New aminotransferase and method for producing amino compound using same

Abstract

The purpose of the present invention is to provide: a new aminotransferase capable of improving the production efficiency of an amino compound; or a transformant having the same. The purpose of the present invention is to also provide a biosynthesis method for an amino compound using said new aminotransferase or a transformant having the same.　The present invention for solving the problem is an aminotransferase that uses a carbonyl compound as a substrate, and that is characterized by having an amino acid sequence that has an identity of 90% or more with respect to any one of the amino acid sequences of SEQ ID NO: 13, 17, 21, 25, and 29. The carbonyl compound is preferably a compound represented by formula (1). The present invention makes it possible to supply bio-derived products and can realize carbon recycling, and thereby provides a contribution in achieving the Goal 12, etc., of the SDGs. In formula (1), R represents a methyl group or an ethyl group. n represents an integer of 2-16.

Description

Novel aminotransferase and method for producing amino compounds using the same

[0001] The present invention relates to a novel aminotransferase and a method for producing an amino compound using the same.

[0002] Amid calls for business restructuring with a strong focus on addressing environmental issues such as global warming and marine plastic pollution, development of bio-based product production technologies that accelerate the realization of carbon recycling is underway. For example, one known bio-based product production technology is a method for biosynthesizing amino compounds via the reaction of a biocatalyst, an aminotransferase. Patent Document 1 discloses genetically engineered cells that can produce more ω-aminocarboxylic acids, ω-aminocarboxylic acid esters, or more lactams derived from ω-aminocarboxylic acids from carboxylic acids or carboxylic acid esters than wild-type cells, and the cells are genetically modified to enhance the activity of a specific aminotransferase. Furthermore, Patent Document 2 and Non-Patent Document 1 disclose methods for synthesizing long-chain amino acids, which are used as raw materials for nylon production, using an ω-amino acid transaminase derived from Pseudomonas bacteria as a biocatalyst.

[0003] However, because naturally occurring aminotransferases have evolved biologically to use carbonyl compounds known to exist in living organisms as substrates, they generally have no activity or very low activity toward carbonyl compounds that do not exist in living organisms. Furthermore, biosynthetic methods for amino compounds have limitations on applicable substrates and low reactivity, which poses challenges for industrial use. To address these issues, efforts are being made to expand the range of applicable substrates and to develop aminotransferases that exhibit high reactivity.

[0004] Special table 2011-505854 publication Special table 2016-521562 publication

[0005] Applied and Environmental Microbiology, 2016 Vol82, 3846-3856

[0006] Conventionally known aminotransferases have had problems in industrial application, such as insufficient transamination activity and low efficiency of amino compound production. Therefore, an object of the present invention is to provide novel aminotransferases that can solve the above problems and improve the efficiency of amino compound production, and transformants containing the same. Another object of the present invention is to provide methods for biosynthesizing amino compounds using these novel aminotransferases or transformants containing the same.

[0007] Under these circumstances, the present inventors have conducted extensive research and have discovered a novel aminotransferase that can significantly improve the efficiency of producing amino compounds, thereby completing the present invention.

