WO2006093322A2 - Method for manufacturing 4-hydroxy-l-isoleucine or a salt thereof - Google Patents

Abstract

Present invention provides a method for manufacturing 4-hydroxy-L-isoleucine. A method for manufacturing 4-hydroxy-L-isoleucine or a salt thereof, characterized by comprising the steps of contacting the biocatalyst having activity in catalyzing a reaction producing 4-hydroxy-L-isoleucine from acetaldehyde and a-ketobutyric acid or a salt thereof in the presence of an amino group donor with acetaldehyde and a-ketobutyric acid or a salt thereof in the presence of an amino group donor, and producing 4-hydroxy-L-isoleucine

Description

DESCRIPTION

Method for Manufacturing 4-hydroxy-L-isoleucine or a salt thereof

Technical Field

The present invention relates to the microbiological industry, and specifically to methods for manufacturing 4-hydroxy-L-isoleucine or a salt thereof.

Technical Background

4-hydroxy-L-isoleucine is an amino acid which can be extracted and purified from fenugreek seeds (Trigonella foenum-graecum L. leguminosae). 4-hydroxy-L-isoleucine displays an insulinotropic activity of great interest because its stimulating effect is clearly dependent on plasma glucose concentration in the medium, as demonstrated both in isolated perfused rat pancreas and human pancreatic islets (Sauvaire, Y. et al, Diabetes, 47: 206-210, (1998)). Such a glucose dependency is not confirmed on sulfonylureas (Drucker, D. J., Diabetes Al: 159-169, (1998)), the only insulinotropic drug currently used to treat type II diabetes [or non-insulin-dependent diabetes (NIDD) mellitus (NIDDM)], and as a consequence, hypoglycemia remains a common undesirable side effect of sulfonylurea treatment (Jackson, J., and Bessler, R. Drugs, 22: 211-245; 295-320, (1981); Jennings, A. et al. Diabetes Care, 12: 203-208, (1989)). Improvement of glucose tolerance (Am. J. Physiol. Endocrinol., Vol. 287, E463-E471, 2004) is also known. This glucometabolism enhancement activity, and its potential application to pharmaceuticals and health foods, have been reported (Japanese Patent Application Laid-Open No. Hei 6-157302).

4-hydroxy-L-isoleucine, which is only found in plants, due to its particular insulinotropic action, might be considered as a novel secretagogue with potential interest for the treatment of type II diabetes, a disease characterized by a defective insulin secretion associated with various degrees of insulin resistance (Broca, C. et al, Am. J. Physiol. 277 (Endocrinol. Metab. 40): E617-E623, (1999)).

A method of oxidizing iron, ascorbic acid, 2-oxyglutaric acid, and oxygen-dependent isoleucine by utilizing dioxygenase activity in fenugreek extract has been reported as a method for manufacturing 4-hydroxy-L-isoleucine (Phytochemistry, Vol. 44, No. 4, pp. 563-566, 1997). However, this method is unsatisfactory as a method of manufacturing 4-hydroxy-L-isoleucine because the activity of the enzyme is inhibited by the substrate at isoleucine concentrations of 20 niM and above, the enzyme has not been identified, the enzyme is derived from plant extracts and is not readily obtained in large quantities, and the enzyme is unstable.

An efficient eight-step synthesis of optically pure (2S,3R,4S)-4-hydroxyisoleucine with

39% overall yield has been disclosed. The key steps of this synthesis involve the biotransformation of ethyl 2-methylacetoacetate to ethyl (2S,3S)-2-methyI-3-hydroxybutanoate with Geotrichum candidum and an asymmetric Strecker synthesis (Wang, Q. et al, Eur. J. Org.

Chem., 834-839 (2002)).

A short six-step chemoenzymatic synthesis of (2S,3R,4S)-4-hydroxyisoleucine with total control of stereochemistry, the last step being the enzymatic resolution by hydrolysis of a N-phenylacetyl lactone derivative using the commercially available penicillin acylase G immobilized on Eupergit C(E-PAC), has also been disclosed (Rolland-Fulcrand, V. et al, J. Org.

Chem., 873-877 (2004)).

But currently, there have been no reports of producing 4-hydroxy-L-isoleucine by enzymatic transamination of 4-hydroxy-3-methyl-2-keto-pentanoic acid or by the other enzymatic conversion from the other starting materials.

Description of the Invention

Accordingly, the problem to be solved by the present invention is how to provide a method for manufacturing 4-hydroxy-L-isoleucine (used to mean including both free form and a salt form thereof, and may referred to as "4HIL"; hereinafter the same) that lends itself to industrial use.

In order to solve the above-stated problem, the present inventors used aldol reaction to obtain 4-hydroxy-3-methyl-2-keto-pentanoic acid (used to mean including both free form and a salt form thereof, and may referred to as "HMKP"; hereinafter the same), a precursor of 4HIL, and focused on methods of using aminotransferase to convert it to 4HTL, examining the use of MhpE aldolase derived from E. coli (Appl. Environ. Microbiol, Vol. 64, No. 10, 4093-4094, 1998) and the aldolase reported by Sugiyama et al. (WO2004-018672). However, the quantity of HMKP produced was consistently less than 1 μM, which was inadequate for use in transamination. Accordingly, further investigation resulted in the discovery of enzymes suited to aldol reaction and transamination, respectively. Moreover, a new microorganism having novel enzymatic activity capable of simultaneously conducting both reactions was discovered. That is, based on the present invention, these biocatalysts can be used to provide a method for conveniently manufacturing 4-hydroxy-L-isoleucine. Accordingly, objects of the present invention include providing an enzymatic method for producing HMKlP by aldol reaction of acetaldehyde and α-ketobutyric acid (used to mean including both free form and a salt form thereof; hereinafter the same) using bacterial aldolase using bacterium. Further objects of the present invention include providing an enzymatic method for producing 4HIL by transamination of HMKP using a bacterial aminotransferase, and providing a method for producing 4HIL from HMKP using a bacterium which has been modified to have an enhanced aminotransferase activity, particularly a branched-chain amino acid aminotransferase.

Still further, objects of the present invention include providing an enzymatic method for producing 4HIL by two steps of aldol "reaction and transamination.

That is, the present invention comprises at a minimum the following:

[1] A method for manufacturing 4-hydroxy-L-isoleucine or a salt thereof, comprising the steps of: contacting a biocatalyst that actively catalyzes a reaction producing 4-hydroxy-L-isoleucine that is shown by the following Formula (I) from acetaldehyde and α-ketobutyric acid in the presence of an amino group donor,

---co

OH NH₂ with acetaldehyde and α-ketobutyric acid in an aqueous solvent containing an amino group donor, and isolating 4-hydroxy-L-isoleucine.

[2] The method according to [1], wherein the reaction is conducted in the presence of

NADH or NADPH.

[3] The method according to any of [1] and [2], wherein the aqueous solvent contains branched-chain amino acid aminotransferase. [4] The method according to any of [1] to [3], wherein the amino group donor is selected from the group of branched-chain amino acids.

[5] The method according to any of [1] to [4], wherein the biocatalyst is comprised of an enzyme having aldolase activity of producing 4-hydroxy-3-methyl-2-keto-pentanoic acid from acetaldehyde and α-ketobutyric acid, and an enzyme having transamination activity of producing 4-hydroxy-L-isoleucine from 4-hydroxy-3-methyl-2-keto-pentanoic acid in the presence of an amino group donor. [6] The method according to any of [1] to [4], wherein the biocatalyst is a bacterium containing an enzyme having aldolase activity of producing 4-hydroxy-3-methyl-2-keto-pentanoic acid from acetaldehyde and α-ketobutyric acid and an enzyme having transamination activity of producing 4-hydroxy-L-isoleucine from 4-hydroxy-3-methyl-2-keto-pentanoic acid in the presence of an amino group donor.

[7] The method according to [6], wherein the bacterium is modified to enhance at least one activities of aldolase and branched-chain amino acid aminotransferase.

[8] The method according to [7], wherein the activities of aldolase and branched-chain amino acid aminotransferase is enhanced by increasing the expression of the aldolase and/or the branched-chain amino acid aminotransferase.

[9] The method according to [8], wherein the expression of the aldolase and/or the branched-chain amino acid aminotransferase is increased by modifying an expression control sequence of the gene encoding said aldolase and/or branched-chain amino acid aminotransferase or by increasing the copy number of the gene encoding said aldolase and/or branched-chain amino acid aminotransferase.

[10] The method according to any of [5] to [10], wherein the enzyme having aldolase activity is an aldolase belonging to the hpcH/hpal family of aldolases.

[11] The method according to any of [5] to [10], wherein the enzyme having transamination activity is an aminotransferase belonging to the branched-chain amino acid aminotransferase. [12] The method according to any of [6] to [7] wherein the bacterium is of the genus Schizosaccharomyces, Arthrobacter, Brevibacterium, Candida, Corynebacterium, Micrococcus, Cellulomonas, Actinoplanes, Chromobacterhim, Rahnella, Rhizobiwn, Erwinia, Hansenula, Torulopsis, Kloeckera, Rhodotorula, Panellus, Mucor, Debaryomyces, Sporobolomyces, Escherichia, Salmonella, Flavobacterium, Bacillus, or Proteus. [13] The method according to any of [6], [7] and [12] wherein the bacterium belongs to Schizosaccharomyces pombe, Arthrobacter simplex, Brevibacterium ammoniagenes, Candida utilis, Micrococcus luteus, Micrococcus flavus, Micrococcus roseus, Corynebacterium glutamicum, Corynebacterium aquaticum, Coiγnebacterium paurometabolum, Arthrobactor globiformis, Arthrobactor sulfureus, Arthrobactor viscosus, Brevibacterium protophormiae, Brevibacterium acetylicum, Brevibacterium stationis, Brevibacterium fuscum, Cellulomonas fimi, Cellulomonas biazotea, Actinoplanes auranticolor, Chromobacterium iodinum, Citrobacter fi-eundii, Erwinia carotovora subsp. cai'otovora, Rahnella aquatilis, Rhizobiwn radiobacter, Hansenula anomala, Hansenula miso, Candida stellata, Hansenula saturnus, Hansenula nonfermentans, Hansenula polymorpha, Torulopsis nitratophila, Candida guilliermondii, Candida lipolytica, Candida macedoniensis, Candida pseudotropicalis, Candida tropicalis van lambica,

Candida soϊani, Candida albicans, Kloeckera africana, Kloeckera japonica, Rhodotorula mucilaginosa, Panellus serotinus, Miicor racemosus f.sp. racemosus, Mucor lamprosporus, Mucor petrinsularis, Debaryomyces vanrijiae, Sporobolotnyces roseus, Escherichia coli K12,

Salmonella typhimurium, Flavobacterium ferrugineum, Bacillus subtilis or Proteus mirabilis.

[14] The method according to any of [6], [7], [12] and [13] wherein the bacterium is bacterial culture products, cells, or treated cells.

[15] A method for manufacturing 4-hydroxy-L-isoleucine or a salt thereof, comprising the steps of: contacting a biocatalyst having activity in the production of 4-hydroxy-L-isoleucine from 4-hydroxy-3-methyl-2-keto-pentanoic acid, in the presence of an amino group donor with

4-hydroxy-3-methyl-2-keto-pentanoic acid in an aqueous medium containing an amino group donor, and isolating 4-hydroxy-L-isoleucine.

[16] The method according to [15] wherein the biocatalyst is an enzyme having transamination activity of producing 4-hydroxy-L-isoleucine from

4-hydroxy-3-methyl-2-keto-pentanoic acid in the presence of an amino group donor.

[17] The method according to [15] wherein the biocatalyst is a bacterium containing an enzyme having transamination activity of producing 4-hydroxy-L-isoleucine from

4-hydroxy-3-methyl-2-keto-pentanoic acid in the presence of an amino group donor.

[18] The method according to any of [15] to [16], wherein the transamination is performed by an isolated branched-chain amino acid aminotransferase.

[19] The method according to [18], wherein the branched-chain amino acid aminotransferase is isolated from a bacterium selected from the group consisting of Escherichia and Bacillus.

[20] The method according to any of [15] to [17], wherein the

4-hydroxy-3-methyl-2-keto-pentanoic acid is obtained by aldol reaction of α-ketobutyric acid and acetaldehyde.

[21] The method according to [17], wherein the bacterium is modified to enhance an activity of branched-chain amino acid aminotransferase.

[22] The method according to [21], wherein the activity of branched-chain amino acid aminotransferase is enhanced by increasing the expression of branched-chain amino acid aminotransferase. [23] The method according to [22], wherein the expression of branched-chain amino acid aminotransferase is increased by modifying an expression control sequence of the gene encoding said branched-chain amino acid aminotransferase or by increasing the copy number of the gene encoding the branched-chain amino acid aminotransferase. [24] The method according to any of [17] and [21] wherein the bacterium is of the genus Schizosaccharomyces, Candida, Arthrobacter, Brevibacterium, Cryptococcus, Pseudomonas, Hansenula, Flavohacterium, Bacillus, Micrococcus, Pichia, Escherichia or Torulopsis. [25] The method according to any of [17], [21] and [24] wherein said bacterium is Schizosaccharomyces pombe, Arthrobacter simplex, Brevibacterium ammoniagenes, Cryptococcus flavus, Candida utilis, Pseudomonas sp., Flavobacterium heparinum, Bacillus thuringiensis, Bacillus subtilis, Micrococcus luteus, Pichia orientalis, Hansenula jadinii, Torulopsis sphaerica, Escherichia coli or Brevibacterium linens.

[26] The method according to any of [17], [21], [24] to [25] wherein the bacterium is bacterial culture products, cells, or treated cells. [27] The method according to any of [1] to [26] wherein the 4-hydroxy-L-isoleucine is selected from at least one member of the group consisting of (2S,3S,4S)-4-hydroxyisoleucine, (2S,3R,4R)-4-hydroxyisoleucine, (2S,3S,4R)-4-hydroxyisoleucine and (2S,3R,4S)-4hydroxy- isoleucine.

Based on the present invention, it is possible to manufacture in an industrially useful manner 4HIL, for which no convenient manufacturing method was previously known.

Brief Description of the Drawings

Figure 1 shows the effects of amino acids as amino group donors on the level of production by Brevibacterium ammoniagenes of 4HIL from acetaldehyde and α-ketobutyric acid. The relative production levels for various amino acids are given when the quantity of 4HIL produced using glutamic acid as the amino group donor was adopted as 100%.

