET-HT-BBR] and BL21 (DE3) [pET-HT-STA] strains. The electrotransformation was done using "Bio-Rad" electroporator (USA) (No .165-2098 , version 2-89) according to the manufacturer's instructions. Cells of the BL21 (DE3) [pET-HT-BBR] and BL21 (DE3) [pET- HT-STA] strains were each grown in LB broth (also referred to as lysogenic broth as described in Sambrook, J. and Russell, D.W. (2001) Molecular Cloning: A Laboratory Manual (3 ^rd ed.). Cold Spring Harbor Laboratory Press) at 37°C up to OD540 ~1 and 150 rpm. Isopropyl-p-D-thio-galactoside (IPTG) was added to a final concentration of 1 mM, and the cell culture was incubated for 2 hours at 37 °C and 150 rpm. Induced cells were harvested from 1 L of cultivation broth, re-suspended in 60-80 mL of HT-I-buffer (20 mM NaH ₂ P0 ₄ , 0.5 M NaCl, 20 mM imidazole, pH 7.4, adjusted with NaOH) , and disrupted under 2000 Psi (~140 bar) using French-press

(Thermo Spectronic) . The debris was removed by

centrifugation for two times at 4°C and 13000 rpm followed by filtration through 0.45 μπι filter (CHROMAFIL Xtra CA- 45/25, MACHEREY-NAGEL GmbH) . A solution of crude proteins was loaded onto HiTrap Chelating column (GE Healthcare) pre-packed with immobilized metal affinity chromatography (IMAC) sorbent of 1 ml total volume and equilibrated with the HT-I-buffer. The IMAC was performed in accordance with the manufacturer's recommendations. The active fractions were collected, combined, and desalted using PD10 columns

(GE Healthcare) equilibrated with SB-buffer (20 mM Tris-HCl, 120 mM NaCl, 1 mM β-mercaptoethanol , 15% glycerol, pH 7.5). The protein preparations were divided into aliquots of volume (200 μΐ,) and stored at -70°C. The protein

concentration was determined using BIO-RAD PROTEIN ASSAY (BIO-RAD, USA) .

[0111]

Example 3. Preliminary analysis of substrate specificity of BBR47_51900 and Staur_4851.

The substrate specificity of BBR47_51900 and

Staur_4851 was studied in the reaction mixture containing the enzyme and 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 salts thereof, of the same or two different kinds. The composition of the reaction mixture of total volume of 50 μΐι was as follows unless otherwise noted:

BBR47_51900 or Staur_4851 4 μς

Tris-HCl, pH 8.0 50 mM

First L-amino acid 10 mM

Second L-amino acid 10 mM

Adenosine 5' -triphosphate (ATP) 10 mM

MgS0 ₄ x7H ₂ 0 10 mM

H ₂ 0 to 50 μΐ,

[0112]

Reactions were carried out at 32 °C for 15 hours. 1-2J ^~ L of reaction mixture was subjected to thin layer

chromatography (TLC) analysis using as mobile phase the mixture of 2-propanol : acetone : 250 mM ammonia : H ₂ 0 as 100 : 100 : 12 : 28. A solution (0.3%, w/v) of ninhydrin in acetone was used as a visualizing reagent. Detection was performed at 540 nm. The new spots on TLC-plates were detected after developing the reaction mixtures, which contained:

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) .

[0113]

The obtained results indicate that BBR47_51900 and Staur_4851 can catalyze ligation of an acidic L-amino acid such as L-Glu and L-Asp with other L-amino acids.

[0114]

Example 4. Determination by HPLC analysis of dipeptides synthesized by BBR47_51900 and Staur_4851.

Dipeptides synthesized by BBR47_51900 and Staur_4851 were determined using HPLC analysis of reaction mixtures of total volume of 400 i , which contained:

BBR47_51900 or Staur_4851 160 pg

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

MgS0 ₄ x7H ₂ 0 10 mM, where Me denote methyl group.

[0115]

Reactions were carried out at 32oC for 15 hours. Then

0.5 mL of reaction mixture was filtered through the Amicon Ultra-0.5 mL, 3K Centrifugal Filters (Millipore,

#UFC500396) and subjected to HPLC analysis.

[0116]

The conditions were as follows:

Equipment: HITACHI L-2000 series.

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

Temperature: 40 °C.

Buffers: ·

A (for mixture L-Asp/L-Val and L-Glu/L-Val) : 0.1 M ^ KH ₂ P0 ₄ (pH 2.2) + 5 mM octanesulfonate, sodium salt : CH ₃ CN as 4 : 1 (v/v) ,

B (for mixture L-Asp/L-Phe) : 0.1 M KH ₂ P0 ₄ (pH 2.2) + 5 mM octanesulfonate, sodium salt : CH ₃ CN as 7 : 3 (v/v) , C (for mixture L-Asp/L-PheOMe, L-Asp/L-Trp, and L- Val/L-Val) : 0.1 M KH ₂ P0 ₄ (pH 2.2) + 5 mM octanesulfonate , sodium salt : CH ₃ CN as 3 : 2 (v/v) ,

D (for mixture L-Phe/L-Phe) : 0.1 M KH ₂ P0 ₄ (pH 2.2) + 5 mM octanesulfonate, sodium salt : CH ₃ CN as 1 : 1 (v/v) . Gradient profile: isocratic.

Flow rate: 1.5 mL/min.

Injection volume: 10 pL. Detection: UV 210 nm .

[0117]

Chemicals used for HPLC analysis were as follows:

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

MgS0 ₄ x7H ₂ 0: Junsei Chemical Co., Ltd. #83580-0301

<xAsp-Phe (H-Asp-Phe-OH) : Bachem G-1620

βΑερ-ΡΙΐθ (H-Asp (Phe-OH) -OH) : Bachem G-4750

aAsp-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

aAsp-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

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

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

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

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

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

βΑ3ρ3^3Γηε: (H-Asp ( Phe-OMe) -OH) : Bachem G-3725

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

[0118]

Solution of aGlu-Val, yGlu-Val, aAsp-Val (10 mM each) and aAsp-Phe, Asp-Phe (5 mM each) were prepared for HPLC analysis. The concentration of a dipeptide formed was determined using corresponding calibration curves. Each solution with standard sample was diluted to 50-folds or 100-folds for LC-QTOF/MS/MS analysis (Example 5) .

[0119]

The results of HPLC analysis of reaction mixtures are shown in Table 1. As it can be seen from the Table 1, BBR47_51900 and Staur_4851 catalyze formation of dipeptide having an acidic L-amino acid such as L-Asp and L-Glu at the N-terminus .

[0120]

Example 5. Determination by LC-QTOF/MS/MS analysis of dipeptides synthesized by BBR47_51900.

Samples of reaction mixtures and standard solutions obtained as described in Example 4 were subjected to LC- QTOF/MS/MS analysis.

[0121]

The conditions were as follows:

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

LC conditions:

Column: Develosil C30 2.0 x 250 mm, 3 μτα (Nomura

Chemical) .

Temperature: 20 °C.

Buffers :

A: H ₂ 0 (0.025% Formic acid),

B: CH ₃ CN (0.025% Formic acid).

Gradient profile:

Time (min) B (%)

0 0

20 22.5

20.1 100

25 100

Flow rate: 0.3 mL/min.

Injection volume: 2-5 L. [0122]

MS conditions:

Capillary voltage: 3.0 kV.

Cone voltage: 20 V.

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

Source temperature: 80oC.

Desolvation temperature: 120oC.

Cone gas flow rate: 50 L/hr.

Desolvation gas flow rate: 700 L/hr.

[0123]

The results of LC-QTOF/MS/MS analysis of reaction mixtures are shown in Figures 7-9. As it can be seen from the Figures 7-9, BBR47_51900 catalyze formation of aAsp-Phe, aAsp-Val, and Glu-Val dipeptides.

[0124]

Example 6. Searching for enzymes which are isofunctional to BBR47_51900 and Staur_4851.

The HMMER method was used for searching in the

sequence databases for homologues of protein sequences, and for making protein sequence alignments. The method uses a , probabilistic model referred to as the profile hidden

Markov model (profile HMM) (Finn R.D. et al., HMMER web server: interactive sequence similarity searching, Nucleic Acids Res., 2011, 39 (Web Server issue ): 29-37 ) .