[0008] The gist of the present invention is as follows: [1] An aminotransferase that uses a carbonyl compound as a substrate, characterized by comprising an amino acid sequence having 90% or more identity to any of the amino acid sequences of SEQ ID NOs: 13, 17, 21, 25, and 29. [2] The aminotransferase according to [1], characterized in that the carbonyl compound is a compound represented by the following formula (1): (In formula (1), R represents a methyl group or an ethyl group, and n is an integer of 2 to 16.) [3] A polynucleotide comprising an amino acid sequence having 90% or more identity to any of the amino acid sequences of SEQ ID NOs: 13, 17, 21, 25, and 29, and comprising a nucleotide sequence encoding a polypeptide having transamination activity using a carbonyl compound as a substrate. [4] A vector comprising the polynucleotide according to [3]. [5] A transformant having the vector according to [4]. [6] A transformant for producing an aminoalkanoic acid ester from an alkanoic acid ester, characterized by comprising the aminotransferase according to [1] or [2]. [7] A transformant for producing an aminoalkanoic acid ester from an alkanoic acid ester, characterized by comprising the polynucleotide according to [3]. [8] A method for producing an amino compound represented by formula (2) below, characterized by contacting a carbonyl compound represented by formula (1) below with an amino group donor in the presence of the aminotransferase according to [1] or [2]. (In the formulas (1) and (2), R represents a methyl group or an ethyl group, and n is an integer of 2 to 16.) [9] A method for producing an amino compound represented by the following formula (2), characterized by contacting a transformant characterized by having the aminotransferase described in [1] or the polynucleotide described in [3] with a carbonyl compound represented by the following formula (1) and an amino group donor. (In the formulas (1) and (2), R represents a hydrogen atom, a methyl group, or an ethyl group. n is an integer of 2 to 16.)

[10] A method for producing an aminotransferase, comprising a step of culturing a transformant having the aminotransferase according to [1] or the polynucleotide according to [3].

[0009] The present invention provides a novel aminotransferase with significantly improved amino compound production efficiency or a transformant containing the same, as well as a method for biosynthesizing amino compounds using the novel aminotransferase of the present invention or a transformant containing the same.

[0010] FIG. 1 is a schematic diagram of the plasmid vector pKEF865. FIG. 2 is a schematic diagram of the plasmid vector pKEF838. FIG. 3 is a diagram comparing the production efficiencies of the transformants of Examples 1 to 5, where the amount of aminoalkanoate produced by the transformant of Comparative Example 1 is set to 1. FIG. 4 is a schematic diagram of the plasmid vector pKEF840. FIG. 5 is a diagram comparing the enzyme activities of the His-tagged proteins of unknown function in Examples 6 to 10, calculated relative to the rate of transamination of methyl 6-oxohexanoate by the His-tagged aminotransferase of Comparative Example 2 set to 1. FIG. 6 is a diagram comparing the enzyme activities of the His-tagged proteins of unknown function in Examples 6 to 10, calculated relative to the rate of transamination of methyl 8-oxooctanoate by the His-tagged aminotransferase of Comparative Example 2 set to 1. FIG. 7 shows the results of comparing the enzyme activities of the His-tag-linked aminotransferase of Comparative Example 2, which was used to calculate the relative transamination reaction rates for methyl 10-oxodecanoate, for the His-tag-linked proteins of unknown function in Examples 6 to 10, when the transamination reaction rate for methyl 10-oxodecanoate was set at 1.

[0011] The present invention is described in detail below. Unless otherwise specified, molecular biological techniques used herein can be performed by methods described in general experimental manuals known to those skilled in the art, or methods based thereon. Terms used herein are to be interpreted as having the meanings commonly used in the relevant technical field, unless otherwise specified. Furthermore, the present invention enables the supply of bio-based products and the realization of carbon recycling, thereby contributing to the achievement of Goal 12 of the SDGs.

[0012] <Aminotransferase> The catalytic activity of generating an amino compound by transferring the amino group of a donor (compound) to a substrate (compound) is called "transamination activity," and a protein having such activity is called an "aminotransferase." The aminotransferase of the present invention is a polypeptide comprising an amino acid sequence having 90% or more identity to any of the amino acid sequences of SEQ ID NOs: 13, 17, 21, 25, and 29, and having transamination activity using a carbonyl compound as a substrate. Because the aminotransferase of the present invention has a structure comprising an amino acid sequence having 90% or more identity to any of the amino acid sequences of SEQ ID NOs: 13, 17, 21, 25, and 29, it is characterized by superior transamination activity and higher amino compound production efficiency than conventional aminotransferases that use a carbonyl compound as a substrate.

[0013] The aminotransferase of the present invention acts on a specific carbonyl compound and an amino group donor such as L-alanine to catalyze the amino group transfer reaction to the carbonyl compound, thereby producing an amino compound with high efficiency.