Figure 2 shows the effects of amino acids as amino group donors on the level of production by Arthrobactor simplex of 4HIL from acetaldehyde and cc-ketobutyric acid. The relative production levels for various amino acids are given when the quantity of 4HIL produced using glutamic acid as the amino group donor was adopted as 100%.

Figure 3 shows accumulation of 4HIL and α-aminobutyric acid analyzed by HPLC.

Figure 4 shows purification of HMKP-aldolase from Arthrobacter simplex AKU 626 (IFO 12069). Figure 5 shows determination of the HPAL oligomeric structure: A - Determination the Mw of the asiHPAL monomer using calibrated SDS - PAGE gel. PageRuler™ Protein Ladder (Fermentas, Lithuania) was used as a set of protein markers. Experimental data (black circle) were fitted (black line) by linear regression analysis using Sigma Plot 8 software. B - Determination native Mw of the asiHPAL using SEC on the Superdex™ 200 HR 10/3 OA (Farmacia) columns calibrated by Molecular Weight Protein Markers (Sigma). Experimental data (black circle) were fitted (black line) by linear regression analysis using Sigma Plot 8 software.

Figure 6 shows alignment of the determined N-terminal sequence with all known proteins (BLAST services). Figure 7 shows construction of the MG1655[mhpD::attL-kan-attR-Ptac-RBS] strain.

Figure 8 shows replacement of regulatory part of yfaU and yhaF genes by artificial Ptac-RBS expression module in the chromosome of E. coli MG 1655.

Figure 9 shows expression of yfaU and yhaF genes under the control of Ptac-RBS expression module. Lane: 1, 4-Mw marker; 2,5- MG1655 crude sell lysate preparation (about 20 μg applied), 3- MG1655-attLΔyfaV-Km-attR Ptac-RBS- yfaU crude sell lysate preparation (about

20 μg applied); 6- MG1655-attLΔyhaU-Km-attR Ptac-RBS- yhaF crude sell lysate preparation

(about 20 μg applied).

Figure 10 shows investigation of HMKP-aldolase activity in crude cell lysates of MG1655[attLΛyfaV-Km-attR Ptac-RBS- yfaU] and MG1655[attLΔyhaU-Km-attR Ptac-RBS- yhaF] strains using TLC analisis. Abbreviation: 4HIL - 4-hydroxy-isoleucine; AABA - α-aminobutyric acid; GLU - L-glutamic acid. Developer: acetoneπsopropanol: (NH₄)OH:H20 = 100:100:25:16. Lanes (2 μl of reaction mixture applied): 1,2,3,4 -MG1655; 5,6,7,8 - MG1655-attLΔyfaV-Km-attR Ptac-RBS- yfaU; Tracks-9, 10, 11, 12 MG1655-attLΔyhaU-Km-attR Ptac-RBS- yhaF. Figure 11 shows purification of YfaU and YhaF aldolases. A) SDS-PAGE gel of final enzyme preparations. Lane: 1-Mw protein marker; 2- YfaU preparation 10 μg protein applied; 3- YhaF preparation 10 μg protein applied. B) Purification steps.

Best Mode of Implementing the Invention The present invention is described in detail below.

1. General definitions and methods usable for practicing the present invention

In the present Specification, the term "4-hydroxy-L-isoleucine" or "4HIL" refers to single compound or diastereomer mixture comprised of two or more selected from the group consisting of (2S,3S,4S)-4-hydroxyisoleucine, (2S,3R,4R)-4-hydroxyisoleucine,

(2S,3 S,4R)-4-hydroxyisoleucine, and (2S,3R,4S)-4-hydroxyisoleucine.

The term "bacterium" as employed in the present Specification includes an enzyme-producing bacteria, a mutant and a genetic recombinant of such bacteria in which the targeted enzymatic activity exists or has been enhanced, and the like.

The phrase "increasing the expression of the gene" means that the expression of the gene is higher than that of a non-modified strain, for example, a wild-type strain. Examples of such modifications include increasing the copy number of expressed gene(s) per cell, increasing the expression level of the gene(s), and so forth. The quantity of the copy number of an expressed gene is 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 gene expression can be measured by various known methods including

Northern blotting, quantitative RT-PCR, and the like. The amount of the protein encoded by the gene can be measured by known methods including SDS-PAGE followed by immunoblotting assay (Western blotting analysis), and the like.

"Transformation of a bacterium with DNA encoding a protein" means introduction of the DNA into a bacterium, for example, by conventional methods. Transformation of this DNA will result in an increase in expression of the gene encoding the protein of present invention, and will enhance the activity of the protein in the bacterial cell. Methods of transformation include any known methods that have hitherto been reported. For example, a method of treating recipient cells with calcium chloride so as to increase permeability of the cells to DNA has been reported for Escherichia coli K-12 (Mandel, M. and Higa, A., J. MoI Biol, 53, 159 (1970)) and may be used.

Methods of enhancing gene expression include increasing the gene copy number. Introducing a gene into a vector that is able to function in a bacterium of the present invention increases the copy number of the gene. For such purposes multi-copy vectors can be preferably used. The multi-copy vector is exemplified by pBR322, pMWl 19, pUC19, pET22b, or the like. Enhancement of gene expression may also be achieved by introduction of multiple copies of the gene into a bacterial chromosome by, for example, homologous recombination, Mu integration, or the like. For example, one act of Mu integration allows for introduction of up to 3 copies of the gene into a bacterial chromosome.

Increasing the copy number of the gene can also be achieved by introducing multiple copies of the gene into the chromosomal DNA of the bacterium. In order to introduce multiple copies of the gene into a bacterial chromosome, homologous recombination is carried out using a sequence which exists in multiple copies as targets in the chromosomal DNA. Sequences having multiple copies in the chromosomal DNA include, but are not limited to repetitive DNA, or inverted repeats existing at the end of a transposable element. Also, as disclosed in US patent No. 5,595,889, it is possible to incorporate the gene into a transposon, and allow it to be transferred to introduce multiple copies of the gene into the chromosomal DNA.

Enhancing gene expression may also be achieved by placing the DNA of the present invention under the control of a potent promoter. For example, the P_tao promoter, the lac promoter, the trp promoter, the trc promoter, the P_R, or the P_L promoter of lambda phage are all known to be potent promoters. The use of a potent promoter can be combined with multiplication of gene copies.

Alternatively, the effect of a promoter can be enhanced by, for example, introducing a mutation into the promoter to increase the transcription level of a gene located downstream of the promoter. Furthermore, it is known that substitution of several nucleotides in the spacer between ribosome binding site (RBS) and the start codon, 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 et al, Annu. Rev. Microbiol, 35, 365-403, 1981; Hui et al, EMBO J., 3, 623-629, 1984). Previously, it was shown that the rhtA23 mutation is an A-for-G substitution at the -1 position relative to the ATG start codon (ABSTRACTS of 17^th International Congress of Biochemistry and Molecular Biology in conjugation with 1997 Annual Meeting of the American Society for Biochemistry and Molecular Biology, San Francisco, California August 24-29, 1997, abstract No. 457).

Moreover, it is also possible to introduce a nucleotide substitution into a promoter region of the gene on the bacterial chromosome, which results in stronger promoter function. The alteration of the expression control sequence can be performed, for example, in the same manner as the gene substitution using a temperature-sensitive plasmid, as disclosed, in International Patent Publication WO 00/18935 and Japanese Patent Application Laid-Open No. 1-215280.

Methods for preparation of plasmid DNA include, but are not limited to digestion and ligation of DNA, transformation, selection of an oligonucleotide as a primer and the like, or other methods well known to 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, Second Edition", Cold Spring Harbor Laboratory Press (1989). 2. Manufacturing method of the present invention

(1) Process I

In the first manufacturing method of the present invention, 4HIL is produced by placing an amino group donor, acetaldehyde and α-ketobutyric acid in a solvent in the presence of a biocatalyst having activity in the production of 4HIL that is shown in Formula (I) below from acetaldehyde and α-ketobutyric acid in the presence of an amino group donor (referred to as "process I" hereinafter).

That is, the process I comprises two steps of enzymatic reaction: (1) a step of enzymatic aldol reaction producing HMKP from acetaldehyde and α-ketobutyric acid, and (2) a step of enzymatic transamination producing 4HIL from HMKP.

Further, the second step of the process I is an another aspect of the present invention, which is later described in the present specification.

The biocatalysts employed in the process I are an enzyme (aldolase) having aldolase activity that mediates the step (reaction formula (III) below) of producing

4-hydroxy-3-methyl-2-pentanoic acid from acetaldehyde and α-ketoburyric acid; and an enzyme

(aminotransferase and/or dehydrogenase) having transamination activity that mediates the step

(reaction formula (IV) below) producing 4HIL from HMKP; or a bacterium incorporating these

In the first step of the process I in the present invention, "aldol reaction" means the reaction where an enolate ion which derives from corresponding keto-compound, such as α-keto butyric acid, reacts with a carbonyl compound such as acetaldehyde to form a β-hydroxyketone, for example, 4-hydroxy-3-methyl-2-keto-pentanoic acid (HMKP).

In the present invention, enzymatic aldol reaction means that an aldol reaction which is caused or catalyzed by an aldolase. Particularly, a bacterial aldolase is preferred. Said enzymatic aldol reaction can be performed by an isolated aldolase, crude enzymatic solution containing an activity of an aldolase, or by cultivating a bacterium having an activity of aldolase in a medium containing α-ketobutyric acid and acetoaldehyde. Examples of such bacteria are described later in detail, but any bacterium having such aldolase activity can be usable in the present invention.

A bacterial aldolase in the present invention is an enzyme which can catalyze the reaction from acetoaldehyde and α-ketobutyric acid to form HMKP. Particularly, aldolases which can be categorized in "HpcH/Hpal aldolase family" are preferable. Preferable aldolase may have a homology of not less than 50%, preferably not less than 55%, and most preferably not less than 60%, with respect to the amino acid sequence in N-terminal shown in SEQ ID NO. 11, as long as such aldolase share the feature of catalyzing the reaction from acetoaldehyde and α-ketobutyric acid to form HMKP.

Traditionally the term "HpcH/Hpal aldolase family" (where HpcH family includes so called HHED aldolases and HKP aldolases) includes 2,4-dihydroxyhept-2-ene-l,7-dioic acid aldolases and 4-hydroxy-2-oxovalerate aldolases. These enzymes are involved into carbohydrate transport and metabolism (catabolism).

Clusters of Orthologous Groups of proteins (COGs) were delineated by comparing protein sequences encoded in complete genomes, representing major phylogenetic lineages. Each

COG consists of individual proteins or groups of paralogs from at least 3 lineages and thus corresponds to an ancient conserved domain (Tatusov, R.L. et al, Science, 278, 5338, 631-637

(1997); http://www.ncbi.nlm.nih.gov/COG).

Protein sequence which is used for characterization of HpcH/Hpal aldolase family is categorized in COG3836 (gnl|CDD|13153) (SEQ ID NO: 5).

Applying the protein sequence of SEQ ID NO: 5 for BLAST protein search gives many proteins sharing relatively high homology, including 2,4-dihydroxyhept~2-ene-l,7-dioic acid aldolases (Accession nos. AAF12475, ZP_00501227, ZP_00467871 etc.), 2-dehydro-3-deoxyglucarate aldolases (Accession nos. ABB11891, ZP_00687019, ZP_00425668, etc.) and some hypothetical proteins (Accession nos. AAN81241, YP_311186, etc.); those are included into the HpcH/Hpal aldolase family according to the present invention. Analysis of homology search result revealed the high level of homology between proteins of HpcH/Hpal aldolase family (2,4-dihydroxyhept-2-ene-l,7-dioic acid aldolases and 4-hydroxy-2-oxovalerate aldolases) and 2-dehydro-3-deoxyglucarate aldolases, for example, 2-dehydro-3-deoxyglucarate aldolase from Escherichia coli (gb]AAN82322.1) coded by yhaF

- W - gene (synonym - garL gene) (SEQ ID NO.:24). The nucleotide sequence of the yhaF gene and the amino acid sequence of the YhaF protein encoded by the yhaF gene are shown in SEQ ID NO: 24 and SEQ ID NO: 25, respectively. Further experimental work (see Example section) proved that both 2,4-dihydroxyhept-2-ene-l,7-dioic acid aldolases/ 4-hydroxy-2-oxovalerate aldolases and 2-dehydro-3-deoxyglucarate aldolases are able to catalyze regioselective reaction of formation of HMKP from α-ketobutyric acid and acetaldehyde. Example of 2,4-dihydroxyhept-2-ene-l,7-dioic acid aldolases include the yfaU gene from E. coli. The yfaU gene encodes the putative 2,4-dihydroxyhept-2-ene-l,7-dioic acid aldolase (synonym — HHED-aldolase) (SEQ ID NO: 26). The nucleotide sequence of the yfaU gene and the amino acid sequence of the YfaU protein encoded by the yfaU gene are shown in SEQ ID NO: 26 and SEQ ID NO: 27, respectively.

So, for the purpose of the present invention term "HpcH/Hpal aldolase family" besides 2,4-dihydroxyhept-2-ene-l,7-dioic acid aldolases and 4-hydroxy-2-oxovalerate aldolases also includes 2-dehydro-3-deoxyglucarate aldolases. Following the aldol reaction, the second step, transamination process can be allowed to proceed.

In the second step of the process I in the present invention, "transamination" means the reaction whereby an amino group is transferred from a donor compound, for example, L-glutamic acid or L-glutamate, to an acceptor compound with a keto-group, for example, 4-hydroxy-3-methyl-2-keto-pentanoic acid or the like.

In the present invention, "enzymatic transamination" means a transamination reaction which is carried out by an -aminotransferase (transaminase) or a dehydrogenase enzyme. Particularly a bacterial aminotransferase (transaminase) is preferred. Said enzymatic transamination can be performed by an isolated aminotransferase, crude enzyme solution containing an activity of an aminotransferase, or by cultivating a bacterium having an activity of aminotransferase in a medium containing 4-hydroxy-3-methyl-2-keto-pentanoic acid. To reduce the cost for producing 4HIL through simplification of the process, adding the substrate directly to the culture solution in the method is most preferable.