[0125]

Compared to BLAST, FASTA, and other sequence alignment and database search tools based on older scoring

methodology, HMMER aims to be significantly more accurate and more reliable to detect remote homologues such as isofunctional proteins because of the strength of its underlying mathematical models. In the past, this strength came at significant computational expense, but in the new HMMER3 project, HMMER has become essentially as fast as BLAST (Finn R.D. et al., HMMER web server: interactive sequence similarity searching, Nucleic Acids Res., 2011, 39(Web Server issue) :W29-37 ) .

[0126]

To search enzymes which are isofunctional to

BBR47_51900 and Staur_4851, the alignment of BBR47_51900 and Staur_4851 (Figure 10) was subjected to the HMMsearch program from HMMER3 suite that allows searching for one or more profiles against a protein sequence database

(http://hmmer.janelia.org/). Based on the sequences alignment for BBR47_51900 and Staur_4851, the profile HMM had been originated (Model 1), which was used for

homologues search. The list of the nearest isofunctional proteins found (hits) is shown on Figure 11). Analysis of the distribution diagram of | Log (E-value) | -values revealed five groups of proteins sharing the | Log (E-value) | -values between the members of a group lower than that values between groups (Figure 12). The first group comprises BBR47_51900 and Staur_4851, the second group comprises DES and BCE, the third group comprises BMY, the fourth group comprises BTH, and the fifth group comprises BUR. The rest proteins are a number of ungrouped homologues having stochastic | Log (E-value) I -values. The AME and SFL proteins were selected from such ungrouped homologues as the negative control.

[0127]

Given the proteins from the first (BBR47_51900 ,

Staur_4851) and second (DES and BCE) groups are

isofunctional, the new profile HMM (Model 2) based on alignment of BBR47_51900, Staur_4851, DES and BCE (Figure 13) can be originated, which can be used for the

isofunctional proteins search using the HMMsearch program as described above. Thus, a new list of isofunctional proteins can be originated (Figure 14), which is described by a distribution diagram of I LoglO (E-value) I -values, which is different from the initial diagram (Figure 15) . The new list of isofunctional proteins can comprise three groups such as the first group comprising BBR47_51900, Staur_4851, DES and BCE; the second group comprising BMY and BTH; and the third group comprising BUR, wherein AME is closer to the first group (the position change from No. 47 (Figure 12) to No. 33 (Figure 15)) and SFL is more distant from the first group (the position change from No. 49 (Figure 12) to No. 62 (Figure 15) ) .

[0128]

Given the proteins from the first (BBR47_51900,

Staur_4851, DES and BCE) and second (such as BMY) groups are isofunctional , the new profile HMM (Model 3) based on alignment of BBR47_51900, Staur_4851, DES, BCE and BMY (Figure 16) can be originated, which can be used for the isofunctional proteins search using the HMMsearch program as described above. Thus, a new list of isofunctional proteins can be originated (Figure 17) . The new list of isofunctional proteins can comprise three groups such as the first group comprising BBR47_51900, Staur_4851, DES, BCE and BMY; the second group comprising BTH; the third group comprising BUR, wherein AME is at the position No. 37 and SFL is at the position No. 65 (Figure 17).

[0129]

Given the proteins from the first (BBR47_51900 ,

Staur_4851, DES, BCE and BMY) and second (BTH) groups are isofunctional, the new profile HMM (Model 4) based on alignment of BBR47_51900, Staur_4851, DES, BCE,. BMY and BTH (Figure 18) can be originated, which can be used for the isofunctional proteins search using the HMMsearch program as described above. Thus, a new list of isofunctional proteins can be originated (Figure 19) . The new list of isofunctional proteins can comprise three groups such as the first group comprising BBR47_51900, Staur_4851, DES, BCE, BMY and BTH; the second group comprising BUR; the third group comprising AME, wherein SFL is at the position No. 73 (Figure 19) .

[0130]

Given the proteins from the first (BBR47_51900 ,

Staur_4851, DES, BCE, BMY and BTH) and second (BUR) groups are isofunctional , the new profile HMM (Model 5) based on alignment of BBR47_51900, Staur_4851, DES, BCE, BMY, BTH and BUR (Figure 20) can be originated, which can be used for the isofunctional proteins search using the HMMsearch program as described above. Thus, a new list of

isofunctional proteins can be originated (Figure 21) . The new list of isofunctional proteins can comprise three groups such as the first group comprising BUR; the second group comprising BBR47_51900, Staur_4851, DES, BCE, BMY and BTH; the third group comprising AME, wherein SFL is at the position No. 104 (Figure 21).

[0131]

Given the proteins from the first (BUR) , second

(BBR47_51900, Staur_4851, DES, BCE, BMY and BTH) and third (AME) groups are isofunctional , the new profile HMM (Model 6) based on alignment of BBR47_51900, Staur_4851, DES, BCE, BMY, BTH, BUR and AME (Figure 22) can be originated, which can be used for the isofunctional proteins search using the HMMsearch program as described above. Thus, a new list of isofunctional proteins can be originated (Figure 23) . The new list of isofunctional proteins can comprise two groups such as the first group comprising BUR; the second group comprising BBR47_51900, Staur_4851, DES, BCE, BMY, BTH and AME, wherein SFL is at the position No. 65 (Figure 23) . [0132]

Given the proteins from the first (BUR) and second (BBR47_51900, Staur_4851, DES, BCE, BMY, BTH and AME) groups, and the SFL protein are isofunctional, the new profile HMM (Model 7) based on alignment of BBR47_51900, Staur_4851, DES, BCE, BMY, BTH, BUR, AME and SFL (Figure 24) can be originated, which can be used for the

isofunctional proteins search using the HMMsearch program as described above. Thus, a new list of isofunctional proteins can be originated (Figure 25). The new list of isofunctional proteins can comprise three groups such as the first group comprising BUR; the second group comprising BBR47_51900, Staur_4851, DES, BCE, BMY, BTH and AME; and the third group comprising SFL is at the position No. 38 (Figure 25) .

[0133]

In the similar manner new lists of isofunctional L- amino acids a-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 a derivative thereof at the C-terminus, can be originated. If the

BBR47_51900 and Staur_4851 proteins are used to originate the profile HMM (Model 1), the | LoglO (E-value) | > 233 can be used for the new isofunctional Lais search; if the

BBR47_51900, Staur_4851, DES and BCE proteins are used to originate the HMM profile (Model 2), the I LoglO (E-value ) | > 196 can be used for the new isofunctional Lais search; if the BBR47_51900, Staur_4851, DES, BCE and BMY proteins are used to originate the HMM profile (Model 3), the |Logl0(E- value) | ≥ 182 can be used for the new isofunctional Lais search; if the BBR47_51900, Staur_4851, DES, BCE, BMY and BTH proteins are used to originate the HMM profile (Model 4), the I LoglO (E-value) | > 175 can be used for the new isofunctional Lais search; if the BBR47_51900, Staur_4851, DES, BCE, BMY, BTH and BUR proteins are used to originate the HMM profile (Model 5), the | LoglO (E-value) | > 162 can be used for the new isofunctional Lais search; if the

BBR47_51900, Staur_4851, DES, BCE, BMY, BTH, BUR and A E proteins are used to originate the HMM profile (Model 6) , the I LoglO (E-value) | ≥ 142 can be used for the new

isofunctional Lais search; and if the BBR47_51900,

Staur_4851, DES, BCE, BMY, BTH, BUR, AME and SFL proteins are used to originate the HMM profile (Model 7), the

I LoglO (E-value) | ≥ 128 can be used for the new

isofunctional Lais search (Figure 26) , wherein the E-value is a parameter of the HMMsearch program (Finn R.D. et al., HMMER web server: interactive sequence similarity searching, Nucleic Acids Res., 2011, 39 (Web Server issue) : W29-37 ) .

[0134]

Example 7. Cloning, expression, and purification of the DES, BUR, BCE, BTH, AME, SFL and BMY enzymes.

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

[0135] The DES, BUR, BCE, BTH, AME, SFL and BMY proteins can be expressed in E. coli, purified, and their activities can be investigated as described for BBR47_51900 and Staur_4851 in Examples 1-5.

[0136]

Example 8. Construction of the E. coli peptidase-deficient 1-6Δ strains.