[0014] The carbonyl compound that is used as a substrate by the aminotransferase of the present invention is preferably a carbonyl compound having an aldehyde group at the alkyl terminal, and more preferably a compound represented by the following formula (1):

[0015] In the above formula (1), R represents a methyl group or an ethyl group, and n is an integer of 2 to 16.

[0016] The R may be either a methyl group or an ethyl group, but is preferably a methyl group. The n may be an integer of 2 to 16, but is preferably an integer of 3 to 14, more preferably an integer of 4 to 12, even more preferably an integer of 4 to 10, and particularly preferably 4, 6, 8, or 10.

[0017] The carbonyl compound used as a substrate by the aminotransferase of the present invention is a methyl oxoalkanoate or an ethyl oxoalkanoate represented by the above formula (1), and specifically, methyl oxobutanoate, ethyl oxobutanoate, methyl oxopentanoate, ethyl oxopentanoate, methyl oxohexanoate, ethyl oxohexanoate, methyl oxoheptanoate, ethyl oxoheptanoate, methyl oxooctanoate, ethyl oxooctanoate, methyl oxononanoate, ethyl oxononanoate, methyl oxodecanoate, or methyl oxobutanoate represented by the above formula (1). methyl oxodecanoate, ethyl oxodecanoate, methyl oxoundecanoate, ethyl oxoundecanoate, methyl oxododecanoate, ethyl oxododecanoate, methyl oxotridecanoate, ethyl oxotridecanoate, methyl oxotetradecanoate, ethyl oxotetradecanoate, methyl oxopentadecanoate, ethyl oxopentadecanoate, methyl oxohexadecanoate, ethyl oxohexadecanoate, methyl oxoheptadecanoate, ethyl oxoheptadecanoate, methyl oxooctadecanoate, or ethyl oxooctadecanoate. Among these, from the viewpoint of the transamination activity of the aminotransferase of the present invention, methyl oxohexanoate, ethyl oxohexanoate, methyl oxooctanoate, ethyl oxooctanoate, methyl oxodecanoate, ethyl oxodecanoate, methyl oxoundecanoate, ethyl oxoundecanoate, methyl oxododecanoate, ethyl oxododecanoate, methyl oxotridecanoate, and ethyl oxotridecanoate are preferred, methyl oxohexanoate, ethyl oxohexanoate, methyl oxooctanoate, ethyl oxooctanoate, methyl oxodecanoate, ethyl oxodecanoate, methyl oxododecanoate, and ethyl oxododecanoate are more preferred, and methyl 6-oxohexanoate, methyl 8-oxooctanoate, methyl 10-oxodecanoate, and methyl 12-oxododecanoate are even more preferred.

[0018] The amino group donor used by the aminotransferase of the present invention may be an amino acid or an amine compound. In the present invention, an ω-amino compound may serve as the amino group donor. Examples of the amino acid include L-alanine (aminopropionic acid), aminopropanoic acid (β-alanine), aminobutyric acid (tyrosine), aminopentanoic acid (norleucine), aminohexanoic acid (enosinic acid), aminoheptanoic acid (glycine), aminooctanoic acid (isoleucine), aminoundecanoic acid (arginine), aminododecanoic acid (ammonium dodecylsalicylic acid), aminoheptanoic acid (norvaline), aminoisobutyric acid (phenylalanine), methionine, tryptophan, asparagine, cysteine, glutamine, serine, threonine, glutamic acid, histidine, lysine, proline, and derivatives thereof. Examples of the amine compound include primary amines such as 1,4-diaminobutane, 1,5-diaminopentane, 1,6-diaminohexane, 6-aminohexan-1-ol, taurine, tyramine, cyclohexylamine, isopropylamine, 2-aminoindan, and derivatives thereof.