The aminotransferases of the present invention include a branched-chain amino acid aminotransferase (BCAT) . For example, aminotransferase encoded by the UvE gene, aromatic aminotransferase encoded by tyrB gene, aspartate aminotransferase encoded by aspC gene, valine-pyruvate aminotransferase encoded by avtA gene, and the like are exemplified. The branched-chain amino acid aminotransferase (BCAT) is preferable. Protein sequence which is used for the characterization of BCAT is categorized in COG0115 (SEQ ID NO: 41). Also, protein belonging to BCAT is categolized in EC 2.6.1.42.

Practically all branched-chain amino acid aminotransferases exhibit broad substrate specificity, so it is expected that most of them can use HMKP as a substrate for the amino-group transfer.

The branched-chain amino acid aminotransferase catalyzes the reaction of transfer of an amino-group from L-glutamic acid to different cc-ketoacids, such as α-ketoisovaleric acid, 2-keto-3-methylvaleric acid, and 2-keto-4-methylpentanoic acid with formation of L-valine, L-isoleucine, and L-leucine, respectively. The branched-chain amino acid aminotransferases from a great majority of microorganisms are known, and the nucleotide sequences of genes encoding these aminotransferases have been disclosed.

The HvE gene encodes the HvE protein, which is a branched-chain amino acid aminotransferase from Escherichia coli, (synonyms include B3770, HvE, branched-chain amino acid: 2-oxoglutaric acid aminotransferase, BCAT, transaminase B, leucine transaminase, valine transaminase, and isoleucine transaminase). The UvE gene is located between the UvM and

UvD genes on the chromosome of E. coli strain K- 12. The nucleotide sequence of UvE gene is known (nucleotide positions: 3950507 to 3951436; GenBank accession no. NC_000913.2; gi:49175990) (SEQ ID NO: 1). The nucleotide sequence of the HvE gene and the amino acid sequence of the HvE protein encoded by the HvE gene are shown in SEQ ID NO: 1 and SEQ ID

NO: 2, respectively.

The ywaA gene encodes the branched-chain amino acid aminotransferase from Bacillus subtilis. The ywaA gene is located between the dltE and HcH genes on the chromosome of B. subtilis strain 168. The nucleotide sequence of the ywaA gene is known (nucleotide positions: 3956412 to3957503; GenBank accession no. NC_000964.2; gi:50812173) (SEQ ID NO: 3). The nucleotide sequence of the ywaA gene and the amino acid sequence of the YwaA protein encoded by the ywaA gene are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

Non-annotated genes encoding branched-chain amino acid aminotransferases from other microorganisms can be identified by homology to known genes encoding branched-chain amino caid aminotransferases, followed by evaluation of the activity of proteins encoded by the genes.

Homology between two amino acid sequences can be determined using well-known methods, for example, the computer program BLAST 2.0, which calculates three parameters: score, identity, and similarity. Therefore, the UvE gene from E. coli and ywaA gene from B. subtilis can be obtained by

PCR (polymerase chain reaction; refer to White, TJ. et al, Trends Genet, 5, 185 (1989)) utilizing primers prepared based on the known nucleotide sequence of the gene. Genes encoding branched-chain amino acid aminotransferases from other microorganisms can be obtained in a similar manner.

Since there may be some differences in DNA sequences between bacterial strains, the above-described genes encoding aldolase or BCAT to be used are not limited to the nucleotide sequences shown in SEQ ID NOS: 1, 3, 24 and 26 but may also include nucleotide sequences similar to those shown in SEQ ID NOS: 1, 3, 24 and 26. Therefore, the protein variants encoded by the above-described genes may have a similarity of not less than 80%, preferably not less than 90%, and most preferably not less than 95%, with respect to the entire amino acid sequences shown in SEQ ID NOS. 2, 4, 25 and 27, as long as the abilities of the proteins to catalyze the said reactions are maintained.

Moreover, the above-described genes may be represented by variants which can hybridize under stringent conditions with the nucleotide sequences shown in SEQ ID NOS: 1, 3, 24 and 26 or with probes prepared based on these nucleotide sequences, provided that they encode functional proteins. "Stringent conditions" include those under which a specific hybrid is formed and a non-specific hybrid is not formed. For example, stringent conditions are exemplified by washing at 60⁰C one time, preferably two or three times, with a solution containing 1 xSSC and 0.1% SDS, preferably O.lx SSC and 0.1% SDS. The length of the probe may be suitably selected, depending on the hybridization conditions, and usually varies from 100 bp to 1 kbp.

The biocatalyst can be employed in any form, such as a bacterium (including a culture product, bacterial cells, or treated cells), a purified enzyme, or a crude enzyme, so long as it incorporates the above aldolase and aminotransferase (or dehydrogenase). Examples of forms of the treated cells of the bacteria employed in the present invention include dried bacterial mass, freeze-dried bacterial mass, products treated with surfactants or organic solvents, enzyme-treated products, ultrasound-treated products, mechanically ground products, solvent-treated products, protein fractions of bacterial mass, immobilized products of bacterial mass, and processed bacterial mass. Any bacterium having activity in the production of 4HEL from acetaldehyde and α-ketobutyric acid in the presence of an amino group donor may be employed.

When a bacterium is used for the process I, such bacterium could be modified to enhance an activity of an aldolase and/or an aminotransferase in a culture medium containing substrates, and isolating the produced 4HDDL from the culture medium. In one embodiment of the process I of the present invention, a bacterium is a recombinant microorganism which contains the amplified and/or expressed aldolase and aminotransferase gene.

In the process I of the present invention, acetaldehyde and α-ketobutyric acid are enzymatically reacted by the aldolase and further transaminated by the BCAT both formed and accumulated in the recombinant microorganism which contains the amplified and expressed aldolase gene and BCAT gene.

When a bacterium is used as a biocatalyst, a bacterium of the genus Schizosaccharomyces, Arthrobacter, Brevibacterium, Candida, Corynebacterium, Micrococcus, Cellulomonas, Actinoplanes, Chromobacterium, Rahnella, Rhizobium, Erwinia, Hansenula, Torulopsis, Kloeckera, Rhodotorula, Pcmellus, Mucor, Debaryomyces, Sporobolomyces, Escherichia, Salmonella, Flavobacteriutn, Bacillus, or Proteus can be employed.

Of these, a bacterium of the genus Schizosaccharomyces, Candida, Arthrobacter, Brevibacterium, Escherichia, or Bacillus is preferably employed. Specific examples are: Schizosaccharomyces pombe (AKU4220 strain, NBRC346 strain), Arthrobacter simplex (AKU626 strain, NBRC 12069 strain), Brevibacterium ammoniagenes (AKU642 strain, NBRC 12072 strain), Candida utilis (AKU4649 strain, IAM12203 strain), Micrococcus luteus (AKU501 strain, AKU504 strain, AKU542 strain, NBRC3232 strain, NBRC3333 strain, NBRC398 strain), Micrococcus flavus (AKU502 strain, ATCC10240 strain), Micrococcus roseus (AKU505 strain, AKU506 strain, NBRC3764 strain, NBRC3768 strain), Corynebacterium glutamicum (AKU507 strain, AKU508 strain, AKU509 strain, AKU652 strain, ATCC13032 strain, ATCC13059 strain, ATCC13060 strain, ATCC14067 strain), Corynebacterium aquaticum (AKU604 strain, NBRC 12154 strain), Corynebacterium paurometabolum (AKU605 strain, NBRCl 6120 strain), Arthrobactor globiformis (AKU625 strain, NBRC12140 strain), Arthrobactor sulfureus (AKU635 strain, NBRC12678 strain), Arthrobactor viscosus (AKU636 strain, NBRC 13497 strain), Brevibacterium protophormiae (AKU647 strain, NBRC12128 strain), Brevibacterium acetylicum (AKU650 strain, NBRC12146 strain), Brevibacterium stationis (AKU655 strain, NBRC 12144 strain), Brevibacterium fiiscum (AKU656 strain, NBRC12127 strain), Cellulomonas fimi (AKU671 strain, IAM12106 strain), Cellulomonas biazotea (AKU674 strain, NBRC12680 strain), Chromobacterium iodinum {Brevibacterium iodinum, AKU814 strain, NBRC3558 strain), Erwinia carotovora subsp. carotovora (AKU40 strain, AKU41 strain, NBRC3830 strain, NBRC 12380 strain), Hansenula anomala (NBRC 149 strain, AKU4303 strain), Hansenula miso (NBRC 146 strain, AKU4307 strain), Candida stellata (NBRC 895 strain, AKU4308 strain), Hansenula saturnus (NBRC 992 strain, AKU4314 strain), Hansenula nonfermentans (NBRC 1473 strain, AKU4332 strain), Hatisennla polymorpha (NBRC 1475 strain, AKU4333 strain), Torulopsis nitratophila (NBRC 10004 strain, AKU4539 strain), Candida guilliermondii (NBRC 566 strain, AKU4580 strain), Candida lipolytica (NBRC 717 strain, AKU4582 strain), Candida macedoniensis (NBRC 706 strain, AKU4587 strain), Candida pseudotropicalis (NBRC 617 strain, NBRC 882 strain, AKU4592 strain, AKU4591 strain), Candida tropicalis var. lambica (NBRC 1213 strain, AKU4610 strain), Candida solani (NBRC 762 strain, AKU4612 strain), Candida albicans (NBRC 1270 strain, AKU4626 strain), Kloeckera africana (NBRC 868 strain, AKU4704 strain), Kloeckera japonica (NBRC 151 strain, AKU4706 strain), Rhodotorula mucilaginosa (NBRC 1100 strain, AKU4819 strain), Panellus serotinus (NBRC 30264 strain, AKU5510 strain), Mucor racemosus f.sp. racemosus (NBRC 4581 strain, AKU3002 strain), Mucor lamprosporus (NBRC 6337 strain, AKU3018 strain), Mucor petrinsularis (NBRC 6751 strain, AKU3019 strain), Debaryomyces vanrijiae (JCM 2170 strain, AKU4362 strain), Sporobolomyces roseus (NBRC 1106 strain, AKU4442 strain), Escherichia coli K12 (NBRC 3992 strain, AKU46 strain), Salmonella typhimurium (NBRC 12529 strain, AKU94 strain), Flavobacterium ferrugineum (IAM 1493 strain, AKU154 strain), Bacillus subtilis (ATCC 23857, NBRC 12210), and Proteus mirabilis (NBRC 3849 strain, AKU83 strain).

Of these, preferred examples are: Brevibacteriwn ammoniagenes (AKU642 strain, NBRC 12072 strain), Arthrobacter simplex (AKU626 strain, NBRC 12069 strain), Micrococcus luteus (AKU501 strain, NBRC3333 strain), Micrococcus flavus (AKU502 strain, ATCC10240 strain), Corynebacterium glutamicum (AKU507 strain, AKU508 strain, AKU509 strain, AKU652 strain, ATCC 13032 strain, ATCC 13059 strain, ATCC 13060 strain, ATCC 14067 strain), Escherichia coli K12 (NBRC 3992 strain, AKU46 strain), Bacillus subtilis (ATCC 23857, NBRC 12210), and Erwinia carotovora subsp. carotovora (AKU40 strain, AKU41 strain, NBRC3830 strain, NBRC 12380 strain).

These bacteria may be cultured by the usual culturing methods. Any medium, whether natural or synthetic, that permits the efficient culturing of the bacterium and contains a carbon source, nitrogen source, inorganic salt source and the like that are utilizable by the bacterium, may be employed in culturing. The carbon source need only be utilizable by the bacterium; carbon sources that are suitable for use include sugars such as glucose, fructose, sucrose, and maltose; starch, starch hydrolysis products, and molasses; organic acids such as acetic acid, lactic acid, and glutamic acid; and alcohols such as ethanol and propanol. Nitrogen sources that are suitable for use, provided they are utilizable by the bacterium, include: ammonia; ammonium salts of various inorganic and organic acids, such as ammonium sulfate, ammonium chloride, ammonium acetate, and ammonium phosphate; other nitrogenous compounds; as well as peptones, meat extracts, yeast extracts, corn steep liquor, casein hydrolysis products, soybeans, soybean hydrolysis products, soybean meal, soybean hydrolysis products, various fermentation bacterial masses, and their digestion products.

Inorganic salts that may be employed provided they are utilizable by the bacterium include: potassium phosphate, ammonium sulfate, ammonium chloride, sodium chloride, magnesium sulfate, ferrous sulfate, and manganese sulfate. Additionally, trace amounts of salts of elements such as calcium, zinc, boron, copper, cobalt, and molybdenum may also be added. As needed, vitamins such as thiamine and biotin; amino acids such as glutamic acid and aspartic acid; and nucleic acid-related compounds such as adenine and guanine, may also be added.

Culturing is conducted under aerobic conditions by shake culturing, deep ventilation stir culturing, or the like. A culturing temperature of 15 to 37⁰C for a culturing period of 10 to 96 hours will suffice. The culture is maintained at pH 5.0 to 9.0. The pH may be adjusted with organic or inorganic acids, alkali solutions, urea, calcium carbonate, or ammonia.

Examples of the amino group donor employed in the present invention are inorganic ammonium salts such as ammonia, ammonium sulfate, ammonium chloride, and urea; and various amino acids such as glutamic acid, valine, leucine, isoleucine, and other branched-chain amino acids. The preferred amino acids can be determined by simple pretests with the actual bacterium being employed. Of these, branched-chain amino acids are preferred from the perspectives of reaction efficiency and availability. Valine, leucine, isoleucine, glutamic acid, and their salts are desirable. Since these amino acids are primarily employed in the present invention in their L-forms, they are desirably employed in the form of L-form free acids or suitable salts. The concentration of the amino group donor is 0.01 to 1,000 g/L, preferably 1 to 100 g/L.

The concentration of acetaldehyde is 0.1 to 50 g/L, preferably 0.5 to 20 g/L in the present invention. The acetaldehyde concentration can be determined by measurement of the prepared solution with an F-Kit Acetaldehyde (made by Roche Diagnostics), or simply by a preparation method of dilution based on the purity of reagents. In process I of the present invention, the concentration of α-ketobutyric acid is not specifically prescribed. However, it is desirably from 1/10 to 10 times the acetaldehyde concentration.

The solvent used in the reaction can be an aqueous solvent such as water or a phosphate, carbonate, acetate, borate, citrate, Tris, or some other buffer; an alcohol such as methanol or ethanol; an ester such as ethyl acetate; a ketone such as acetone; an amide such as acetamide; some other organic solvent; or an aqueous solvent containing one or more of the above. As needed, a surfactant such as Triton X-IOO or Nonion HS204 (made by NOF Corporation), or an organic solvent such as toluene or xylene, can be added in a proportion of about 0.1 to 20 g/L.