8.1. Construction of the E. coli peptidase-deficient 1-5Δ strains .

The iadA gene was deleted in the E. coli BW25113 strain (KEIO collection, strain No. ME9062) having the

ApepB mutation (KEIO collection, strain No. J 2507; The E. coli Genetic Stock Center, Yale University, New Haven, USA, the accession No. CGSC9995) {E. coli 1Δ strain) . For this purpose, the DNA fragment bearing the AattL-cat-XattR cassette was PCR (polymerase chain reaction) amplified using the primers PI (SEQ ID NO: 28) and P2 (SEQ ID NO: 29), and the pMW118-XattR-cat-XattL plasmid as the template

(Katashkina Zh.I. et al., Mol . Biol. (Mosk.), 2005,

39 (5) : 823-831) . The resulting DNA fragment was introduced into the E. coli BW25113 /pKD46 strain by

electrotransformation using "Bio-Rad" electroporator (USA) (No .165-2098 , version 2-89) according to the manufacturer's instructions. The recombinant plasmid pKD46 (Datsenko K.A. and Wanner B.L., Proc. Natl. Acad. Sci. USA, 2000, 97:6640- 6645) with the temperature-sensitive replicon was used as the donor of the phage λ-derived genes responsible for the ARed-mediated recombination system. The pKD46 plasmid can be integrated into E. coli BW25113 (ΔρβρΒ) by the described method (Datsenko K.A. and Wanner B.L., Proc. Natl. Acad. Sci. USA, 2000, 97:6640-6645) to obtain the E. coli

BW25113 (ΔρβρΒ) /pKD46 strain. Alternatively, the E. coli BW25113 (ΔρβρΒ) strain containing the recombinant plasmid pKD46 can be obtained from the E. coli Genetic Stock Center, Yale University, New Haven, USA, the accession number is CGSC7739.

[0137]

The E. coli BW25113 (ΔρβρΒ, iadA: : AattR-ca t-AattL) transformant is resistant to chloramphenicol (Cm) encoded by the cat gene and harbors in chromosome the "excisable" chloramphenicol-resistance marker (Cm ^R -marker) instead of iadA. The Cm ^R -marker was excised as described in

(Katashkina Zh.I. et al., Mol. Biol. (Mosk.), 2005,

39 (5) : 823-831) to construct the E. coli B 25113 (ΔρβρΒ, iadA: : AattB) strain (E. coli 2Δ strain).

[0138]

The pepE gene was deleted in the E. coli B 25113

(ΔρβρΒ, iadA: :AattB) strain. A DNA fragment bearing the AattL-cat-AattR cassette was PCR amplified using the

primers P3 (SEQ ID NO: 30) and P4 (SEQ ID NO: 31), and the pMW118-AattR-cat-AattL plasmid as the template. The

resulting DNA fragment was introduced into the E. coli

BW25113 (ApepB, iadA: : AattB) /pKD46 strain by

electrotransformation as described above. The Cm ^R -marker was excised from the E. coli BW25113 (ApepB, iadA: :AattB, pepE: :AattR-cat-AattL) transformant to construct the E.

coli B 25113 (ApepB, iadA: :AattB, pepE: : AattB) strain (E. coli 3Δ strain) .

[0139]

The ybiK gene was deleted in the E. coli BW25113 (ΔρβρΒ, iadA: :AattB, pepE: : AattB) strain. A DNA fragment bearing the AattL-c t-AattR cassette was PCR amplified using the primers P5 (SEQ ID NO: 32) and P6 (SEQ ID NO: 33), and the pMW118-AattR-cat-AattL plasmid as the template. The

resulting DNA fragment was introduced into the E. coli

BW25113 (ΔρβρΒ, iadA: :AattB, pepE: :AattB) /pKD46 strain by electrotransformation as described above. The Cm -marker was excised from the E. coli BW25113 (ΔρβρΒ, iadA: :AattB, pepE: : AattB, ybiK: : AattR-cat-AattL) transformant to

construct the E. coli BW25113 [ApepB, iadA: : AattB,

pepE: : AattB, ybiK: : AattB) strain (E. coli 4Δ . strain).

[0140]

The dapE gene was deleted in the E. coli BW25113 (ΔρβρΒ, iadA: :AattB, pepE: : AattB, ybiK: : AattB) strain. A DNA fragment bearing the AattL-cat-AattR cassette was PCR amplified using the primers P7 (SEQ ID NO: 34) and P8 (SEQ ID NO: 35), and the pM 118-AattR-cat-AattL plasmid as the template.. The resulting DNA fragment was introduced into the E. coli BW25113 (ApepB, iadA: : AattB, pepE: : AattB, ybiK: : AattB) /pKD46 strain by electrotransformation as described above. The Cm ^R -marker was excised from the E.

coli BW25113 (ΔρβρΒ, iadA: :AattB, pepE: : AattB, ybiK: : AattB, dapE: : AattR-cat-AattL) transformant to construct the E. coli BW25113 (ApepB, iadA:: AattB, pepE: : AattB, ybiK: : AattB, dapE: : AattB) strain (E. coli 5Δ strain).

[0141]

Thus the E. coli strains having one to five deleted the peptidases encoding genes (the E. coli 1-5Δ strains) were constructed.

[0142]

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

To analyze the specific aspartic peptide-hydrolyzing activity, the artificial dipeptide DP3 (L-Asp-L-5- Fluorotryptophan) was synthesized (the Branch of the

Institute for Bioorganic Chemistry (BIBCh) of the Russian Academy of Sciences, Pushchino, Russian Federation) . The peptide hydrolyzing activity was investigated in vitro and in vivo. [0143]

For in vitro studies, cells of E. coli B 25113 and E. coli 5Δ strain were grown on LB and M9-salts + Glucose

(0.2%, w/v) media (Sambrook, J. and Russell, D.W. (2001) Molecular Cloning: A Laboratory Manual (3 ^rd ed.). Cold

Spring Harbor Laboratory Press) at 37°C to cells density of OD^nm ~1. Grown cells were harvested by centrifugation

(4°C, 10000 rpm) , re-suspended in buffer E (50 mM Tris-HCl pH 8.0, 20 mM NaCl) , disrupted by sonication followed by centrifugation (14000 g, 4°C, 20 min) to remove cell debris. The crude protein concentration can be determined using the Bradford protein assay (Bradford M.M., Anal. Biochem. , 1976, 72:248-254) using bovine serum albumin as a standard. The obtained crude proteins preparations were used to

investigate DP3-hydrolyzing activity.

[0144]

The reaction mixture contained:

50 mM Tris-HCl pH 8.0,

20 mM NaCl,

5 mM DP3 dipeptide,

1 mM ZnS0 ₄ or MnCl ₂ ,

24 μg of crude proteins preparation,

H ₂ 0 to a total volume of 10 μL.

[0145]

Reaction mixtures were incubated at 37 °C for different time. The DP3-hydrolyzing activity was measured by

quantitative TLC analysis of the 5-fluorotryptophan

released (Figure 27, Table 4). As a mobile phase, the mixture of 2-propanol : acetone : H ₂ 0 as 25 : 25 : 4 was used. A solution (0.3%, w/v) of ninhydrin in acetone was used as a visualizing reagent. The obtained results

indicate that aspartic peptide-hydrolyzing activity can be determined in 5Δ strain suggesting that there are unknown peptidases having DP3-hydrolyzing activity in the E. coli 5Δ strain.

[0146]

For in vivo studies, toxicity of DP3 dipeptide for constructed peptidase-deficient E. coli 5Δ strain was investigated (Figure.28). Cells of E. coli B 25113 and E. coli 5Δ strains were grown on M9-salts medium supplemented with D-glucose or glycerol (0.4%, w/v) to cells density of OD55 _5nm ~2. The cells biomass was diluted and about 106 cells were plated onto M9-salts agar supplemented with D- glucose or glycerol (0.4%, w/v), and DP3 dipeptide. Plates were incubated at 37°C for 48 hours (for D-glucose) or 72 hours (for glycerol) (Table 5). Visual analysis showed that deletion of five known aspartic peptide-hydrolyzing enzymes encoded by the pepB, iadA, pepE, ybiK and dapE genes decreases DP3 toxicity for E. coli 5Δ strain (from 6 μΜ for B 25113 to 30 μΜ for 5Δ strain grown on M9-salts + D- glucose medium) . Increasing DP3 concentration up to 50 μΜ resulted in growth arrest of the E. coli 5Δ strain

suggesting the residual intracellular DP3 peptidase

activity.