[0019] The amino compounds obtainable using the aminotransferase of the present invention as a catalyst are aminoalkanoate esters, specifically methyl aminobutanoate, ethyl aminobutanoate, methyl aminopentanoate, ethyl aminopentanoate, methyl aminohexanoate, ethyl aminohexanoate, methyl aminoheptanoate, ethyl aminoheptanoate, methyl aminooctanoate, ethyl aminooctanoate, methyl aminononanoate, ethyl aminononanoate, methyl aminodecanoate, ethyl aminodecanoate, methyl aminoundecanoate, ethyl aminoundecanoate, methyl aminododecanoate, ethyl aminododecanoate, methyl aminotridecanoate, ethyl aminotridecanoate, methyl aminotetradecanoate, ethyl aminotetradecanoate, methyl aminopentadecanoate, ethyl aminopentadecanoate, methyl aminohexadecanoate, ethyl aminohexadecanoate, methyl aminoheptadecanoate, ethyl aminoheptadecanoate, methyl aminooctadecanoate, or ethyl aminooctadecanoate. Among these, from the viewpoint of the transamination activity of the aminotransferase of the present invention, methyl aminohexanoate, ethyl aminohexanoate, methyl aminooctanoate, ethyl aminooctanoate, methyl aminodecanoate, ethyl aminodecanoate, methyl aminoundecanoate, ethyl aminoundecanoate, methyl aminododecanoate, ethyl aminododecanoate, methyl aminotridecanoate, and ethyl aminotridecanoate are preferred, methyl aminohexanoate, ethyl aminohexanoate, methyl aminooctanoate, ethyl aminooctanoate, methyl aminodecanoate, ethyl aminodecanoate, methyl aminododecanoate, and ethyl aminododecanoate are more preferred, and methyl 6-aminohexanoate, methyl 8-aminooctanoate, methyl 10-aminodecanoate, and methyl 12-aminododecanoate are even more preferred.

[0020] The transaminase of the present invention is a polypeptide comprising an amino acid sequence having 90% or more identity to any of the amino acid sequences of SEQ ID NOs: 13, 17, 21, 25, and 29. The transaminase of the present invention may be a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 13, a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 17, a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 21, a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 25, or a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 29, and from the viewpoint of improving transaminase activity, a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 25 is preferred.

[0021] Here, an amino acid sequence having 90% or greater identity to any of the amino acid sequences of SEQ ID NOs: 13, 17, 21, 25, and 29 may be identical to the amino acid sequence represented by each SEQ ID NO, or may be an amino acid sequence having 90% or greater identity to the amino acid sequence represented by each SEQ ID NO, with one or more amino acids deleted, substituted, inserted, and / or added from the amino acid sequence represented by each SEQ ID NO, as long as the amino acid sequence has transamination activity. As described above, when amino acids are deleted, substituted, inserted, and / or added, the position, number, and type of the deletion, substitution, insertion, or addition are not particularly limited, as long as the amino acid sequence has transamination activity. It is already known that proteins having an amino acid sequence modified by deletion, substitution, insertion, and / or addition of one or more amino acids from a certain amino acid sequence maintain their biological activity (e.g., Mark, D.F. et al., Proc. Natl. Acad. Sci. USA (1984) 81, 5662-5666).

[0022] The amino acid transaminase of the present invention is preferably a polypeptide comprising an amino acid sequence having 95% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence of any one of SEQ ID NOs: 13, 17, 21, 25, and 29, and having transamination activity using a carbonyl compound as a substrate; more preferably a polypeptide comprising an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted, and / or added in the amino acid sequence of any one of SEQ ID NOs: 13, 17, 21, 25, and 29, and having transamination activity using a carbonyl compound as a substrate; and even more preferably a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 13, 17, 21, 25, and 29, and having transamination activity using a carbonyl compound as a substrate.