The concentration of the bacterium employed in process I is 0.1 to 700 g/L, preferably 10 to 300 g/L (based on the bacterial mass (wet weight)). The bacterium, amino group donor, acetaldehyde, and α-ketobutyric acid can be added in the above-stated concentrations to the aqueous medium and reacted at a temperature of 15 to 6O⁰C, preferably 20 to 5O⁰C, and a pH of 5 to 12, preferably 7 to 11, for from 10 minutes to 80 hours to produce 4HIL.

In process I, the amino group donor, acetaldehyde and α-ketobutyric acid can be added in the above-stated concentrations either to the initial culture or during culturing of the bacterium employed as the biocatalyst to produce 4HTL.

During the reaction, in addition to the reaction substrate and the bacterium employed as the reaction catalyst, the above-described BCAT can be added to the aqueous solvent in which the reaction is being conducted. In this process, a gene encoding BCAT can be incorporated into the bacterium employed as biocatalyst to transform the bacterium, or a transformant can be obtained by incorporating a gene encoding BCAT into a suitable host cell such as various bacteria such as E. coli or insect cells. BCAT can then be employed in the form of the bacterial mass or processed bacterial mass of the transformant obtained, or in the form of a purified enzyme or crude enzyme.

When the reaction of forming 4HIL is conducted using a crude enzyme solution, the cultured microorganism is recovered by centrifugation etc., and then the cells are disrupted or lysed to prepare a crude enzyme solution containing aldolase and aminotransferase and/or dehydrogenase. To disrupt the cells, a method such as ultrasonic disruption, French press disruption, glass bead disruption, or the like can be used, while to lyse the cells, a method such as treatment with albumen lysozyme or peptidase or with a suitable combination of these is used. When the reaction of forming 4HIL is conducted using a purified enzyme solution, the crude enzyme solution containing aldolase and aminotransferase and/or dehydrogenase is purified by usual techniques such as precipitation, filtration, column chromatography, etc.

Separation and recovery of the microorganism from the culture solution and preparation of the enzyme solution can be typically carried out by a combination of known methods such as centrifugation, ultrasonic disruption, an ion-exchange resin method, a precipitation method, etc.. (2) Process II

In the second manufacturing method of the present invention, an amino group donor and 4-hydroxy-3-methyl-2-keto-pentanoic acid (HMKP) are placed in an aqueous solvent in the presence of the biocatalyst having transamination activity producing 4HEL from HMKP that is denoted by formula (II) below in the presence of an amino group donor

!)

to produce 4HTL (referred to as process PI hereinafter).

The biocatalyst employed in the process II is one or more enzymes (aminotransferase and/or dehydrogenase) having transamination activity that mediate the step (reaction formula (W) above) of producing 4HIL from HMKP in the presence of an amino group donor, or a bacterium incorporating these enzymes. In the process II, the terms "amino group transamination activity" and "aminotransferase and/or dehydrogenase" are defined as in the second stage reaction of process I above.

The biocatalyst can be employed in any form, such as a bacterium (including a culture product, bacterial cells, treated cells), purified enzyme, or crude enzyme, so long as it contains the above-described enzymes. Any bacterium may be employed so long as it has dehydrogenase and/or aminotransferase activity that converts optically active HMKP into 4HIL in the presence of an amino group donor.

When a bacterium is used for the process II, such bacterium could be modified to enhance an activity of aminotransferase in a culture medium containing HMKP by aminotransferase such as BCAT formed and accumulated in the recombinant microorganism which contains the amplified and expressed BCAT gene.

Examples of bacteria that can be employed as biocatalysts are bacteria of the genera Schizosaccharomyces, Candida, Arthrobacter, Brevibacterium, Cotynebacterium, Pseudomonas, Hansenula, Flavobacteriwn, Bacillus, Micrococcus, Torulopsis, Cryptococcus, Escherichia and Pichia. From the perspectives of activity and availability, the use of a bacterium of the genus Schizosaccharomyces, Candida, Arthrobacter, Brevibacterium, Pseudomonas, Hansenula, Flavobacteriwn, Bacillus, Escherichia, or Pichia is desirable.

A bacterium belonging to the genus Escherichia or Bacillus is preferred. Here, the phrase "a bacterium belonging to the genus Escherichia" means that the bacterium is classified into the genus Escherichia according to the classification known to a person skilled in the art of microbiology. Examples of a bacterium belonging to the genus Escherichia as used in the present invention include, but are not limited to, Escherichia coli (E. colϊ). The bacterium belonging to the genus Escherichia that can be used in the present invention is not particularly limited; however, e.g., bacteria described by Neidhardt, F.C. et al. (Escherichia coli and Salmonella typhimurium, American Society for Microbiology, Washington D.C., 1208, Table 1) are encompassed by the present invention.

The phrase "a bacterium belonging to the genus Bacillus" means that the bacterium is classified into the genus Bacillus according to the classification known to a person skilled in the art of microbiology. Examples of a bacterium belonging to the genus Bacillus as used in the present invention include, but are not limited to, Bacillus subtilis (B. suhtilis) and Bacillus amyloliquefaciens (B. amyloliquefaciens) .

Specific examples are: Schi∑osaccharomyces pombe (AKU4220 strain, NBRC346 strain), Arthrobacter simplex (AKU626 strain, NBRC 12069 strain), Brevibacteriwn ammoniagenes (AKU642 strain, NBRC12072 strain), Candida utilis (AKU4570 strain, AKU4649 strain, IAM12203 strain, NBRC396 strain), Pseudomonas sp. (AKU839 strain, NBRC12691 strain), Flavobacterium heparinum (AKU150 strain, NBRC12017 strain), Bacillus thuringiensis

(AKU238 strain, NBRC3951 strain), Micrococcus luteus (AKU543 strain, NBRC3066 strain),

Pichia orientalis (AKU4256 strain, NBRC1279 strain), Hansenula jadinii (AKU4324 strain, NBRC987 strain), Torulopsis sphaerica (AKU4530 strain, NBRC648 strain), Brevibacteriwn linens (AKU653 strain, NBRC12171 strain), Escherichia coli K12 (NBRC 3992 strain, AKU46 strain), Bacillus subtilis (ATCC 23857, NBRC 12210), and Ciyptococcus flavus (AKU 4802 strain, NBRC 710 strain).

Of these, preferred examples are: Schizosaccharomyces pombe (AKU4220 strain, NBRC346 strain), Arthrobacter simplex (AKU626 strain, NBRC 12069 strain), Brevibacterium ammoniagenes (AKU642 strain, NBRC12072 strain), Candida utilis (AKU4570 strain, AKU4649 strain, IAM12203 strain, NBRC396 strain), Pseudomonas sp. (AKU839 strain, NBRC12691 strain), Flavobacterium heparinum (AKU 150 strain, NBRC 12017 strain), Bacillus thuringiensis (AKU238 strain, NBRC3951 strain), Pichia orientalis (AKU4256 strain, NBRC1279 strain), Hansenula jadinii (AKU4324 strain, NBRC987 strain), Escherichia coli Kl 2 (NBRC 3992 strain, AKU46 strain), Bacillus subtilis (ATCC 23857, NBRC 12210), and Brevibacterium linens (AKU653 strain, NBRC12171 strain).

Of the above bacteria, those strains the designation of which begins with AKU may be obtained from the Laboratory of Fermentation Physiology and Applied Microbiology, Division of Applied Life Sciences, Faculty of Agriculture, Graduate School, Kyoto University.

Those strains the designation of which begins with ATCC may be obtained from the American Type Culture Collection (ATCC, Address: P.O. Box 1549, Manassas, VA 20108, United States of America) . The registration numbers corresponding to the individual strains are recorded in the ATCC catalog (http://www.atcc.org/common/catalog/bacteria/bacterialndex.cfrn).

Those strains the designation of which begins with JCM are kept at the Riken Wako Institute (2-1 Hirosawa, Wako-shi, Saitama-ken) and can be obtained by registration number. Registration numbers corresponding to individual strains are recorded in the JCM catalog (http://www.jcm.riken.jp/JCM/catalogue.htm).

Those strains the designation of which begins with IAM are kept in the IAM Culture

Collection, Bioresearch Laboratory, Institute of Molecular and Cellular Biosciences, University of

Tokyo (1-1-1 Yayoi, Bunkyo-ku, Tokyo, Postal Code 113-0032) and can be obtained using their registration numbers. The registration numbers of individual strains are recorded in the IAM catalog (IAM Catalogue of Strains, Third Edition, 2004).

Those strains the designation of which begins with NBRC (formerly IFO) can be obtained from the National Institute of Technology and Evaluation (2-5-8 Kazusakamatari, Kisarazu-shi, Chiba, Postal Code 292-0818). The registration numbers of individual strains are recorded in the NBRC catalog: (http://www.nbrc.nite.go.jp/NBRC2/NBRCDispSearchSerylet?lang=en).

The various conditions under which these bacteria are cultured are identical to those described for the process I. In the process II, the amino group donor and optically active HMKP can be added in the above-stated concentrations either to the initial culture or during culturing of the bacterium employed as the biocatalyst to produce optically active 4HIL. In the process II of the present invention, the starting material HMKP can be employed in the form of a purified preparation, crude product, or the like.

HMKP can be obtained either chemically or enzymatically. Chemically HMKP can be obtained, for example, as a result of an aldol reaction of α-ketobutyric acid and acetaldehyde.

When a chemical aldol reaction is applied for obtaining HMKP, a reaction is preferably carried out under standard alkaline conditions. As for reaction solvent, polar solvents such as water, methanol, ethanol, propanol, acetonitrile and dimethylformamide or mixed solvents thereof are preferable. Particularly, water and a mixed solvent (hydrous organic solvent) of water and polar solvents are preferable. The pH of the solvent is within a range of preferably 8 to 12, more preferably 9 to 11.

When the pH is too high or too low, the yield is likely to decrease. Bases may satisfactorily be used to achieve such pH under alkaline conditions, and include, for example, inorganic bases such as alkali metal salts and alkali earth metal salts including alkali earth metal hydroxides and carbonates, e. g., lithium hydroxide, sodium hydroxide, potassium hydroxide sodium carbonate, potassium carbonate and calcium carbonate, and organic bases such as triethylamine or cadaverine.

The amount of α-ketobutyric acid or acetaldehyde has no specific limitation. When the amount of α-ketobutyric acid or acetaldehyde is used in excess, the reaction yield is likely to be improved. The preferable molecular ratio of α-ketobutyric acid and acetaldehyde is from 1:1 to 1:3.

The reaction can be carried at a reaction temperature within a range of preferably -10 to

70⁰C₅ more preferably 0 to 15°C. The reaction time has no specific limitation, and is generally

0.1 to 48 hours, preferably 0.5 to 6 hours. Proline can be added to the reaction mixture to control the chirality of 4-hydroxy-3-methyl-2-keto-pentanoate {Tetrahedron Letters, Volume 41, Issue 36,

September 2000, Pages 6951-6954).

When an enzymatic aldol reaction is applied for obtaining HMKJP, any aldolase which catalyzes the intended reaction can be used without any limitation. Preferably, aldol reaction using aldolase as described in the step 1 of the process I above can be used. Such aldolase can be used as a form of a purified enzyme solution, a crude enzyme solution containing aldolase, purified by usual techniques such as precipitation, filtration, column chromatography, etc. or as a form of microorganism containing the same. Additionally, a reaction solution containing

HMKP produced by enzymatic reaction derived from a bacterium with 4HIL as substrate

(Japanese Patent Application Laid-Open Hei 6-340578) can be employed. The concentration of 4-hydroxy-3-methyl-2-keto-pentanoic acid employed is 0.5 to 700 g/L, preferably 10 to 500 g/L.

The process II of the present invention includes the steps of cultivating a bacterium which has been modified to enhance an activity of a branched-chain amino acid aminotransferase in a culture medium containing HMKP, and isolating the produced 4HIL from the culture medium. In one embodiment of the process II of the present invention, HMKP is enzymatically transaminated using the aminotransferase, preferably BCAT, formed and accumulated in the recombinant microorganism which contains the amplified and expressed aminotransferase gene (preferably BCAT gene). When the reaction of forming 4HIL is allowed to proceed by adding HMKP directly to the culture solution while culturing the transformant which has amplified and expressed the BCAT gene, the reaction is conducted in a stationary state or with gentle stirring. Preferably, the reaction temperature is controlled at 10 ⁰C to 60 ⁰C, preferably 25 ⁰C to 45 ⁰C, and the pH at 3 to 11, preferably pH 6 to 9. The pH of the culture can be adjusted with ammonia, calcium carbonate, various acids, various bases, and buffers. Usually, 1 to 5-day cultivation leads to accumulation of the target 4HIL in the liquid medium. Insofar as the reaction proceeds, the substrate HMKP may be added, and when necessary, can be reacted in a necessary amount for a necessary time. Either a synthetic or natural medium may be used in the present invention, so long as the medium includes a carbon source and a nitrogen source and minerals and, if necessary, appropriate amounts of nutrients which the bacterium requires for growth. Such nutrients are the same as described in previous section.

When the reaction of forming 4HEL is conducted using a crude enzyme solution, the cultured microorganism is harvested by centrifugation etc., and then the cells are disrupted or lysed to prepare a crude enzyme solution containing aminotransferase, preferably BCAT. To disrupt the cells, a method such as ultrasonic disruption, French press disruption, glass beads disruption, or the like can be used; while to lyse the cells, a method such as treatment with albumen lysozyme or peptidase or with a suitable combination of these is used. When the reaction of forming 4HIL is conducted using a purified enzyme solution, the crude enzyme solution containing aminotransferase, preferably BCAT, is purified by usual techniques such as precipitation, filtration, column chromatography, etc.

Separation and recovery of the microorganism from the culture solution and preparation of the enzyme solution can be typically carried out by a combination of known methods such as centrifugation, ultrasonic disruption, an ion-exchange resin method, a precipitation method, etc..

Isolated BCAT can be obtained by traditional methods including expressing in vivo or in vitro the gene encoding the branched-chain amino acid aminotransferase, followed by isolating the obtained enzyme using chromatography with fractionation, purifying an antibody to BCAT, expressing the modified gene encoding for the branched-chain amino acid aminotransferase having His₆-tag followed by nickel-column isolation using pET expression system (Novagen), and the like.

Activity of branched-chain amino acid aminotransferases can be detected and measured by, for example, the method described by Lee-Peng, RC. et al (J Bacteriol. 139(2): 339-345 (1979)).