[0147]

8.3. Identification of residual peptidases in the E. coli 5Δ strain having specific aspartic peptide-hydrolyzing activity.

To identify the residual intracellular peptidases having specific aspartic peptide-hydrolyzing activity, the following procedure was used. Cells of the E. coli 5Δ strain were grown at 37 °C overnight in 4 L of M9-salts media supplemented with D-glucose (0.4%, w/v). Grown cells were harvested by centrifugation (4°C, 10000 rpm) and re- suspended in 100 mL of buffer F (20 mM Tris-HCl pH 7.5, 20 mM NaCl) . [0148] .

Purification protocol was as follows:

Step 1. Cells were disrupted by 2 passages through French-press (Thermo Spectronic) followed by centrifugation (14000 g, 4°C, 20 min) to remove cell debris. The obtained crude proteins preparation was loaded onto DEAE FF 16/10 column (20 mL) (GE Healthcare) equilibrated with buffer F (20 mM Tris-HCl pH 7.5, 20 mM NaCl). The elution was carried out at flow rate of 1 mL/min by applying the liner gradient of NaCl (from 20 to 600 mM in 20 column volumes) in buffer F. Fractions (10 mL each) were collected and analyzed as described in Example 8.2. Active fractions 16- 21 were found.

[0149]

Step 2. Proteins from collected fractions 16-21 were precipitated by saturated (60%) (NH ) ₂ S0 ₄ , re-suspended in 2 mL of buffer F and loaded onto standard Superdex 200 HR 10/30A column (GE Healthcare) equilibrated with buffer G (20 mM Tris-HCl pH 7). Isocratic elution was carried out at flow rate of 0.5 mL/min by applying buffer G. 0.5 ml

Fractions (0.5 mL each) were collected and analyzed as described in Example 8.2. Active fractions (12-13) were found .

[0150]

Step 3. Proteins from collected fractions 12-13 (Step

2) were loaded onto Soursel5Q column (1.6 mL) (GE

Healthcare) equilibrated with buffer G (20 mM Tris-HCl pH 7). The elution was carried out at flow rate of 0.5 mL/min by applying the liner gradient of NaCl (from 0 to 400 mM in 20 column volumes) in buffer G. Fractions (0.5 mL each) were collected and analyzed as described in Example 8.2. Active fractions (15-17) were found. Purification data are summarized in Table 6. [0151]

Step 4. To identify the peptidases having specific aspartic peptide-hydrolyzing activity the proteins from several fractions (15-17) were subjected to SDS-PAGE

(Laemmli U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 1970, 227:680-685). The profile of activity elution was compared with. that of proteins elution. Only one protein was found, for which activity and elution profiles were identical.

[0152]

The purified protein was extracted from SDS-gel and digested with trypsin (Govorun V.M. et al., Biochemistry (Mosc), 2003, 68 (1) : 42-49) . The digestion mixture was mass-analyzed using MALDI-TOF as described in (Govorun V.M. et al., Biochemistry (Mosc), 2003, 68 ( 1 ) : 42-49 ) . The resulted mass-spectrum of the isolated protein matched with that obtained for aminopeptidase A/I (PepA) of E. coli. Thus the sixth peptidase was found in E. coli 5Δ strain.

[0153]

8.4. Construction of the E. coli peptidase-deficient 6Δ strain .

The pepA gene was deleted in the E. coli BW25113 (ApepB, iadA: :AattB, pepE: : AattB, ybiK: : AattB, dapE: : AattB) strain as described in Example 8.1. A DNA fragment bearing the AattL-cat-AattR cassette was PCR amplified using the primers P9 (SEQ ID NO: 36) and P10 (SEQ ID NO: 37), and the pMW118-AattR-cat-AattL plasmid as the template. The

resulting DNA fragment was introduced into the E. coli BW25113 (ApepB, iadA: : AattB, pepE: : AattB, ybiK: : AattB, dapE: : AattB) /pKD46 strain by electrotransformation as described above to construct the E. coli B 25113 (ApepB, iadA: :AattB, pepE: : AattB, ybiK: : AattB, dapE: : AattB,

pepA: : AattL-ca -AattR) strain (E. coli 6Δ strain). [0154]

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

The specific aspartic peptide-hydrolyzing activity was analyzed in vitro as described in Example 8.2. The obtained results (Table 4) indicate that aspartic peptide- hydrolyzing activity can be determined in 6Δ strain, which is lower as compared with 4-5Δ strains.

[0155]

Example 9. Fermentative production of dipeptides having an acidic L-amino acid such as L-Asp or L-Glu at the N- terminus using the modified E. coli 5Δ and 6Δ strains having Lai activity.

The dipeptides having an acidic L-amino acid such as L-Asp or L-Glu at the N-terminus is produced using a bacterium of the family Enterobacteriaceae, more

specifically a bacterium belonging to the genus Escherichia such as E. coli having dipeptide-producing ability, in a medium supplemented with or devoid of, for example, but not limited to required amino acids. A dipeptide-producing bacterium is the E. coli 5Δ or 6Δ strain as described above deficient of peptidase activity, further modified to have L-amino acid -ligase activity. The dipeptide-producing strain is further The gene(s) encoding Lal(s) selected from the group consisting of BBR47_51900, Staur_4851, DES, BUR, BCE, BTH, AME, SFL and BMY is (are) introduced into

chromosome of the E. coli or introduced into the bacterial cell on a plasmid having the gene encoding the Lai. The gene(s) encoding Lal(s) is (are) placed under a promoter.

[0156]

The modified E. coli 5Δ or 6Δ harboring gene(s) encoding Lal(s) and the control 5Δ or 6Δ strains are each cultivated at 28-3 °C for 18-72 hours in Luria-Bertani broth (also referred to as lysogenic broth as described in Sambrook, J. and Russell, D.W. (2001) Molecular Cloning: A Laboratory Manual (3 ^rd ed. ) · Cold Spring Harbor Laboratory Press). The E. coli 5Δ or 6Δ strain harboring gene(s) encoding Lal(s) is inoculated into 2 mL of a fermentation medium in 20 ^χ 200 mm test-tubes and cultivated at 28-37 °C for 18-72 hours on a rotary shaker at 250 rpm.

[0157]

The composition of the fermentation medium is (g/L) : Glucose 5-40

NaCl 0.8

(NH ₄ ) ₂ S0 ₄ . 22

K ₂ HP0 ₄ 2.0

MgS0 ₄ x7H ₂ 0 0.8

MnS0 ₄ x5H ₂ 0 0.02

FeS0 ₄ x7H ₂ 0 0.02

Thiamine hydrochloride 0.002

Yeast extract 1.0-2.0

CaC0 ₃ 30

L-Phe 0 - 100 (mM)

L-Asp 0 - 100 (mM)

[0158]

The fermentation medium is sterilized at 116°C for 30 min, except that glucose and CaC0 ₃ are sterilized

separately and as follows: glucose at 110°C for 30 min,

CaC0 ₃ at 116°C for 30 min. The pH is adjusted to 5-8 by KOH solution.

[0159]

After cultivation, accumulated dipeptide is measured using thin-layer chromatography (TLC) . TLC plates (10 x 20 cm) are coated 'With 0.11 mm layers of . Sorbfil silica gel containing non-fluorescent indicator ( Sorbpolymer,

Krasnodar, Russian Federation) . The TLC plates are developed with a mobile phase consisting of 2-propanol : acetone : 250 mM ammonia : H ₂ 0 as 100 : 100 : 12 : 28. A solution (0.3%, w/v) of ninhydrin in acetone is used as a visualizing reagent. Detection is performed at 540 ran.

[0160]

Auxiliary example 1.

The multiple alignments of the BBR47_51900 and

Staur_4851 proteins with known L-amino acid a-ligases

(Lais) are shown in Table 2 (identity) and Table 3

(similarity) . As it can be seen from the Tables 2 and 3, the BBR47_51900 and Staur_4851 proteins have the identity value of not higher than 25% (Table 2) and the similarity value of not higher than 43% (Table 3) with known Lais.

[0161]

Auxiliary example 2.