[0023] The term "amino acid sequence in which one or more amino acids have been deleted, substituted, inserted, and / or added" also encompasses amino acid sequences in which, for example, 1 to 40 amino acids have been deleted, substituted, inserted, and / or added, preferably 1 to 20 amino acids, more preferably 1 to 15 amino acids, even more preferably 1 to 10 amino acids, even more preferably 1 to 5 amino acids, particularly preferably 1 to 3 amino acids, and even more particularly preferably 1 to 2 amino acids. These amino acid mutations can be performed by any method known to those skilled in the art, such as chemical synthesis, genetic engineering, or mutagenesis.

[0024] In the present invention, a polypeptide refers to a compound formed by two or more amino acids linked by peptide bonds. The number of constituent amino acids is not particularly limited, and includes, for example, a dipeptide consisting of two amino acids, a tripeptide consisting of three amino acids, a tetrapeptide consisting of four amino acids, an oligopeptide consisting of about 10 amino acids, and a peptide or protein consisting of 20 or more amino acids.

[0025] Here, the "identity" between two amino acid sequences refers to the percentage (%) of identical amino acid residues appearing at corresponding positions when the two amino acid sequences are aligned optimally (maximizing amino acid identity). The "identity" between two amino acid sequences can be determined using various programs commonly available to those skilled in the art, such as the Protein BLAST program in BLAST (Basic Local Alignment Search Tool, Altschul et al., J. Mol. Biol., (1990), 215(3):403-10)).

[0026] The aminotransferases of the present invention can also be identified by specific nucleic acid sequences that encode these aminotransferases. There are many nucleic acid sequences that correspond to the amino acid sequences of SEQ ID NOs: 13, 17, 21, 25, and 29, depending on codon usage, but preferred examples include the base sequences of SEQ ID NOs: 10, 14, 18, 22, and 26, respectively.

[0027] The aminotransferase of the present invention can be expressed in host cells such as bacteria by synthesizing DNA having a specific nucleic acid sequence encoding the aminotransferase (e.g., the nucleic acid sequences shown in SEQ ID NOs: 10, 14, 18, 22, and 26) using a known DNA synthesis method, and then expressing the enzyme in a host cell such as a bacterial cell according to the method described below.

[0028] The aminotransferase of the present invention may be used in the form of an isolated polypeptide (isolated enzyme), or may be expressed in a host cell and used in a transamination reaction using the host cell (transformant) as a catalyst. When the host cell (transformant) is used as a catalyst, the host cell (transformant) expressing the aminotransferase of the present invention can be cultured in a culture medium containing an amino group acceptor and an amino group donor, or dispersed in a reaction solution containing an amino group acceptor and an amino group donor and reacted therewith, thereby allowing the transamination reaction from a carbonyl compound to an amino compound to proceed within the host cell (transformant).

[0029] The host cell may be a prokaryote or a eukaryote. Examples of the host cell include microorganisms such as fungi (e.g., bacteria, yeast, fungi, etc.), mammalian cells (e.g., human-derived cells), and plant cells. Among these, microorganisms such as fungi are preferred, and bacteria and yeast are more preferred.

[0030] Specific examples of the host cells include Corynebacterium, Brevibacterium, Bacillus, Lactobacillus, Lactococcus, Candida, Pichia, Kluveromyces, Saccharomyces, Escherichia coli, Zymomonas, Yarrowia, Methylobacterium, Ralstonia, Pseudomonas, Burkholderia, and Clostridium. Among these, Escherichia coli, Corynebacterium glutamicum, and Pseudomonas putida are preferred, with Escherichia coli being more preferred.

[0031] The method for producing an amino compound by promoting a transamination reaction using the host cell (transformant) as a catalyst can be described in the section "Method for producing an amino compound" below.