When the reaction of forming 4HTL is allowed to proceed by use of the crude enzyme solution or purified enzyme containing BCAT, the reaction is allowed to proceed while a reaction solution containing the substrate HMKP and the crude enzyme solution or purified enzyme is controlled at 10 ⁰C to 60 ⁰C, preferably 25 ⁰C to 45 ⁰C, and the pH at 3 to 11, preferably pH 6 to 9. Insofar as the reaction proceeds, the substrate HMKP may be added, and when necessary, can be reacted in a necessary amount for a necessary time.

The phrase "modified to enhance an activity of a branched-chain amino acid aminotransferase (BCAT)" means that the activity per cell is higher when compared to that of a non-modified strain, for example, a wild-type strain. Examples of such modifications include increasing the number of BCAT molecules per cell, increasing the specific activity per BCAT molecule, and so forth. Furthermore, a wild-type strain that may be used for comparison purposes includes, for example, Escherichia coli K-12.

Enhancing BCAT activity in a bacterial cell can be attained by increasing the expression of the gene encoding BCAT. Any BCAT gene derived from bacteria may be used as the BCAT gene of the present invention. BCAT genes derived from bacteria belonging to the genus Escherichia and Bacillus are preferred.

The aminotransferase and/or dehydrogenase moderating above-recorded reaction formula (IV) that is optionally contained in the biocatalyst employed in process II and in step 2 of process I do not hinder the manufacturing method of the present invention even when one of the enzymes functions within the bacterium to produce 4HIL. However, since it is necessary to add nicotinamide-adenine dinucleotide (NADH) or nicotinamide-adenine dinucleotide phosphate (NADPH) when the above reaction is mediated by dehydrogenase, the above reaction will be mediated by aminotransferase when neither NADH nor NADPH is present in the reaction solution. The decision of whether to conduct the reaction under dehydrogenase reaction conditions (in the presence of NADH or NADPH) or under aminotransferase reaction conditions (in the absence of NADH or NADPH) can readily be made by one skilled in the art by conducting suitable preliminary tests using the bacterium to be employed in the reaction to determine the optimum conditions. When conducting the reaction under dehydrogenase reaction conditions, the concentration of the NADH or NADPH employed is 0.05 to 10 mg/L. During the reaction, the joint use of a reaction activating a microbe-derived enzyme such as glucose dehydrogenase to regenerate the NAD⁺ or NADP⁺ produced by the reaction is desirable.

The 4HIL produced by process I or II can be isolated by the usual amino acid purification methods. For example, operations such as processing with an ion exchange resin or membrane and crystallization can be used in combination to isolate 4HIL from the supernatant of a reaction solution from which centrifugation has been used to remove the solid matter.

Examples

The present invention is described in greater detail below through Examples. However, the present invention is not limited by these Examples. [Example 1]

Fifty mL quantity of medium containing 10 g/L of glucose, 3 g/L OfK₂HPO₄, 0.2 g/L of MgSO₄-TH₂O, 15 g/L of peptone, 1 g/L of yeast extract, and 2 g/L of sodium chloride that had been adjusted to pH 7 was poured into a 500 mL Sakaguchi flask and sterilized. The various microbes shown in Table 1 were then inoculated and shake cultured for 1 to 3 days at 28°C.

Centrifugation was used to collect the bacteria of each strain from 5 mL of the culture solution. Each of the bacterial strains obtained was suspended in 1 mL of a reaction solution (a) (10 g/L of HMKP, 10 g/L of L-glutamic acid, 20 g/L of ammonium chloride, 100 g/L of glucose, 0.6 g/L of NADH, 0.6 g/L of NADPH, 20 U/mL of glucose dehydrogenase (SIGMA), potassium phosphate buffer solution (pH 7.0)) and reacted for 2 to 5 days at 3O⁰C. Upon completion of the reaction, the bacterial mass was removed by centrifugation and the presence or absence of 4HIL production in the supernatant was determined by TLC. Method of preparing 4HIL

The extraction and purification of 4FHL was conducted according to the method of L. Fowden, H. M. Pratt, and A. Smith (Phytochemistry, Vol. 12, 1707, 1973). Four grams of purified 4HIL were isolated from 4 kg of seeds of Trigonella foenumgraecum L.

Method of preparing 4-hydroxy-3-methyl-2-keto-pentanoic acid Purified 4HIL was dissolved to 10 mg/mL in pH 8.0 buffer solution, 0.43 U of L-amino acid oxidase was added per mg of 4HIL, and oxidation was conducted with thorough stirring for 20 hours at 37⁰C. Ninhydrin reagent and the like were employed to confirm adequate oxidation, after which freeze-drying was conducted. The purification of

4-hydroxy-3-methyl-2-keto-pentanoic acid was conducted according to Japanese Patent Application Laid-Open No. Hei 6-340578. That is, the freeze-dried residue was extracted with methanol and methyl teλt-butyl ether was added to precipitate a sodium salt of 4-hydroxy-3 -methyl-2-keto-pentanoic acid. TLC conditions Thin-layer silica gel (60F254, Merck Corp.) spotted with 1 μL of reaction solution was developed with a developing solvent (n-butanol:acetic acid:water = 4: 1:1) and 4HIL was detected with Ninhydrin reagent. The generation of a spot having an Rf value identical to that of purified 4HIL was determined and the absence of a spot corresponding to 4HIL was confirmed in reaction product not containing HMKP as a negative control.

Results

Transamination activity from HMKP to 4HIL was found in 17 microbes, as shown in Table 1. This reaction was presumed to have been mediated by aminotransferase and dehydrogenase.

Table 1 shows activity in the production of 4HIL from HMKP. [Table 1]

[Example 2]

Fifty mL quantity of medium containing 10 g/L of glucose, 3 g/L Of K₂HPO₄, 0.2 g/L of MgSO₄- 7H₂O, 15 g/L of peptone, 1 g/L of yeast extract, and 2 g/L of sodium chloride that had been adjusted to pH 7 was poured into a 500 mL Sakaguchi flask and sterilized. The various microbes shown in Table 2 were then inoculated and snaked for 1 to 3 days at 28⁰C.

Centrifugation was used to collect the bacteria of each strain from 5 mL of the culture solution. Each of the bacterial strains obtained was suspended in 1 mL of reaction solution (1) (10 g/L of acetaldehyde, 10 g/L of α-ketobutyrate, 10 g/L of L-glutamic acid, 20 g/L of ammonium chloride, 100 g/L of glucose, 0.6 g/L of NADH, 0.6 g/L of NADPHO, 20 U/mL of glucose dehydrogenase (SIGMA), potassium phosphate buffer (pH 7.0)); reaction solution (2) (10 g/L of acetaldehyde, 10 g/L of α-ketobutyrate, 10 g/L of L-glutamic acid, 20 g/L of ammonium chloride, 100 g/L of glucose, 0.6 g/L of NADH, 0.6 g/L of NADPH, 20 U/mL of glucose dehydrogenase (SIGMA), borate buffer (pH 9.0)); or reaction solution (3) (2g/L of acetaldehyde, 5 g/L of α-ketobutyrate, 10 g/L of L-glutamic acid, 20 g/L of ammonium chloride, 100 g/L of glucose, 0.6 g/L of NADH, 0.6 g/L of NADPH, 20 U/mL of glucose dehydrogenase (SIGMA), borate buffer (pH 9.0)) and reacted for 2 to 5 days at 3O⁰C. At the After the reaction, the bacterial mass was collected and 4HEL production in the supernatant was measured by amino acid analysis using HPLC.

HPLC analysis: High pressure chromatograph (Waters, USA) with spectrofluorometer 1100 series (Agilent, USA) was used. The chosen detection wave range: excitation wavelength at 250 nm, range of emission wavelengths were 320-560 nm. The separation by accq-tag method was performed in a column Nova-Pak™ C18 150 x 3,9 mm, 4μm (Waters, USA) at +400 ⁰C. Injection volume of the sample was 5μl. The formation of amino acid derivatives and their separation was performed according to Waters manufacturer's recommendation (Liu, H. et al, J. Chromatogr. A₃ 828, 383-395 (1998); Waters accq-tag chemistry package. Instruction manual. Millipore Corporation, pp.1-9 (1993)). To obtain amino acid derivatives with 6-aminoquinolil-N-hydroxysuccinymidyl carbamate, the kit Accq-Fluor™ (Waters, USA) was used. The analysis by accq-tag method was performed using concentrated Accq-tag Eluent A (Waters, USA). All solutions were prepared using Milli-Q water, standard solutions were stored at + 4 ⁰C.

Results

The maximum values of 4HIL produced in reaction solutions (1) to (3) are shown in Table 2. 4HIL production presumed to have occurred by aldol reaction and transamination from acetaldehyde and α-ketobutyric acid was observed in 30 strains of bacteria. Arthrobactor simplex exhibited the highest production level. Even for the bacteria with the lowest production level, it was anticipated that the production level could be increased by optimizing reaction conditions.

Table 2 shows activity in the production of 4HIL from acetaldehyde and α-ketobutyric acid. [Table 2]

[Example 3]

Fifty niL quantity of medium containing 10 g/L of glucose, 3 g/L Of K₂HPO₄, 0.2 g/L of MgSO₄- 7H₂O, 15 g/L of peptone, 1 g/L of yeast extract, and 2 g/L of sodium chloride that had been adjusted to pH 7 was poured into a 500 mL Sakaguchi flask and sterilized. The various microbes shown in Table 3 were then inoculated and shake cultured for 1 to 3 days at 28⁰C.

Centrifugation was used to collect the bacteria of each strain from 5 mL of the culture solution. Each of the bacterial strains obtained was suspended in 1 mL of reaction solution (4) (10 g/L of acetaldehyde, 10 g/L of α-ketobutyrate, 10 g/L of L-glutamic acid, potassium phosphate buffer (pH 7.0)); reaction solution (5) (10 g/L of acetaldehyde, 10 g/L of α-ketobutyrate, 10 g/L of L-glutamic acid, borate buffer (pH 9.0)); or reaction solution (6) (2g/L of acetaldehyde, 5 g/L of α-ketobutyrate, 10 g/L of L-glutamic acid, borate buffer (pH 9.0)) and reacted for 2 to 5 days at 3O⁰C. After the reaction, the bacterial pellet was collected and the synthesis of 4HTL in the supernatant was measured by amino acid analysis using HPLC. HPLC analysis was performed as described in the example 2.

Results

The quantities of 4HTL produced that were measured in the reaction solution for the various aminotransferase reaction conditions are shown in Table 3.

4HIL production presumed to have occurred by aldol reaction and aminotransferase reaction from acetaldehyde and α-ketobutyrate was observed in 23 strains of bacteria.

Table 3 shows 4HIL production activity from acetaldehyde and α-ketobutyrate under aminotransferase reaction conditions. [Table 3]

[Example 4]

Fifty mL quantity of medium containing 10 g/L of glucose, 3 g/L Of K₂HPO₄, 0.2 g/L of MgSO₄-7H₂O, 15 g/L of peptone, 1 g/L of yeast extract, and 2 g/L of sodium chloride that had been adjusted to pH 7 was poured into a 500 mL Sakaguchi flask and sterilized. Brevibacteriwn ammoniagenes or Arthrobactor simplex was inoculated and cultured for 1 to 3 days at 28⁰C.

Centrifugation was used to collect the bacteria of each strain from 5 mL of the culture solution. Each of the bacterial strains obtained was suspended in 1 mL of reaction solution (2 g/L of acetaldehyde, 5 g/L of α-ketobutyrate, 10 g/L of amino acid, 20 g/L of ammonium chloride, 100 g/L of glucose, borate buffer (pH 9.0)) and reacted for 2 to 5 days at 3O⁰C. The amino acids shown in Figures 1 and 2 were employed as amino group donors. When the reaction had concluded, the bacterial mass was removed by centrifugation, and the presence or absence of 4BDDL production in the supernatant was measured by amino acid analysis using HPLC.

HPLC analysis was performed as described in the example 2.

Results

Figures 1 and 2 show the effect of amino group donors on 4HIL produced under aminotransferase reaction conditions from acetaldehyde and α-ketobutyrate. The use of branched-chain amino acids such as leucine, valine, and isoleucine as amino group donors was found to tend to increase the level of 4HIL production relative to glutamic acid. Thus, branched amino acids were presumed to be good amino group donors for increasing the level of production in the manufacturing of 4HIL by the present invention.

[Example 5]

Five mL quantities of medium containing 10 g/L of glucose, 3 g/L OfK₂HPO₄, 0.2 g/L of MgSO₄-7H₂O, 15 g/L of peptone, 1 g/L of yeast extract, and 2 g/L of sodium chloride that had been adjusted to pH 7 were inoculated with, the various bacteria shown in Table 4 and shake cultured for 1 to 2 days at 28°C.

BCAT from Escherichia coli was cloned and expressed using the pET expression system (Novagen, Madison, WI, USA) as hisβ-tag derivatives.

To construct the pET-HT-IlvE-ECO plasmid, the UvE gene from E. coli was amplified by PCR using chromosomal DNA of E. coli strain MG1655 as a template and primer Pl (SEQ ID NO: 6) and primer P2 (SEQ ID NO: 7) as "upstream" and "downstream" primers respectively.

Primer Pl contains the Ncol restriction site and six codons coding for histidine at the 5 '-end thereof, primer P2 contains the BamΗl restriction site at the 5 '-end thereof. The resulting PCR fragment was digested with Ncol and BamHl restrictases and ligated into vector pET-15(b+) which had been previously treated with the same restrictases. Thus plasmid pET-HT-IlvE-ECO was obtained.