The pair-wise alignment data for the BBR47_51900, Staur_4851, DES , BUR, BCE, BTH, AME, SFL and BMY proteins are shown in Table 7 (identity) and Table 8 (similarity) . As it can be seen from the Tables 7 and 8, the BBR47_51900, Staur_4851, DES, BUR, BCE, BTH, AME, SFL and BMY proteins have the identity value of not higher than 25% (Table 7) and the similarity value of not higher than 44% (Table 8).

[0162]

Table 1. Dipeptides synthesized by BBR47_51900 and

Staur 4851.

Dipeptides (mM)

Enzyme

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 [0163]

Table 2. Multiple alignment (identity, in %) of BBR47 and Staur 4851 with known Lais.

acids residues among all ungapped positions between the pairs .

Protein abbreviation:

I - BBR47_51900 (NCBI Reference Sequence: YP_002774671.1) ; 2 - Staur_4851 (NCBI Reference Sequence: AD072629.1);

3 - TDE2209 (NCBI Reference Sequence: NP_972809.1 ) ;

4 - BL00235 (NCBI Reference Sequence: YP_081312.1 ) ;

5 - plul218 (NCBI Reference Sequence: NP_928530.1 ) ;

6 - YwfE (UniProtKB/Swiss-Prot : P39641.1);

7 - Rspl486 (NCBI Reference Sequence: NP_523045.1 ) ;

8 - NP_900476 (NCBI Reference Sequence: NP_900476.1 ) ;

9 - Aple02000835 (NCBI Reference Sequence: ZP_00134462.2 ) ;

10 - SMU1321c (NCBI Reference Sequence: NP_721690.1 ) ;

II - YP_816266 (NCBI Reference Sequence: YP_816266.1 ) ;

12 - YP_001544794 (NCBI Reference Sequence:

YP_001544794.1) ;

13 - YP_077482 (NCBI Reference Sequence: YP_077482.1 ) ;

14 - BAH56723 (GenBank: BAH56723.1);

15 - NP_358563 (NCBI Reference Sequence: NP_358563.1 ) ;

16 - YP_910063 (NCBI Reference Sequence: YP_910063.1 ) ;

17 - BAG72134 (GenBank: BAG72134.1);

18 - plul440 (NCBI Reference Sequence: NP_928738.1 ) ;

19 - AAZ37741 (GenBank: AAZ37741.1). [0164]

Table 3. Multiple alignment (similarity, in

BBR47 51900 and Staur 4851 with known Lais.

Similarity can be defined as percentage of identical plus similar amino acid residues

Protein abbreviation:

1 - BBR47_51900 (NCBI Reference Sequence: YP_002774671.1) ;

2 - Staur_4851 (NCBI Reference Sequence: AD072629.1 ) ;

3 - TDE2209 (NCBI Reference Sequence: NP_972809.1 ) ;

4 - BL00235 (NCBI Reference Sequence: YP_081312.1 ) ;

5 - plul218 (NCBI Reference Sequence: NP_928530.1 ) ;

6 - YwfE (UniProtKB/Swiss-Prot : P39641.1);

7 - Rspl486 (NCBI Reference Sequence: NP__523045.1) ;

8 - NP_900476 (NCBI Reference Sequence: NP_900476.1 ) ;

9 - Aple02000835 (NCBI Reference Sequence: ZP_00134462.2) ;

10 - SMU1321c (NCBI Reference Sequence: NPJ721690.1 ) ;

11 - YP_816266 (NCBI Reference Sequence: YP_816266.1 ) ;

12 - YP_001544794 (NCBI Reference Sequence:

YP_001544794.1) ;

13 - YP_077482 (NCBI Reference Sequence: YP_077482.1) ;

14 - BAH56723 (GenBank: BAH56723.1);

15 - NP_358563 (NCBI Reference Sequence: NP_358563.1 ) ;

16 - YP_910063 (NCBI Reference Sequence: YP_910063.1 ) ;

17 - BAG72134 (GenBank: BAG72134.1);

18 - plul440 (NCBI Reference Sequence: NP_928738.1 ) ;

19 - AAZ37741 (GenBank: AAZ37741.1) [0165]

Table 4. The specific aspartic peptide-hydrolyzing activity (A, in nmoles/mg min) in the E. coli 4-6Δ strains.

Standard Deviation: <5%

Abbreviations:

WT - BW25113;

4Δ - BW25113 (ΔρβρΒ, iadA: : AattB, pepE: : AattB, ybiK : AattB) ;

5Δ - BW25113 (ΔρβρΒ, iadA: :AattB, pepE: : AattB, ybiK: : AattB, dapE: : AattB;:

6Δ - BW25113 (ΔρβρΒ, iadA: :AattB, pepE: : AattB, ybiK: : AattB, dapE: :AattB, pepA: : AattL-cat-AattR) .

[0166]

Table 5. Investigation of the DP3 toxicity due to the specific aspartic peptide-hydrolyzing activity in the E. coli 5Δ strain.

++: the level of growth is equal to that of the wild-type strain observed at 0 mM DP3

+: the level of growth is lower compared to that of the wild-type strain observed at 0 mM DP3

+-: the level of growth could be observed but is very low compared to that of the wild-type strain observed at 0 mM DP3

-: growth could not be observed [0167]

Table 6. Purification of peptidases having specific aspartic peptide-hydrolyzing (DP3-hydrolyzing) activity.

[0168]

Table 7. Pair-wise alignment (identity, in of Lais.

Identity can be defined as percentage of identical amino acids residues among all ungapped positions between the pairs. BBR means BBR47 51900, and STA means Staur 4851.

[0169]

Table 8. Pair-wise alignment (similarity, in % of Lais .

Similarity can be defined as percentage of identical plus similar amino acid residues among all ungapped positions between the pairs. BBR means BBR47_51900, and STA means Staur 4851.

[0170]

Example 10. Enzymatic production of Asp-Phe with

BBR47_51900 and Staur_4851 using ATP regeneration system The product yield of Asp-Phe was studied in the reaction mixture of BBR47_51900 and Staur_4851 containing phosphoenolpyruvate and pyruvate kinase for regeneration of ATP in order to prevent highly concentrated ATP from inhibiting the enzyme reaction. The composition of the reaction mixture of total volume of 1 ml was as follows.

BBR47_51900 or Staur_ 4851 0.15 U

Tris-HCl pH 9.0 50 mM

L-AspNa 100 mM

L-Phe 100 mM

A P 10 mM

Phosphoenolpyruvate 100 mM

MgS0 ₄ x7H ₂ 0 10 mM

Pyruvate kinase 25 U

H ₂ 0 to a total volume of 1 mL [0171]

Reactions were carried out at 37°C for 1-48 hours. 100 μΐ, out of the 1 mL reaction mixture was sampled at each reaction time. Each reaction mixture, into which 10 μΐ, of 1 M EDTA (pH9.0) was added to stop the reaction, was

subjected to HPLC analysis. The condition was as described in Example . As a result, BBR47_51900 and Staur_4851 could produce Asp-Phe in the ATP regeneration system (Table 9) .

[0172]

Table 9.

[0173]

Example 11. Analysis of the specific Asp-Phe hydrolyzing activity in the E. coli 7Δ strain

11.1. Construction of the E. coli Asp-Phe hydrolysing peptidase-deficient 7Δ strains

The pepD gene was deleted in the E. coli J 109 strain. A DNA fragment bearing the AattL-ca t-AattR cassette was PCR amplified using the primers Pll (SEQ ID NO: 38) and P12 (SEQ ID NO: 39), and the pMW118-AattL-ca t-AattR plasmid as the template. The resulting DNA fragment was introduced into the E. coli JM109/pKD46 strain as described above. The Cm ^R - marker was excised from the E. coli JM109 (pepD: : AattR- cat-AattL) transformant to construct the E. coli JM109 {pepD: : AattB) strain.

[0174]

The pepE gene was deleted in the E. coli JM109 {pepD: : AattB) strain. A DNA fragment bearing the AattL-ca t-AattR cassette was PCR amplified using the primers P13 (SEQ ID NO: 40) and P14 (SEQ ID NO: 41), and the pMW118-AattL-ca.t- AattR plasmid as the template. The resulting DNA fragment was introduced into the E. coli JM109 (pepB: : AattB) / pKD46 strain as described above. The Cm ^R -marker was excised from the E. coli JM109 (pepD: : AattB, pepE: : AattR-cat- AattL) transformant to construct the E. coli JM109 (pepD: : AattB, pepE: : AattB) strain.