[0032] <Polynucleotide> The present invention also includes a polynucleotide encoding the above-mentioned amino acid transferase of the present invention. The polynucleotide of the present invention comprises an amino acid sequence having 90% or more identity to any of the amino acid sequences of SEQ ID NOs: 13, 17, 21, 25, and 29, and a nucleotide sequence encoding a polypeptide having transamination activity using a carbonyl compound as a substrate. Furthermore, the polynucleotide of the present invention preferably comprises an amino acid sequence having 95% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence of any one of SEQ ID NOs: 13, 17, 21, 25, and 29, and comprises a nucleotide sequence encoding a polypeptide having transamination activity using a carbonyl compound as a substrate; more preferably comprises an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted, and / or added in the amino acid sequence of any one of SEQ ID NOs: 13, 17, 21, 25, and 29, and encodes a polypeptide having transamination activity using a carbonyl compound as a substrate; and even more preferably comprises a nucleotide sequence encoding a polypeptide having transamination activity using a carbonyl compound as a substrate, and comprises the amino acid sequence of any one of SEQ ID NOs: 13, 17, 21, 25, and 29. There are many nucleic acid sequences encoding polypeptides consisting of the amino acid sequences of SEQ ID NOs: 13, 17, 21, 25, and 29, depending on codon usage, and preferred examples include the nucleotide sequences of SEQ ID NOs: 10, 14, 18, 22, and 26, respectively. The polynucleotide of the present invention is preferably a polynucleotide that contains the above-mentioned nucleic acid sequence or hybridizes under stringent conditions to a polynucleotide consisting of a DNA sequence complementary to a polynucleotide consisting of the above-mentioned base sequence, and encodes a polypeptide that has transamination activity using a carbonyl compound as a substrate.

[0033] As used herein, the term "polynucleotide" refers to a biological polymer formed by the covalent linkage of nucleotide monomers in a chain, and is used interchangeably with "nucleic acid," "gene," or "nucleic acid molecule." As used herein, the term "base sequence" is used interchangeably with "nucleic acid sequence" or "nucleotide sequence," and is represented as a sequence of deoxyribonucleotides (abbreviated as A, G, C, and T). For example, a "polynucleotide comprising the nucleotide sequence of SEQ ID NO: 10" refers to a polynucleotide comprising the sequence represented by each of the deoxynucleotides A, G, C, and / or T of SEQ ID NO: 10.

[0034] As used herein, "stringent conditions" may refer to low stringency conditions, moderate stringency conditions, or high stringency conditions. "Low stringency conditions" refer to, for example, 5x SSC, 5x Denhardt's solution, 0.5% SDS, 50% formamide, and 32°C. "Moderate stringency conditions" refer to, for example, 5x SSC, 5x Denhardt's solution, 0.5% SDS, 50% formamide, and 42°C, or 5x SSC, 1% SDS, 50 mM Tris-HCl (pH 7.5), 50% formamide, and 42°C. "Highly stringent conditions" include, but are not limited to, the following: (1) 5xSSC, 5xDenhardt's solution, 0.5% SDS, 50% formamide, 50°C; (2) 0.2xSSC, 0.1% SDS, 60°C; (3) 0.2xSSC, 0.1% SDS, 62°C; (4) 0.2xSSC, 0.1% SDS, 65°C; or (5) 0.1xSSC, 0.1% SDS, 65°C. Under these conditions, the higher the temperature, the more efficiently antisense oligomers with higher sequence identity can be obtained. However, several factors, such as temperature, probe concentration, probe length, ionic strength, time, and salt concentration, can affect the stringency of hybridization. Those skilled in the art can achieve similar stringency by appropriately selecting these factors. Here, "base sequence identity" refers to the identity over the entire range of the base sequences being compared for a pair of two nucleic acids, and is expressed as the percentage (%) of matching bases in an optimal alignment of the base sequences created using a mathematical algorithm known in the technical field of the present invention.

[0035] The polynucleotide of the present invention may include a promoter sequence (transcriptional regulatory region) operably linked to the above-described base sequence. Here, "operably linked" refers to a juxtaposition in which the described components are in a relationship that allows them to function in their intended manner. In one embodiment, this term refers to a functional link between a nucleic acid expression control sequence (e.g., a promoter and / or enhancer) and a polynucleotide sequence of interest, which may be a direct link or an indirect link (when another polynucleotide sequence is interposed between them). The promoter sequence is capable of directing transcription of the linked polynucleotide of interest.