It was found that the hisβ-tag-IlvE-ECO protein was localized in the soluble fraction of total cellular protein, when expressed in the pET-system from the pET-HT-IlvE-ECO plasmid under standard induction conditions (1 mM IPTG: isopropyl-thio-β-D-galactopyranoside, 2-3 hour at 37 ⁰C). The hybrid protein was purified by IMAC (Immobilized-Metal-Affinity-Chromatography). The following procedures were carried out: 1) 200 ml of the E. coli B strain BL21(DE3)[pET-HT-IlvE-ECO] cell culture was grown in the four 500 ml flasks until OD₅₅₅=I was reached. Synthesis of the his6-tag-ilvE protein was induced by adding EPTG to a final concentration of 1 mM, followed by incubation for 2 hours. Then, cells were harvested by centrifugation, washed twice in 100 ml of 100 mM NaCl solution, pelleted again, resuspended in the 20 ml of buffer I (20 mM Tris-HCl, 500 mM NaCl, ImM PMSF, 1OmM imidazole, 5% (w/v) glycerol ; pH 8) and disrupted by one passage through a French pressure cell; 2) the resulting lysate was centrifuged and supernatant proteins were applied to a 1 ml Hitrap® (Pharmacia) column; then, the column was washed with 10 ml of buffer I and the bound proteins were eluted by buffer II (20 mM Tris-HCl, 40OmM imidazole; pH 8). The resulting ilvE preparation was obtained by gel filtration using a 10 ml BioGel PlO (BioRad) column equilibrated with buffer III (20 mM potassium phosphate buffer pH 7, 1 mM DTT₅ 10 μM PLP, 10% (w/v) glycerol). The various bacterial strains shown in Table 4 were collected by centrifugation from 5 mL of the culture solutions and washed with physiological saline. To these, a culture solution of E. coli B strain BL21(DE3)[pET-HT-IlvE-ECO] was added, and the mixture was again washed with physiological saline. Each of the bacterial strains thus obtained was suspended in 1 mL of reaction solution (250 mM acetaldehyde, 75 mM α-ketobutyrate, 1% of L-glutamic acid, 50 mM potassium phosphate buffer (pH 7.0)) and reacted for 12 to 15 hours at 28⁰C. After the reaction, the cells were removed by centrifugation and the synthesis of 4HTL in the supernatant was measured by HPLC.

HvE may be employed in the form of an E. coli variant by which it is strongly expressed, as is set forth above, or by the addition of 0.1 to 10 μg of purified HvE. The source of the enzyme employed is not limited so long as it has transamination activity toward HMKP.

HPLC analysis was performed as described in the example 2.

Results

Table 4 shows the maximum values of 4HIL produced by a combination reaction of

BCAT and a bacterium. Activity in the production of 4HIL presumed to be the result of aldol reaction from acetaldehyde and α-ketobutyric acid and an amination reaction was observed in 40 strains of bacteria. A synergistic increase in the production level by employing HvE was confirmed relative to reaction conditions employing the bacterium alone.

Table 4 shows activity in the production of 4HTL from acetaldehyde and α-ketobutyric acid. [Table 4]

- : 4HIL production level not exceeding 100 μM

[Example 6] Cloning and efficient expression of the BCAT from Escherichia coli and Bacillus subtilis.

BCAT from Escherichia coli and Bacillus subtilis was cloned and expressed using the pET expression system (Novagen, Madison, WI, USA) as his₆-tag derivatives.

Construction of the pET-HT-IlvE-ECO plasmid and preparation of ilvE protein were performed as described in the example 5.

To construct the pET-HT-IlvE-BSU plasmid, the ywaA gene from Bacillus subtilis encoding BCAT aminotransferase (Berger, BJ et al, J Bacteriol, 185(8), 2418-31 (2003)) was amplified by PCR using chromosomal DNA of B. subtilis strain 168 as a template and primers P5

(SEQ ID NO: 8) and P6 (SEQ ID NO: 9) as "upstream" and "downstream" primers, respectively.

Primer P5 contains the Ncol restriction site and six codons coding for histidine at the 5 '-end thereof, primer P6 contains the Notl restriction site at the 5 '-end thereof. The resulting PCR fragment was digested with Ncol restrictase and ligated with plasmid pET-15(b+) which had been previously digested with the same restrictase. Then, the linear ligated DΝA fragment was used as a template for PCR-amplifϊcation using oligonucleotides T7 (Νovagen, SEQ ID NO: 10) and P6

(SEQ ID NO: 9) as a primers. The resulting PCR fragment containing the ywaA gene under the control of T7 promoter of the plasmid pET-15(b+) was digested by Xbάl and Notl restrictases and ligated into the pET-22(b+) vector which had been previously treated with the same restrictases. Thus, plasmid pET-HT-IlvE-BSU was obtained.

The corresponding protein bsuBCAT was expressed as a hybrid hise-tagged protein. It was found that the his₆-tag-IlvE protein from B. subtilis was localized in the soluble fraction of total cellular protein, when it was expressed in the pET-system from the pET-HT-IlvE-BSU plasmid under standard induction conditions (see above). Hybrid hisβ-tag-bsuBCAT was purified as described above.

Activities of the hisβ-tag enzymes in the crude cell lysate of the E. coli B strains BL21(DE3)[pET-HT-ilvE-ECO] and BL21(DE3)[pET-HT-ilvE-BSU] were determined after 2 hours of expression induction.

The specific activities of BCAT aminotransferases were estimated by measuring the V_max forward parameter of the reaction: α-keto-precursor + glutamate = α-amino acid + α-ketoglutarate catalyzed by said enzymes. Reaction conditions: 100 mM K₂HPO₄, 100 mM L-glutamate, pH 7.4 (adjusted by adding KOH); α-keto-precursor - 20 mM; proteins - 2,5-10 μg/reaction; temperature - 37 ⁰C.

Initial velocity of the transamination reaction (V₀) was determined by quantitative TLC analysis (developer - n-propanol : acetone : NH₃ : H₂O = 25 : 25 : 6 : 2) of the time dependence formation of the detectable reaction products (aminobutyrate, isoleucine, leucine, valine). Data of specific activities of BCAT amintransferases are presented in Table 5.

* Abbreviations: KB - α -ketobutyric acid; KMV - ketomethylvalerate; KTV - ketoisovalerate; KIC - ketoisocaproate.

[Example 7] Transamination of the HMKP using strains with enhanced activity of different BCAT aminotransferases and isolated ecoBCAT and bsuBCAT aminotransferases.

In order to investigate of the 4HIL synthesis in the two-step biotransformation process using homologues of BCAT aminotransferases, the following procedures were carried out:

1) HMKP preparation: 0.5 ml of 1 M α-ketobutyrate in water supplemented 60 mM KOH was slowly mixed with 0.5 ml of 1 M acetaldehyde in water at 4 ⁰C and incubated for 3 hours at 10 ⁰C. This solution is defined as 'HMKP solution'.

2) To provide the in vivo biotransformation process, the E. coli B strains BL21(DE3)[pET-HT-ilvE-ECO] and BL21(DE3)[pET-HT-ilvE-BSU] were each cultured in LB broth at 34 ⁰C until OD₅₅₅=I was reached. Then, the IPTG was added to final concentration of 1 mM and cultivation was continued for 3 hours at 34 ⁰C. Then, cells were harvested from 1 ml of the culture by centrifugation, washed with 0.1 M NaCl solution and resuspended in the 1 ml of reaction buffer (0.5 ml of HMKP solution plus 0.5 ml of 0.2 M Glutamate, pH 7.0). Reactions were carried out for about 17 hours at 34 ⁰C. Accumulation of 4HIL and α-aminobutyrate was analyzed by HPLC (Fig. 3).

3) To provide the in vitro biotransformation process, his₆-tag enzymes ecoBCAT and bsuBCAT purified as described above (see Example 8) were added to 100 μl of the same reaction buffer (mixture of 0.5 ml of HMKP solution and 0.5 ml of 0.2 M Glutamate, pH 7.0) up to a final concentration of 0.1 mg/ml. Reaction mixtures were incubated for about 17 hours at 34 ⁰C. Accumulation of 4HIL and α-aminobutyrate was analyzed by HPLC (Fig. 3). HPLC analysis was performed as described in the example 2. Data of 4HIL accumulation are presented in the Table 6.

[Table 6]

* calculated as Y = (produced 4HIL[mM]/(supplied α-ketobutyrate [mM]+supplied acetaldehyde [mM]))*100.

[Example 8] Identification of HMKP-aldolase from Arthrobacter simplex AKU 626 (IFO 12069) (asiHPAL). 1. Purification of asiHPAL.

Purification protocol includes the following procedures.

Stepl: 1 ml of overnight bacterial culture (grown for 12 h at 34 ⁰C) was used to inoculate 5 liters of LB-broth [8X(375 ml in 1 L flask)]. Cells were cultivated at optimal temperature for about 24 hours. Then, cells were harvested by centrifugation (16000 x g) at 4 ⁰C, and re-suspended in 30 ml of buffer A [50 mM KH₂PO₄ (pH 7.4 adjusted by KOH)] supplemented by ImM PMSF (phenylmethylsulfonyl fluoride). Step2: Cells were disrupted by 3-5 passages through French pressure cell (max P = 2,5

Psi) followed by centrifugation to remove debris. Protein preparation was passed through

Sephadex G-15 column (2.6 X 28 cm) equilibrated with buffer A.

Step3: Anion-exchange chromatography (AEC 1) was carried out using AKTAbasiclOO system supplemented with 50 ml DEAE (fast flow) column (d = 1.6 cm). Usually 40 - 50 ml of protein preparation obtained from Step2 were applied to column equilibrated with buffer A. The elution was carried out at flow rate 2.5 ml/min by liner gradient 0 - 0.5 M NaCl in buffer A (IO

CV (column volumes)). Each lOml-fraction was collected. Active fractions were pooled and desalted as well as described in "step2 " item.

Step4: Anion-exchange FPLC (AEC 2) was carried out using AKTAbasiclOO system supplemented with 1.6 ml "Soursel5Q" column (Amersham Pharmacia Biotech). Protein preparation obtained from StepS was applied to column equilibrated with buffer A. The elution was carried out at flow rate 1 ml/min by liner 0 - 0.5 M NaCl in buffer A (40 CV). Each 2 ml fraction was collected. Active fractions were pooled (Table 7, 8).

Step5: Hydrophobic interaction chromatography (HIC) was carried out using AKTAbasiclOO system supplemented with 1 ml "Resource PHE"column (Amersham Pharmacia

Biotech). Protein concentration in protein preparation obtained from Step 5 was adjusted to 0.8 mg/ml and then ammonium sulphate was added up to final concentration 1.5 M. Protein solution was applied to column equilibrated with buffer A supplemented with 1.5 M ammonium sulfate.

Elution was carried out at flow rate 1 ml/min by liner gradient from 1.5 M to 0 M ammonium sulfate in buffer A (30 CV). Each 1 ml fraction was collected. Active fractions were pooled

(Table7, 8).

Stepό: Size exclusion chromatography (SEC) was carried out using AKTAbasiclOO system supplemented with Superdex™ 200 HR 10/3 OA (Amersham Pharmacia Biotech) column.

Protein preparation obtained from Step5 was applied to column equilibrated with buffer A supplemented by 100 mM NaCl. Isocratic elution was down at 0.5 ml/min flow rate. Each 1 ml fraction was collected. Active fractions were pooled (Table 7, 8; Fig.4).

Table.7. Purification of the asiHPAL. Specific ^υ

Protein Total Total

Volume activity Purificatio

Step cone. protein Activity²⁵ Yield % ³⁾ (ml) (nmoles/mg/ n rate ^'

(mg/ml) (mg) (nmoles/min) min)

Lysate 120 12.,6 1512 0.6 903 100.00 1

AECl 20 0.7 14 30 401 45 48

AEC2 4 1 4 100 402 45 167

HIC 1 0.1 0,1 1419 142 16 2365

SEC 1 0.04 0.04 1490 60 7 2483

¹^ Specific activity was determined by HPLC monitoring of the time-dependent 4HIL formation in the bsuBCAT/asiHPAL bi-enzymatic reaction with following composition [10OmM L-glutamate (pH 8 ajusted by pH 8.0), 10OmM α-ketobutyrate, 100 mM acetaldehyde ], ImM ZnCl₂, and 0,5 μg purified bsuBCAT) and aliquot of active fraction of asiHPAL separation. All reactions were carried out at 37⁰C.

²⁾ Calculated as (Total protein) x (Specific activity).

³⁾ Calculated as 100% x (Total activity/Total activity in crude lysate).

⁴⁾ Calculated as (Specific activity/Specific activity in crude cell lysate).

¹ Table 8. Chromatographic elution parameters. ^a)

Chromatographic staj

AEC l AEC 2 me SEC NaCl, M NaCl, M (NHLO₂SO₄, M VZV₀

0.35 - 0.37 0.31 -0.35 0.19 - 0.1 1.5 ^a) Data are present as a salts concentration interval within HPAL activity is eluted

Specific activity of asiHPAL was determined by HPLC monitoring of the time-dependent 4HTL formation in the bsuYwaA/asiHPAL bi-enzymatic reaction with following composition [10OmM L-glutamate (pH 8 adjusted by pH 8.0), 10OmM α-ketobutyrate, 100 mM acetaldehyde ], ImM ZnCl₂, and 0.5 μg purified His-tag-bsuBCAT protein (branched-chain amino acid aminotransferase from Bacillus subtilis, obtained in Example 6) and aliquot of elution fraction. All reactions were carried out at 37 ⁰C. Cloning the branched-chain amino acid aminotransferase from Bacillus subtilis (bsuBCAT) and HPLC measuring the 4HIL formation are described in the example 7. Molecular weight of asiHPAL monomer was determined using SDS- PAGE (Fig. 5, A).

Its average value was estimated as 27 kDa. Molecular weight of native asiHPAL was determined using analytical SEC on the Superdex™ 200 HR 10/3 OA (Pharmacia) columns calibrated by

Molecular Weight Protein Markers (Sigma) (Fig. 5, B). Its average value was estimated as 186 kDa. So, asiHPAL is assumed to be the hexamer.

Dependence of HMKP-aldolase from metal ions was investigated. It was established that asiHPAL activity strictly depends on the Zn ²⁺, Mg ²⁺ and Mn ²⁺ ions and is completely blocked in the presence of EDTA. So, it was assumed that asiHPAL belongs to the TypeII aldolases (Table 9).

Table 9. asiHPAL activity depending on the metal ²⁺ ions.

Cofactor (2mM concentration)

Protein

EDTA Zn²⁺ Mg²⁺ Mn²⁺ asiHPAL ND. ^a) 1 ^b) 0.5 0.5

^ non detected ^b) Activities were measured as well as described in the Table 7 footnote. The value of the asiHPAL activity measured in the presence of the Zn ²⁺ ion is taken as 1.

2. Determination of asiHPAL N-terminal sequence.

2.1 Western blotting of the asiHPAL. asiHPAL was immobilized on the Sequi-Blot PVDF membrane (Bio-Rad) using trans-Blot SD sell (Bio-Rad). Optimized blotting conditions: (Dun carbonate transfer buffer: 10 mM NaCHO₃, 3 mM Na₂CO₃ without methanol, six pieces extra thick filter paper/membrane sandwich, starting current 5.5 mA/cm², transfer time — 1 hour).