[0175]

The iadA gene was deleted in the E. coli JM109 {pepD: : AattB, pepE: : AattB) strain. A DNA fragment bearing the AattL-cat-AattR cassette was PCR amplified using the

primers P15 (SEQ ID NO: 42) and P16 (SEQ ID NO: 43), and the pMWH8-AattL-cat-AattR plasmid as the template. The resulting DNA fragment was introduced into the E. coli

JM109 {pepD: : AattB, pepE: : AattB) / pKD46 strain as

described above. The Cm ^R -marker was excised from the E.

coli JM109 (pepD: : AattB, pepE: : AattB, iadA:: AattR-cat- AattL) transformant to construct the E. coli JM109 (pepD: : AattB, pepE: : AattB, iadA:: AattB) strain.

[0176]

The pepA gene was deleted in the E. coli JM109 (pepD: :

AattB, pepE: : AattB, iadA: : AattB) strain. A DNA fragment bearing the AattL-cat-AattR cassette was PCR amplified using the primers P17 (SEQ ID NO: 44) and P18 (SEQ ID NO: 45), and the pMW118-AattL-cat-AattR plasmid as the template. The resulting DNA fragment was introduced into the E. coli JM109 (pepD: : AattB, pepE: : AattB, iadA: : AattB) / pKD46 strain as described above. The Cm ^R -marker was excised from the E. coli J 109 (pepD: : AattB, pepE: : AattB, iadA: : AattB, pepA: : AattR-cat-AattL) transformant to construct the E.

coli J 109 {pepD: : AattB, pepE: : AattB, iadA: : AattB, pepA: : AattB) strain.

[0177]

The pepB gene was deleted in the E. coli JM109 {pepD: : AattB, pepE: : AattB, iadA: : AattB, pepA: : AattB) strain. A DNA fragment bearing the AattL-cat-AattR cassette was PCR amplified using the primers P19 (SEQ ID NO: 46) and P20 (SEQ ID NO: 47), and the pMW118-AattL-cat-AattR plasmid as the template. The resulting DNA fragment was introduced into the E. coli J 109 {pepD: : AattB, pepE: : AattB, iadA: : AattB, pepA: : AattB) / pKD46 strain as described above. The Cm ^R -marker was excised from the E. coli JM109 (pepD: : AattB, pepE: : AattB, iadA: : AattB, pepA: : AattB, pepB: : AattR-cat- AattL) transformant to construct the E. coli JM109 {pepD: : AattB, pepE: : AattB, iadA: : AattB, pepA: : AattB, pepB: :

AattB) strain.

[0178]

The iaaA gene was deleted in the E. coli JM109 (pepD: : AattB, pepE: : AattB, iadA: : AattB, pepA: : AattB, pepB: :

AattB) strain. A DNA fragment bearing the AattL-c t-AattR cassette was PCR amplified using the primers P21 (SEQ ID NO: 48) and P22 (SEQ ID NO: 49), and the pMW118-AattL-cat- AattR plasmid as the template. The resulting DNA fragment was introduced into the E. coli JM109 {pepD: : AattB, pepE: : AattB, iadA:: AattB, pepA: : AattB, pepB: : AattB) / pKD46 strain as described above. The Cm ^R -marker was excised from the E. coli JM109 (pepD: : AattB, pepE: : AattB, iadA: : AattB, pepA: : AattB, pepB: : AattB, iaaA: : AattR-cat-AattL)

transformant to construct the E. coli J 109 (pepD: : AattB, pepE: : AattB, iadA: : AattB, pepA: : AattB, pepB: : AattB, iaaA: : AattB) strain.

[0179]

The dpp gene operon {dppA, dppB, dppC, dppD, dppF) was deleted in the E. coli JM109 {pepD: : AattB, pepE: : AattB, iadA: : AattB, pepA: : AattB, pepB: : AattB, iaaA: : AattB) strain. A DNA fragment bearing the AattL-cat-AattR cassette was PCR amplified using, the primers P23 (SEQ ID NO: 50) and P24 (SEQ ID NO: 51), and the pMW118-AattL-cat-AattR plasmid as the template. The resulting DNA fragment was introduced into the E. coli JM109 (pepD: : AattB, pepE: : AattB, iadA: : AattB, pepA: : AattB, pepB: : AattB, iaaA:: AattB) / pKD46 strain as described above. The Cm ^R -marker was excised from the E. coli JM109 (pepD: : AattB, pepE: : AattB, iadA: : AattB, pepA: : AattB, pepB: : AattB, iaaA: : AattB, dpp: : AattR-cat- AattL) transformant to construct the E. coli JM109 (pepD: : AattB, pepE: : AattB, iadA: : AattB, pepA: : AattB, pepB: :

AattB, iaaA: : AattB, dpp: : AattB) strain.

[0180]

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

Cells of E. coli J 109 and E. coli 7Δ strain were grown on LB agar medium at 37 °C for 16 hours. Grown cells were inoculated into 20 mL of MS medium and grown at 37 °C to cells density of ODgionm ~20. And then, the authentic Asp-Phe was added into the culture (final concentration of 2 mM) , further 32 hours cultivation was carried out. The resulting culture at each cultivation time was centrifuged to obtain a culture supernatant. The residual Asp-Phe in the culture supernatant was analyzed by HPLC. The results are shown in Table 10. Asp-Phe hydrolyzing activity in the culture of E. coli 7Δ strain was much lower as compared with that of E. coli JM109.

The composition of the MS medium is (g/L)

Glucose 20

(NH ₄ ) ₂ S0 ₄ 8

KH ₂ P0 ₄ 0.5

FeS0 ₄ x7H ₂ 0 0.005

MnS0 ₄ x7H ₂ 0 0.005

Yeast extract 1

L-Tyr 0.05 MgS0 ₄ x7H ₂ 0 0.5

CaC0 ₃ 30

[0181]

The fermentation medium is sterilized at 121 °C for 20 minutes, except that glucose, MgS0 ₄ 7H ₂ 0 and CaC0 ₃ are

sterilized separately and as follows: glucose and MgS0 ₄ 7H ₂ 0 at 121°C for 20 min, CaC0 ₃ at 180°C for 2 hours . The pH is adjusted to 7 by KOH solution. As a result, the specific Asp-Phe hydrolyzing activity was lowered in the E. coli 7Δ strain compared with that in the E. coli JM109 (Table 10).

[0182]

Table 10.

[0183]

Example 12. Evaluation of productivity of Asp-Phe by E.

coli 7Δ strain overexpressing a Lai gene

The primary structure of the genes encoding

BBR47_51900 and Staur_4851 was further optimized for

expression in E. coli. The genes encoding BBR47_51900 from Brevibacillus brevis NBRC 100599 and Staur_4851 from

Stigmatella aurantiaca DW4/3-1 were synthesized by the

GenScript and delivered as a set of pUC57 plasmids (pUC57- cBBR and pUC57-cSTA) . The nucleotide sequences of the prepared cBBR and cSTA are represented by SEQ ID NO: 68 and SEQ ID NO: 69, respectively.

[0184]

12.1. Construction of pSF12-cBBR and pSF12-cSTA

A DNA fragment bearing BBR47_51900 was PCR amplified using the primers P25 (SEQ ID NO: 52) and P26 (SEQ ID NO: 53), and the pUC57-cBBR plasmid as the template. The PCR was carried out using the following step program: 98 °C, 30 seconds; (98°C, 15 seconds; 58°C, 10 seconds; 72°C, 1 minute) x 30 cycles; 72°C, 5 minutes with 50 \iL of a

reaction mixture comprising 0.04 pg of the plasmid DNA, 0.2 μΓηο Ι/L each of the primers, 1.0 unit of Phusion High-

Fidelity DNA Polymerase (New England Labs), 10 μΐ of 5 x Phusion HF buffer and 0.2 mmol/L each dNTPs. The amplified DNA fragment was purified by inElute PCR Purification Kit (Qiagen) .

[0185]

A DNA fragment bearing Staur_4851 was PCR . amplified using the primers P27 (SEQ ID NO:54) and P28 (SEQ ID NO:55), and the pUC57-cSTA plasmid as the template. The PCR

condition and purification method was as described above.