2.2 N-terminal sequencing.

Determination of asiHPAL N-terminal sequence was done using 49IcLC Protein Sequencer (Applied Biosystems, USA). 14 cycles were done. Results: NH₂- Pro-Phe-Pro-Val-Glu-Leu-Pro-Asp-Asn-Phe-Ala-Lys-Arg-Val... (SEQ ID NO: 11 )

Alignment of the determined N-terminal sequence with all known proteins (BLAST services) revealed the single protein with similar N-terminal sequence. It is HHDE-aldolase (2,4-dihydroxyhept-2-ene-l,7-dioic acid aldolase) from Brevibacterium linens BL2

(HHDE_BLI)(Fig. 6). So, the matched enzyme is TypeII aldolase and its native substrate,

2,4-dihydroxyhept-2-ene-l,7-dioic acid, is structurally similar to HMKP (indeed, both have hydroxyl group at C₄ position, carbonyl group at C₂ position and carboxyl group at Cj position). Moreover, the Mw of HHDE-BLI subunit is 27 kDa which is in a good agree with the experimentally obtained subunit Mw of asiHPAL. Also Brevibacterium linens is closely related to Arthrobacter simplex. Thus it could be assumed that asiHPAL purified from Arthrobacter simplex is a homologue of HHDE aldolase from Brevibacterium linens BL2.

[Example 9] YfaU and YhaF aldolases from E. coli.

HHDE_BLI is a part of so-called hpcH/hpal aldolase family which incorporates two functional subgroups: 2,4-dihydroxyhept-2-ene-l, 7-dioic acid aldolases and

4-hydroxy-2-oxovalerate aldolases. Simple BLAST searching of HHDE_BLI homologues revealed that subgroup including 2-keto-3-deoxyglucarate aldolases also belongs to hpcH/hpal aldolases family.

Based on the results of BLAST search, it was suggested that any protein belonging to the 2,4-dihydroxyhept-2-ene-l, 7-dioic acid aldolases , 4-hydroxy-2-oxovalerate aldolases or 2-keto-3-deoxyglucarate aldolases groups may be utilized as a HMKP aldolases.

To prove that suggestion, ability of two enzymes from E. coli belonging to the hpcH/hpal aldolase family to exhibit a HMKP aldolase activity was tested. There are YfaU, assigned as putative 2,4-dihydroxyhept-2-ene-l,7-dioic acid aldolases, and YhaF, identified as 2-keto-3-deoxyglucarate aldolase. But authors of the present invention failed to detect HMKP aldolase activity in crude cell lysate of E. coli strain MG 1655. So it was supposed that expression of YfaU and YhaF under examined cultivation conditions is rather low.

To increase expression level of YfaU and YhaF, strong promoter P_tao jointed with effective RBS was inserted upstream of coding regions of yfάU and yhaF genes.

1. Construction of MG1655[mhpD::attL-kan-attR-Ptac-RBS] strain.

1.7 kb DNA fragment containing kan-marker and P_tac promoter with effective RBS was constructed as follows.

At first, DNA fragment containing kan gene flanked with λattL and λattR sites was obtained by PCR using plasmid pMW118-(λα#Z-Km^r-λα#R) as a template and oligonucleotides svs45

(5 ' -gct-ttc-aat-caa-ctg-gtg-ctg-aat-ttc-ctc-gca-cgc-cct-taa-gga-tga-agc-ctg-ctt-ttt-tat-act-aag-ttg-S ' :

SEQ ID NO:12) and P_Bgiii (cgtacagatctgttacaggtcactaat: SEQ ID NO:13) as a primers. Primer svs45 contains at the 5 '-end thereof the part homologous to upstream region of mhpD gene, necessary for further integration into chromosome of E. coli strain MG 1655. Primer contains BgHl site at the 5 '-end thereof. Construction of plasmid pMWl l8-(λattL-Km^τ-λattK) is described in the Reference Example. Then, Ptao promoter with steaky BgRl site was obtained by annealing two oligonucleotides P_tac5 and P_tac3- The 5'-end of oligonucleotide P_taC5 was phosphorylated before annealing by kinase reaction. P_tac5 5 '-pGATCTCCCTGTTGACA ATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCG-3 ' (SEQ ID NO: 14) P_tac3 5'-CGCTCACAATTCCACACATTATACGAGCCGATGATTAATTGTCAACAGGGA-S'

(SEQ ID NO: 15).

Then, DNA fragment obtained by PCR was treated with BgM restrictase and ligated with P_tao promoter with steaky BgHl site/ Resulted mixture was used as a template for PCR reaction conducted with primers svs45 and svs46

(5 ' -aga-acc-gat-aat-ggc-gac-ttt-acg-ctt-act-cat-atg-tat-atc-tcc-ttc-cgc-tca-caa-ttc-cac-aca-tta-tac-S ' : SEQ ID NO: 16). Primer svs46 contains at the 5 '-end thereof the part homologous to downstream region of mhpD gene, necessary for further integration into chromosome of E. coli strain MG1655, and effective RBS in the middle thereof. Obtained DNA-fragment was inserted into MG 1655 chromosome with using routine Red-integration procedure (Datsenko, K. A. and Wanner, B.L., Proc. Natl. Acad. ScL USA, 97, 12, p 6640-6645 (2000)). As a result, mhpD gene was deleted and mhpF and mhpE genes were put under the control of the artificial regulatory region (Fig. 7). 2. Construction of MG1655[attLΔyfaV-kan-attR Ptac-RBS- yfaU] strain.

The nucleotide sequence of the yfaU gene and the amino acid sequence of the YfaU protein encoded by the yfaU gene are shown in SEQ ID NO: 26 and SEQ ID NO: 27, respectively. 1.8 kb DNA fragment containing kan-marker and P_tac-RBS expression module was amplified by PCR with using oligonucleotides SVS_81 (5^l-taa-acg-ttc-ttt-aaa-ggg-att-gct-taa-taa-tgc-gtt-cat~atg-tat-atc-tcc-ttc~cgc-tca-caa-ttc-cac-aca-3^l: SEQ ID NO: 17) and SVS_82

(S'-caa-ttt-gaa-acg-ccc-cta-cag-cca-cta-atc-act-ccg-ggc-gtt-gct-tga-agc-ctg-ctt-ttt-tat-act-aag-ttg-S' : SEQ ID NO: 18) as primers and chromosomal DNA of MGl 655 [mhpD::attL-kan-attR-Ptac-RBS] strain as a template. Then, obtained DNA-fragment was inserted into MGl 655 chromosome with using routine Red-integration procedure (Datsenko, K.A. and Wanner, B.L., Proc. Natl. Acad. Sci. USA, 97, 12, p 6640-6645 (2000)). As a result, MG1655[attLΔyfaV-kan-attR-Ptac-RBS- yfaU] strain was constructed (Fig. 8). 3. Construction of MG1655[attLΔyhaU-kan-attR Ptac-RBS- yhaF] strain.

The nucleotide sequence of the yhaF gene and the amino acid sequence of the YhaF protein encoded by the yhaF gene are shown in SEQ ID NO: 24 and SEQ ID NO: 25, respectively. 1.8 kb DNA fragment containing kan-marker and P_tac-RBS expression module was amplified by PCR with using oligonucleotides SVS 89

(S'-tgc-ggc-ttt-gaa-ttt-att-cgg-gaa-aac-atc-gtt-att-cat-atg-tat-atc-tcc-ttc-cgc-tca-caa-ttc-cac-aca-S¹:

SEQ DD NO:19) and SVS_90 (S'-tcc-cca-taa-taa-taa-aaa-tca-gca-taa-gta-ccc-gag-gta-aat-aaa-tga-agc-ctg-ctt-ttt-tat-act-aag-ttg-S'

: SEQ ID NO:20) as primers and chromosomal DNA of MG1655

[mhpD::attL-kan-attR-Ptac-RBS] strain as a template. Then, obtained DNA-fragment was inserted into MG 1655 chromosome with using routine Red-integration procedure (Datsenko, K. A. and

Wanner, B.L., Proc. Natl. Acad. Sci. USA, 97, 12, p 6640-6645 (2000)). As a result, MGl 655 [attLΔyhaU-Km-attR Ptac-RBS- yhaF] strain was constructed (Fig. 8).

The increased amount of both enzymes was confirmed by SDS_PAGE analysis of crude cell lysate proteins of corresponding strain (Fig. 9).

To investigate HMKP-aldolase activity in crude cell lysates of

MG1655[attLΔyfaV-Km-attR P_t30-RBS- yfaU] and MG1655[attLΔyhaU-Km-attR P_tac-RBS- yhaF] strains following procedure was carried out:

1) 50 μl of overnight bacterial culture (grown for 12 h at 37 ⁰C) was used to inoculate 4 ml of LB-broth supplemented with kanamycin (100 μg/ml). Cells are cultivated at 37 °C for about 1-2 hours until OD₅₅₅=I was reached. Then, cells were harvested by centrifugation (16000 x g) at 4 ⁰C, and resuspended in 0.5 ml of buffer A [50 mM KH₂PO₄ (pH 7.4 adjusted by KOH)] supplemented with ImM PMSF (phenylmethylsulfonyl fluoride). Then cells were routinely disrupted by ultrasonic treatment at 4 ⁰C and cell's debris was removed by centrifugation (16000 xg) at 4 ⁰C.

2) 11 μl of obtained crude cell lysate was added to 9μl of reaction mixture containing 100 mM L-glutamate (diluted from 0.5M stock solution (pH 7.4 adjusted by NaOH)); 100 mM ketobutyrate, 100 mM acetaldehyde, 2 mM MgCl₂ (or MnCl₂, or ZnCl₂, or EDTA), purified bsuBCAT (14 μg). Reaction incubated at 37 ⁰C for 2 hours. 4HEL synthesis was tested by TLC and HPLC analyses (Fig. 10; Table 10).

Table 10. Investigation of HMKP-aldolase activity in crude cell lysates of MG1655[attLΔyfaV-Km-attR P_43C-RBS- yfaU] and MG1655[attLΔyhaU-Km-attR Ptac-RBS- yhaF] strains using HPLC analysis.

[Example 10] Experimental study of the 4HIL yield in the AL/AT-dependent biotransformation process.

For experimental study of the 4HEL biotransformation processes, YfaU and YhaF aldolases were purified from crude cell lysates of E. coli strains MG1655[attLΔyfaV-Km-attR P_tac-RBS- yfaU] and MG1655[attLΔyhaU-Km-attR Ptac-RBS- yhaF], correspondingly.

Purification protocol includes the following procedures.

Stepl: 1 ml of overnight bacterial culture (grown for 12 h at 37 ⁰C ) was used to inoculate 3 liters of LB-broth . Cells are cultivated at 37 ⁰C for about 12 hours. Then, cells are harvested by centrifugation (16000 x g) at 4 ⁰C, and re-suspended in 30 ml of buffer A [50 mM

KH₂PO₄ (pH 7.4 adjusted by KOH)] supplemented with ImM PMSF (phenylmethylsulfonyl fluoride).

Step2: Cells were disrupted by 3 passages through French pressure cell (max P = 2 Psi) followed by centrifugation to remove debris. Protein preparation was passed through Sephadex. G-15 column (2.6 X 28 cm) equilibrated with buffer A.

Step3: Anion-exchange chromatography was carried out using AKTAbasiclOO system supplemented with 50 ml DEAE (fast flow) column (d = 1.6 cm). Usually 40 - 50 ml of protein preparation obtained from Step! were applied to column equilibrated with buffer A. The elution was carried out at flow rate 2.5 ml/min by liner gradient 0 - 0.5 M NaCl in buffer A (10 CV (column volumes)). Each 10 ml fraction was collected.

Active fractions were pooled and passed through Sephadex G-15 column (2.6 X 28 cm) equilibrated with buffer B (10 mM KH₂PO₄ (pH 7.0 adjusted by KOH).

Step4: Protein preparation obtained from Step3 was passed through Hydroxylapatite (Bio-Rad; DNA-Grade, Bio-Gel HTP) column (2.5X1.5 cm) equilibrated with buffer B. Flow through fractions were collected.

Step5: Anion-exchange FPLC was carried out using AKTAbasiclOO system supplemented with 1,6 ml "Soursel5Q" column (Amersham Pharmacia Biotech). Protein preparation obtained from Step4 was applied to column equilibrated with buffer B. The elution was carried out at flow rate 1 ml/min by liner 0 M - 0.5 M NaCl in buffer B (20 CV). Each-2 ml fraction was collected. Active fractions were pooled.

Stepό: (NHt)₂SO₄ was added to protein preparation obtained from step5 up to 1.5 M concentration. Protein precipitate was collected by centrifugation (16000 g, 4 ⁰C, 15 min) and resuspended in buffer B.

Protein preparation obtained from stepό was used in experiments (Fig. 11).

Cloning of mhpFE

As set forth above, adequate biosynthesis of 4HIL was not achieved under conditions where E. cø//-derived MhpE aldolase (Appl. Environ. Microbiol., Vol. 64, No. 10, 4093-4094,

1998) was additionally employed. The nucleotide sequence of the mhpFE gene is shown in SEQ

ID NO: 23. For mhpFE gene derived from E. coli, we amplified the target gene using

5'-cgaattcttatttgttgttgcgcagatcca-3'(SEQ ID No.: 21) as sense-primer and 5'-cgaattcttattt- gttgttgcgcagatcca-3'(SEQ ID No.: 22) as anti-sense-primer and incorporated it into a pET-21(a+)/Nhe-lEcoRl vector. BL21/pET-21a(+)mhpFE transformed with this plasmid was shake cultured in a 500 mL Sakaguchi flask in which 50 mL of LB medium had been dispensed, and expression of the targeted protein was induced with 0.5 mM IPTG when OD₅₅₅ = 1 was reached.

The expression in a soluble fraction was possible only when mhpE jointly expressed with mhpF. Since no tag was present on the mhpFE expressed in this system, the 4HTT, production test was conducted in a system employing cell lysate.

4HUL synthesis.

To study the 4HTL formation in two-step biotransformation process using purified YhaF, YfaU and bsuBCAT enzymes the set of reaction mixtures was prepared. Each of them had 20 μl volume and included 300 mM acetaldehyde, 100 mM α-ketobutyrate, 100 mM L-glutamate (diluted from 0.5M stock solution (pH 7.5 adjusted by NaOH)), 20 mM KH₂PO₄ZNa₂HPO₄ buffer (diluted from 0.2 M stock solution (pH 7), 2 mM MgCl₂, 14 μg purified bsuBCAT, 40 μg purified YfaU (or 120 μg purified YhaF). Each reaction was incubated at 37 ⁰C for 4 hours. Then 4HEL concentration was carried out using routinely HPLC-analysis (Table 11). To study the 4HIL formation in two-step biotransformation process using purified asiHPAL and bsuBCAT enzymes, the reaction mixture (reaction volume = 20μl) containing 100 mM L-glutamate (diluted from 1 M stock solution (pH 8.0 adjusted by NaOH), 100 mM acetaldehyde, 100 mM α-ketobutyrate, 2 mM ZnCl₂, 14 μg of purified bsuBCAT, 0.6 μg purified asiHPAL was prepared. Reaction was incubated at 37 ⁰C for 2 hours. Then 4HIL concentration was carried out using routinely HPLC-analysis (Table 11).

To study the 4HIL formation in two-step biotransformation process using purified mhpFE and ecoBCAT enzymes, E. coli expressing mhpFE. BL21(DE3)[pET-HT-ilvE-ECO] or BL21/pET-21a(+)mhpFE were induced to express the target protein in the manner set forth above and the bacteria were collected from 1 mL of culture solution and washed. They were then reacted with 200 μL of reaction solution (100 mM of acetaldehyde, 100 mM of α-ketobutyrate, 10OmM of L-glutamate, 5OmM phosphate buffer (pH7)). The reaction temperature was 30⁰C, the reaction was conducted overnight,, and the cell lysate were obtained by sonication prior to the reaction. Following the reaction, centrifugation (10,000 rpm, 5 min) was conducted to obtain a supernatant. The supernatant was diluted 25-fold and subjected to amino acid analysis by HPLC. Untransformed E. coli was employed as the control (Table 11).

HPLC analysis was carried out as described in the example 2.

Table 11. Investigation of 4HLL production in two-step biotransformation process using purified YhaF, YfaU, asfflPAL and bsuBCAT enzymes

[Reference example] Construction of pMWl 1 S-(λattL-Km^τ-λattR) plasmid. pMWll8-(λattL-Km^τ-λattR) plasmid was constructed on the basis of pMW118-attL-Tc-attR (WO2005/010175) plasmid by substitution of tetracycline resistance marker gene by kanamycin resistance marker gene from pUC4K plasmid (Vieira, J. and Messing,

J., Gene, 19(3): 259-68 (1982)).

For that purpose large EcoRI - HindΩl fragment of pMWl 18-attL-Tc-attR plasmid was ligated to two fragments of pUC4K plasmid: HindΩl - Pstl fragment (676 bp) and EcoBI —

Hindlϊl fragment (585 bp). Basic pMWl 18-attL-Tc-attR was obtained by ligation of the following four DNA fragments:

1) the BglE-EcoRl fragment (114 bp) carrying attL (SEQ ID NO: 28) which was obtained by PCR amplification of the corresponding region of the E. coli W3350 (contained λ prophage) chromosome using oligonucleotides Pl and P2 (SEQ ID NOS: 29 and 30) as primers (these primers contained the subsidiary recognition sites for BgRl and EcoRl endonucleases);

2) the Pstl-HincRll fragment (182 bp) carrying attR (SEQ ID NO: 31) which was obtained by PCR amplification of the corresponding region of the E. coli W3350 (contained λ prophage) chromosome using the oligonucleotides P3 and P4 (SEQ ID NOS: 32 and 33) as primers (these primers contained the subsidiary recognition sites for Pstl and HindlU endonucleases);

3) the large BgKL-Hindlll fragment (3916 bp) of pMWl 18-ter_trnB. The plasmid pMW118-ter_?-raB was obtained by ligation of the following three DNA fragments: • the large DNA fragment (2359 bp) carrying the Aatϊl-EcoPΛ fragment of pMW118 that was obtained by the following way: pMWl 18 was digested with EcoPl restriction endonuclease, treated with Klenow fragment of DNA polymerase I, and then digested with AatW restriction endonuclease;

• the small Aatll-BgRl fragment (1194 bp) of pUC19 carrying the bla gene for ampicillin resistance (Ap^R) was obtained by PCR amplification of the corresponding region of the pUC19 plasmid using oligonucleotides P5 and P6 (SEQ ID NOS: 34 and 35) as primers (these primers contained the subsidiary recognition sites for Aatll and BgIR endonucleases);

• the small Bglϊl-Pstlpol fragment (363 bp) of the transcription terminator ter_mxδ was obtained by PCR amplification of the corresponding region of the E. coli MGl 655 chromosome using oligonucleotides P7 and P8 (SEQ ID NOS: 36 and 37) as primers (these primers contained the subsidiary recognition sites for BgIU. and Pstl endonucleases);

4) the small EcoPl-Psfl fragment (1388 bp) (SEQ ID NO: 38) of pML-Tc-ter_tfwZ bearing the tetracycline resistance gene and the ter_thrL transcription terminator; the pML-Tc-ter_//»-Z plasmid was obtained in two steps:

• the pML-ter_thrL plasmid was obtained by digesting the pML-MCS plasmid (Mashko, S.V. et al., Biotekhnologiya (in Russian), 2001, no. 5, 3-20) with the Xbal and BamHl restriction endonucleases, followed by ligation of the large fragment (3342 bp) with the Xbal-BamHl fragment (68 bp) carrying terminator XQXJhrL obtained by PCR amplification of the corresponding region of the E. coli MG 1655 chromosome using oligonucleotides P9 and PlO (SEQ ID NOS: 39 and 40) as primers (these primers contained the subsidiary recognition sites for the Xbal and BamHl endonucleases); the pML-Tc-ter_t/??*Z plasmid was obtained by digesting the pML~ter_£/zrL plasmid with the Kpnl and Xbal restriction endonucleases followed by treatment with Klenow fragment of DNA polymerase I and ligation with the small EcoRl~Van91l fragment (1317 bp) of pBR322 bearing the tetracycline resistance gene (pBR322 was digested with EcoBl and Van91l restriction endonucleases and then treated with Klenow fragment of DNA polymerase I).

Industrial Applicability

The present invention provides a method for manufacturing 4HIL by means of an enzymatic process or chemical and enzymatic combination process, for which only vegetable extract enzymes have previously been reported. The present invention is extremely useful in the field of pharmaceuticals and drugs manufacturing.

Claims

Claims

1. A method for manufacturing 4-hydroxy-L-isoleucine or a salt thereof, comprising the steps of: contacting a biocatalyst that actively catalyzes a reaction producing 4-hydroxy-L-isoleucine that is shown by the following Formula (I) from acetaldehyde and α-ketobutyric acid in the presence of an amino group donor,

with acetaldehyde and α-ketobutyric acid in an aqueous solvent containing an amino group donor, and isolating 4-hydroxy-L-isoleucine.

2. The method according to claim 1, wherein the reaction is conducted in the presence of NADH or NADPH.

3. The method according to any of claim 1 and 2, wherein the aqueous solvent contains branched-chain amino acid aminotransferase.

4. The method according to any of claims 1 to 3, wherein the amino group donor is selected from the group of branched-chain amino acids.

5. The method according to any of claims 1 to 4, wherein the biocatalyst is comprised of an enzyme having aldolase activity of producing 4-hydroxy-3-methyl-2-keto-pentanoic acid from acetaldehyde and α-ketobutyric acid, and an enzyme having transamination activity of producing 4-hydroxy-L-isoleucine from 4-hydroxy-3-methyl-2-keto-pentanoic acid in the presence of an amino group donor.

6. . The method according to any of claims 1 to 4, wherein the biocatalyst is a bacterium containing an enzyme having aldolase activity of producing 4-hydroxy-3-methyl-2-keto-pentanoic acid from acetaldehyde and α-ketobutyric acid and an enzyme having transamination activity of producing 4-hydroxy-L-isoleucine from 4-hydroxy-3-methyl-2-keto-pentanoic acid in the presence of an amino group donor.

7. The method according to claim 6, wherein the bacterium is modified to enhance at least one activities of aldolase and branched-chain amino acid aminotransferase.

8. The method according to claim 7, wherein the activities of aldolase and branched-chain amino acid aminotransferase is enhanced by increasing the expression of the aldolase and/or the branched-chain amino acid aminotransferase.

9. The method according to claim 8, wherein the expression of the aldolase and/or the branched-chain amino acid aminotransferase is increased by modifying an expression control sequence of the gene encoding said aldolase and/or branched-chain amino acid aminotransferase or by increasing the copy number of the gene encoding said aldolase and/or branched-chain amino acid aminotransferase.

10. The method according to any of claims 5 to 10, wherein the enzyme having aldolase activity is an aldolase belonging to the hpcH/hpal family of aldolases.

11. The method according to any of claims 5 to 10, wherein the en2yme having transamination activity is an aminotransferase belonging to the branched-chain amino acid aminotransferase.

12. The method according to any of claims 6 to 7 wherein the bacterium is of the genus Schizosaccharomyces, Arthrobacter; Brevibacterium, Candida, Corynebacterium, Micrococcus, Cellulomonas, Actinoplanes, Chromobacterium, Rahnella, Rhizobium, Erwinia, Hansenula, Torulopsis, Kloeckera, Rhodotorula, Panellus, Mucor, Debaryomyces, Sporobolomyces, Escherichia, Salmonella, Flavobacterhim, Bacillus, or Proteus.

13. The method according to any of claims 6, 7 and 12 wherein the bacterium belongs to Schizosaccharomyces pombe, Arthrobacter. simplex, Brevibacterium ammoniagenes, Candida utilis, Micrococcus luteus, Micrococcus flavus, Micrococcus roseus, Corynebacterium glutamicum, Corynebacterium aquaticum, Corynebacterium paicrometabolurn, Arthrobactor globiformis, Arthrobactor sulfureus, Arthrobactor viscosus, Brevibacterium protophormiae, Brevibacterium acetylicum, Brevibacterium stationis, Brevibacterium fuscum, Cellulomonas fimi, Cellulomonas biazotea, Actinoplanes auranticolor, Chromobacterium iodinum, Citrobacter freundii, Erwinia carotovora subsp. carotovora, Rahnella aquatilis, Rhizobium radiobacter, Hansenula anomala, Hansenula miso, Candida stellata, Hansenula saturnus, Hansenula nonfermentans, Hansenula polymorpha, Torulopsis nitr'atophila, Candida guilliermondii, Candida lipolytica, Candida macedoniensis, Candida pseudotropicalis, Candida tropicalis var. lambica, Candida solani, Candida albicans, Kloeckera africana, Kloeckera japonica, Rhodotorula mucilaginosa, Panellus serotinus, Mucor racemosus f.sp. racernosus, Mucor lamprosporus, Mucor petrinsularis, Debaiγomyces vanrijiae, Sporobolomyces roseus, Escherichia coli KJ 2, Salmonella typhimwiwn, Flavobacteriumferrugineum, Bacillus subtilis or Proteus mirabϊlis.

14. The method according to any of claims 6, 7, 12 and 13 wherein the bacterium is bacterial culture products, cells, or treated cells.

15. A method for manufacturing 4-hydroxy-L-isoleucine or a salt thereof, comprising the steps of: contacting a biocatalyst having activity in the production of 4-hydroxy-L-isoleucine from 4-hydroxy-3-methyl-2-keto-pentanoic acid, in the presence of an amino group donor with 4-hydroxy-3-methyl-2-keto-pentanoic acid in an aqueous medium containing an amino group donor, and isolating 4-hydroxy-L-isoleucine.

16. The method according to claim 15 wherein the biocatalyst is an enzyme having transamination activity of producing 4-hydroxy-L-isoleucine from

4-hydroxy-3-methyl-2-keto-pentanoic acid in the presence of an amino group donor.

17. The method according to claim 15 wherein the biocatalyst is a bacterium containing an enzyme having transamination activity of producing 4-hydroxy-L-isoleucine from 4-hydroxy-3-methyl-2-keto-pentanoic acid in the presence of an amino group donor.

18. The method according to any of claims 15 to 16, wherein the transamination is performed by an isolated branched-chain amino acid aminotransferase.

19. The method according to claim 18, wherein the branched-chain amino acid aminotransferase is isolated from a bacterium selected from the group consisting of Escherichia and Bacillus.

20. The method according to any of claims 15 to 17, wherein the 4-hydroxy-3-methyl-2-keto-pentanoic acid is obtained by aldol reaction of α-ketobutyric acid and acetaldehyde.

21. The method according to claim 17, wherein the bacterium is modified to enhance an activity of branched-chain amino acid aminotransferase.

22. The method according to claim 21, wherein the activity of branched-chain amino acid aminotransferase is enhanced by increasing the expression of branched-chain amino acid aminotransferase.

23. The method according to claim 22, wherein the expression of branched-chain amino acid aminotransferase is increased by modifying an expression control sequence of the gene encoding said branched-chain amino acid aminotransferase or by increasing the copy number of the gene encoding the branched-chain amino acid aminotransferase.

24. The method according to any of claims 17 and 21 wherein the bacterium is of the genus

Schizosaccharomyces, Candida, Arthrobacter, Brevibacterium, Cryptococcus, Pseudomonas, Hansenula, Flavobacterium, Bacillus, Micrococcus, Pichia, Escherichia or Torulopsis.

25. The method according to any of claims 17, 21 and 24 wherein said bacterium is Schi∑osaccharomyces pombe, Arthrobacter simplex, Brevibacterium ammoniagenes, Cryptococcυs flavus, Candida utilis, Pseudomonas sp., Flavobacterimn heparinum, Bacillus thuringiensis, Bacillus subtilis, Micrococcus luteus, Pichia orientalis, Hansemda jadinii, Torulopsis sphaerica, Escherichia coli or Brevibacterium linens.

26. The method according to any of claims 17, 21, 24 to 25 wherein the bacterium is bacterial culture products, cells, or treated cells.

27. The method according to any of claims 1 to 26 wherein the 4-hydroxy-L-isoleucine is selected from at least one member of the group consisting of (2S,3S,4S)-4-hydroxyisoleucine_s (2S,3R,4R)-4-hydroxyisoleucine, (2S,3S,4R)-4-hydroxyisoleucine and (2S,3R,4S)-4hydroxy- isoleucine.