[0186]

The thus obtained solutions were subjected to reaction to cleave the amplified DNA with restriction enzyme Nde I and Pst I, and then each 1.3 kb fragments was purified with MinElute Reaction Cleanup Kit (Qiagen) .

[0187]

pSF12-ggt vector was constructed from pUC18 vector and harbours the rpoH promoter and ggt gene encoding gamma- glutamyltranspeptidase from E. coli 3110 strain

(WO2013051685A1) . The pSF12-ggt vector was cleaved with Nde I and Pst I. DNA fragments were separated by agarose gel electrophoresis, and a 3.0 kb DNA fragment was recovered by QIAguick Gel Extraction Kit (Qiagen) .

[0188]

The 1.3 kb DNA fragment containing BBR47_51900 gene or Staur_4851 and the 3.0 kb DNA fragment obtained above were subjected. to ligation reaction using TaKaRa Ligation Kit Ver.2.1 (TaKaRa) at 16°C for 30 minutes. E. coli JM109 competent cell (TaKaRa) was transformed by a heat shock method using the ligation reaction mixture, spread on LB agar medium containing 100 g/mL ampicillin, and cultured overnight at 30 °C. A plasmid was extracted from a colony of the transformant that grew on the medium according to a known method, whereby pSF12-cBBR and pSF12-cSTA were obtained. The DNA sequence of the vectors was confirmed using 3130 Genetic Analyzer (Applied Biosystems) .

[0189]

12.2. Fermentative production of Asp-Phe using the modified E. coli 7Δ strains having Lai activity

A dipeptide-producing bacterium is the E. coli 7Δ strain as described above deficient of peptidase and dipeptide permease activity, further modified to have L- amino acid a-ligase activity. The dipeptide-producing strains harbour the gene encoding BBR47_51900 or Staur_4851 introduced into the bacterial cell on a plasmid, pSF12-cBBR or pSF12-cSTA, respectively. Each genes encoding Lais is placed under the rpoH promoter. The modified E. coli 7Δ strains harboring the gene encoding Lai and the control 7Δ strain were each cultivated at 25°C for 24 hours on LB agar medium (containing 100 g/ml ampicillin for the modified E. coli 7Δ strains harboring the gene encoding Lai) . The E. coli 7Δ strains harboring gene encoding Lai and the control 7Δ strain were inoculated into 20 mL of a MS medium

supplemented with 100 mM L-Asp and 100 mM L-Phe in 500 mL Sakaguchi flask and cultivated at 25°C for 32 hours on a reciprocal shaker at 120 rpm. The resulting culture was centrifuged to obtain a culture supernatant. Accumulated Asp-Phe in the culture supernatant was analyzed by HPLC. The results are shown in Table 11. [0190]

Table 11.

[0191]

As it can be seen from Table 11, Asp-Phe was not produced by use of the 7Δ strain, whereas Asp-Phe was produced by use of 7Δ strains harbouring gene encoding Lai.

[0192]

Example 13. Evaluation of Asp-Phe productivity by E. coli 9A/pMGALl/pHSG-cLal

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

First, to derepress the synthesis of enzymes involved in the biosynthesis of aromatic amino acids, the tyrR gene was deleted in the E. coli JM109 (pepD: : AattB, pepE: :

AattB, iadA: : AattB, pepA: : AattB, pepB: : AattB, iaaA: :

AattB, dpp: : AattB) strain. A DNA fragment bearing the AattL-cat-AattR cassette was PCR amplified using the primers P29 (SEQ ID NO: 56) and P30 (SEQ ID NO: 57), and · the pM 118-AattL-cat-AattR plasmid as the template. The resulting DNA fragment was introduced into the E. coli JM109 (pepD: : AattB, pepE: : AattB, iadA: : AattB, pepA: :

AattB, pepB: : AattB, iaaA:: AattB-, dpp:: AattB) / pKD46 strain as described in Example 11.1. The Cm ^R -marker was excised from the E. coli JM109 (pepD: : AattB, pepE: : AattB, iadA: : AttB, pepA: : AattB, pepB: : AattB, iaaA: : AattB, dpp: : AattB, tyrf?: : AattR-cat-AattL) transformant to construct the E. coli JM109 [pepD: : AattB, pepE: : AattB, iadA: : AattB, pepA: : AattB, pepB: : AattB, iaaA: : AattB, dpp: : AattB, tyrR: : AattB) strain.

[0193]

Next, the tyrA gene was deleted in the E. coli JM109 {pepD: : AattB, pepE : AattB, iadA: : AattB, pepA: : AattB, pepB: : AattB, iaaA: : AattB, dpp: : AattB, tyrR: : AattB) strain so that a prephenate, the common intermediate in the biosynthesis of Phe and Tyr, wouldn't be utilized for Tyr biosynthesis. A DNA fragment bearing the AattL-cat-AattR cassette was PCR amplified using the primers P31 (SEQ ID NO: 58) and P32 (SEQ ID NO: 59), and the pMW118-AattL-cat- AattR plasmid as the template. The resulting DNA fragment was introduced into the E. coli JM109 (pepD: : AattB, pepE: : AattB, iadA: : AattB, pepA: : AattB, pepB: : AattB, iaaA: :

AattB, dpp: : AattB, tyrR: : AattB) / pKD46 strain as

described above. The Cm ^R -marker was excised from the E.

coli JM109 [pepD: : AattB, pepE: : AattB, iadA: : AattB, pepA: : AattB, pepB: : AattB, iaaA: : AattB, dpp: : AattB, tyrR: : AattB, tyrA: : AattR-ca -AattL) transformant to construct the E. coli JM109 (pepD: : AattB, pepE: : AattB, iadA: : AattB, pepA: : AattB, pepB: : AattB, iaaA: : AattB, dpp:: AattB, tyrR:: AattB, tyrA:: AattB) strain.

[0194]

13.2. Construction of pHSG-cBBR and pHSG-cSTA

To construct the pHSG-cBBR plasmid, the corresponding EcoRI - SphI fragment containing of the rpoH promoter and BBR47_51900 gene of the pSF12-cBBR plasmid were excised by digestion with EcoRI and SphI and then ligated with the pHSG396 vector (TaKaRa) digested by the same restrictases .

To construct the pHSG-cSTA, a DNA fragment bearing Staur_4851 under the rpoH promoter was PCR amplified using the primers P33 (SEQ ID NO: 60) and P34 (SEQ ID NO: 61), and the pSF12-cSTA plasmid as the template. The PCR was carried out using the following step program: 98 °C, 30 seconds; (98°C, 15 seconds; 58°C, 10 seconds; 72°C, 1 minute) x 30 cycles; 72 °C, 5 minutes with 50 μΐ, of a reaction mixture comprising 0.04 yg of the plasmid DNA, 0.2 μπιοΙ/L each of the primers, 1.0 unit of Phusion High-Fidelity DNA

Polymerase (New England Labs) , 10 pL of 5 x Phusion HF buffer and 0.2 mmol/L each dNTPs. Then 1.5 kb fragment digested by BamHI and Xhol was ligated with pHSG396 vector digested by the same restrictases .

[0195]

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

pMGALl vector was constructed from pMW19 (Wako) and harbours three genes involved in Phe biosynthesis in E.

coli; pheA, aroG4 and aroL encoding chorismate mutase- prephenate dehydratase (CM-PD) , 3-deoxy-D- arabinoheptulosonate-7-phosphate synthetase (DAHP

synthetase) and shikimate kinase (SK) , respectively

(JP3225597). Each of pheA and aroG4 genes was mutated from the corresponding original genes to avoid negative feedback by Phe biosynthesized.

[0196]

pMGALl and pHSG-cLal vector was simultaneously

introduced into E. coli 9Δ strain by electroporation method using LB agar medium containing 100 pg/mL ampicillin and 25 g/mL chloramphenicol. Thus obtained strain was named E. coli 9A/pMGALl/pHSG-cLal.

[0197]

13.4. Fermentative production of Asp-Phe by E. coli

9A/pMGALl/pHSG-cLal

The modified E. coli 9A/pMGALl/pHSG-cLal and the control 9Δ strain/pMGALl were each cultivated at 25°C for 24 hours on LB agar medium (containing 100 g/ml ampicillin and 25 yg/ml chloramphenicol for the E. coli 9A/pMGALl/pHSG-cLal) . The E. coli 9A/pMGALl/pHSG-cLal and the control 9A/pMGALl strain were inoculated into 20 mL of a MS medium supplemented with 100 mM L-Asp in 500 mL

Sakaguchi flask and cultivated at 25°C for 72 hours on a reciprocal shaker at 120 rpm. The resulting culture was centrifuged to obtain a culture supernatant. Accumulated Phe and Asp-Phe in the culture supernatant was analyzed by HPLC. The results are shown in Table 12.

[0198]

Table 12.

[0199]

As it can be seen from Table 12, Asp-Phe was not produced by use of the 9A/pMGALl strain, whereas Asp-Phe was produced by use of 9A/pMGALl strains harboring gene encoding Lai.

[0200]

Example 14. Analysis of substrate specificity of DES

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

14.1.1. Construction of the ancillary plasmid pELAC

The <PlacUV5> DNA-fragment was PCR-amplified using oligoprimers P35 (SEQ ID NO: 62), P36 (SEQ ID NO: 63), and DNA of pUC18 plasmid (GenBank: L08752.1) as a template.

Resulting DNA fragment was digested by Bglll and Xbal and cloned into pET22 (b+) plasmid (Novagen, Cat. No. 69744- 3) digested by the same endonucleases thus constructing the pELAC plasmid.

[0201]

14.1.2. Construction of the ancillary plasmid pELAC-MBP-HT The malE gene (without signal peptide sequence) was PCR-amplified using oligoprimers P37 (SEQ ID NO: 64), P38 (SEQ ID NO:65), and E. coli MG1655 chromosome as a template. Obtained DNA-fragment was digested by Xbal and BamHI and cloned into pE AC-/Xbal-BamHI vector thus constructing pELAC-MBP-HT plasmid.

[0202]

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

To construct the pELAC-MBP-DES-HT plasmid, we

amplified DNA fragment containing DES gene by using

oligoprimers P39 (SEQ ID NO: 66), P40 (SEQ ID NO :67), and DNA of pUC57-DES (described in dipeptide patent

application) as a template. Obtained DNA fragment was digested with BamHI and NotI and ligated with pELAC-MBP- HT/BamHI-NotI vector thus constructing the pELAC-MBP-DES-HT plasmid.

[0203]

14.2. Expression and purification of MBP fusion His ₆ ~tagged DES

Cells ^' of E. coli 7Δ harboring pELAC-MBP-DES-HT was grown in LB medium containing 100 μg/mL ampicillin in a test tube at 37 °C up to OD ₆ ionm ~2. 2 mL of the resulting culture was inoculated into 100 mL of LB medium

supplemented with IPTG (final concentration of 0.1 mmol/L) in a 500 ml Sakaguchi flask at 30°C, for 8 hours. Induced cells were harvested from 1.6 L of cultivation broth, re- suspended 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 using sonicator (INSONATOR 201M, KUBOTA) . The debris was removed by centrifugation at 4°C and 14,000 rpm for 15 minutes followed by filtration through 0.45 mm filter (Millipore) . A solution of crude proteins was loaded onto HisTALON Superflow cartridge, 5 ml (Clontech) using AKTA avant 25 (GE Healthcare) in accordance with the manufacturer's recommendations. Fractions containing MBP fused His6-tagged DES were combined, and desalted using PD- 10 columns (GE Healthcare) equilibrated with SC-buffer (50 mM Tris-HCl, pH8.0, 0.3 M NaCl, 15% glycerol).

[0204]

14.3. Analysis of Asp-Phe synthesizing activity of MBP fused His6-tagged DES

Dipeptides synthesized by MBP fused His6~tagged DES were determined using HPLC analysis of reaction mixture of total volume of 400 pL, which contained:

DES 160 iq

Tris-HCl, pH 9.0 50 mM

L-Asp 100 mM

L-Phe 100 mM

Adenosine 5' -triphosphate (ATP) 10 mM

MgS0 ₄ x7H ₂ 0 10 mM, where Me denote methyl group.

Reactions were carried out at 37 °C for 15 hours. Then the reaction mixture, into which 10 μΐ of 1 M EDTA, pH9.0 was added to stop the enzymatic reaction, was subjected to HPLC analysis. The conditions were as described in Example . As a result, DES catalyzes formation of 0.30 mM Asp-Phe.

[0205]

While the invention has been described in detail with reference to preferred embodiments thereof, it will be apparent to the one skilled in the art that various changes can be made, and equivalents employed, without departing from the scope of the invention. All the cited references herein are incorporated as a part of this application by reference.

Description of Sequences

SEQ ID NO:l shows the BBR47 SEQ ID NO: 2 shows the BBR47_51900 protein

SEQ ID NO: 3 shows the Staur_4851 gene

SEQ ID NO: 4 shows the Staur_4851 protein

SEQ ID NO: 5 shows the DES gene

SEQ ID NO: 6 shows the DES protein

SEQ ID NO: 7 shows the BUR gene

SEQ ID NO: 8 shows the BUR protein

SEQ ID NO: 9 shows the BCE gene

SEQ ID NO: 10 shows the BCE protein

SEQ ID NO: 11 shows the BTH gene

SEQ ID NO: 12 shows the BTH protein

SEQ ID NO: 13 shows the AME gene

SEQ ID NO: 14 shows the TAME protein

SEQ ID NO: 15 shows the SFL gene

SEQ ID NO: 16 shows the SFL protein

SEQ ID NO: 17 shows the BMY gene

SEQ ID NO: 18 shows the BMY protein

SEQ ID NO: 19 shows the Xbal -EcoRI fragment harboring

BBR47_51900

SEQ ID NO: 20 shows the Xbal -EcoRI fragment harboring

Staur_J 1851

SEQ ID NO: 21 shows the Xbal -EcoRI fragment harboring DES

SEQ ID NO: 22 shows the Xbal -EcoRI fragment harboring BUR

SEQ ID NO: 23 shows the Xbal -EcoRI fragment harboring BCE

SEQ ID NO: 24 shows the Xbal -EcoRI fragment harboring BTH

SEQ ID NO: 25 shows the Xbal -EcoRI fragment harboring AME

SEQ ID NO: 26 shows the Xbal -EcoRI fragment harboring SFL

SEQ ID NO: 27 shows the Xbal -EcoRI fragment harboring BMY

SEQ ID NO: 28 shows the Primer PI

SEQ ID NO: 29 shows the Primer P2

SEQ ID NO: 30 shows the Primer P3

SEQ ID NO: 31 shows the Primer P4

SEQ ID NO: 32 shows the Primer P5 SEQ ID NO: 33 shows the Primer P6

SEQ ID NO: 34 shows the Primer P7

SEQ ID NO: 35 shows the Primer P8

SEQ ID NO: 36 shows the Primer P9

SEQ ID NO: 37 shows the Primer P10

SEQ ID NO: 38 shows the Primer Pll

SEQ ID NO: 39 shows the Primer P12

SEQ ID NO: 40 shows the Primer P13

SEQ ID NO: 41 shows the Primer P14

SEQ ID NO: 42 shows the Primer P15

SEQ ID NO: 43 shows the Primer P16

SEQ ID NO: 44 shows the Primer P17

SEQ ID NO: 45 shows the Primer P18

SEQ ID NO: 46 shows the Primer P19

SEQ ID NO: 47 shows the Primer P20

SEQ ID NO: 48 shows the Primer P21

SEQ ID NO: 49 shows the Primer P22

SEQ ID NO: 50 shows the Primer P23

SEQ ID NO: 51 shows the Primer P24

SEQ ID NO: 52 shows the Primer P25

SEQ ID NO: 53 shows the Primer P26

SEQ ID NO: 54 shows the Primer P27

SEQ ID NO: 55 shows the Primer P28

SEQ ID NO: 56 shows the Primer P29

SEQ ID NO: 57 shows the Primer P30

SEQ ID NO: 58 shows the Primer P31

SEQ ID NO: 59 shows the Primer P32

SEQ ID NO: 60 shows the Primer P33

SEQ ID NO: 61 shows the Primer P34

SEQ ID NO: 62 shows the Primer P35

SEQ ID NO: 63 shows the Primer P36

SEQ ID NO: 64 shows the Primer P37

SEQ ID NO: 65 shows the Primer P38 SEQ ID NO: 66 shows the Primer P39

SEQ ID NO: 67 shows the Primer P40

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

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