[0036] The promoter sequence contained in the polynucleotide of the present invention can be one that functions in host cells. When the host cell is a bacterium, particularly Escherichia coli, the promoter sequence can include, but is not limited to, the T7 promoter, trp promoter, lac promoter, thr promoter, tac promoter, trc promoter, tet promoter, araBAD promoter, rpoH promoter, msrA promoter, and the Pm1 promoter, PR promoter, and PL promoter derived from Bifidobacterium. When the host cell is a budding yeast, the promoter can include, but is not limited to, the TEF1 promoter, ADH1 promoter, TPI1 promoter, TDH3 promoter, PGK1 promoter, and PYK1 promoter. When the host cell is a mammalian cell, the promoter can include, but is not limited to, the CMV promoter, EF1α promoter, PGK promoter, CAGGS promoter, hSyn promoter, and Tet promoter. When the host cell is a plant cell, examples of promoters include the CaMV35S promoter (derived from cauliflower mosaic virus), the ZmUbi promoter (derived from maize), the AtUbq1 promoter (derived from Arabidopsis thaliana), and the GmHsp17.3 promoter (derived from soybean). In either case, the promoter is preferably one that can induce expression at any time during culture using an inducer, although this is not particularly limited. Examples of such induction methods include antibiotics (tetracycline), sugar analogs (isopropyl-β-thiogalactopyranoside (IPTG)), heat shock, and stress. IPTG is particularly desirable when Escherichia coli is used as the host.

[0037] The polynucleotides of the present invention can be chemically synthesized using known DNA synthesis methods.

[0038] <Vector> The vector of the present invention is characterized by comprising the polynucleotide of the present invention described above. The vector of the present invention is used to introduce the polynucleotide of the present invention into a host cell. The explanation in the section on polynucleotides above can be applied as is to the polynucleotide of the present invention comprised in the vector of the present invention. Those skilled in the art can appropriately select and use a vector that can be expressed in a host cell.

[0039] The vector of the present invention preferably comprises, in addition to the polynucleotide of the present invention, i.e., a polynucleotide encoding a specific aminotransferase (enzyme III), a polynucleotide comprising a nucleotide sequence encoding an alkane monooxygenase (enzyme I) for hydroxylating the alkyl group terminal of an alkanoate ester, and a polynucleotide comprising a nucleotide sequence encoding an alkane monooxidase, alcohol dehydrogenase, or alcohol oxidase (enzyme II) for converting a hydroxyalkanoate ester into an aldehyde. The polynucleotides for enzymes I to III may each be introduced into a separate vector. The vector of the present invention includes both vectors comprising the polynucleotides for enzymes I to III, and combinations of multiple vectors each comprising a polynucleotide for each enzyme. Transformants obtained by transformation with these vectors can efficiently produce aminoalkanoate esters intracellularly by adding readily available raw materials, such as alkanoate esters, and an amino group donor, such as L-alanine, to the culture medium or reaction solution.

[0040] The enzyme I is an enzyme (alkane monooxygenase) that catalyzes the reaction of hydroxylating the alkyl terminal of an alkanoic acid ester to form the corresponding hydroxyalkanoic acid ester. The enzyme I is preferably an alkane monooxygenase, and specific examples thereof include alkane monooxygenase encoded by the alkBGT gene derived from Pseudomonas putida GPO1, cytochrome P450 monooxygenase derived from Candida (e.g., Candida tropicalis), and cytochrome P450 monooxygenase derived from plants (e.g., chickpea (Cicer arietinum L.)).

[0041] The enzyme II is an enzyme (alkane monooxidase, alcohol dehydrogenase, or alcohol oxidase) that catalyzes the reaction for converting the hydroxyalkanoic acid ester into the corresponding oxoalkanoic acid ester. The enzyme II is preferably an alcohol dehydrogenase, specifically, for example, an alcohol dehydrogenase (EC 1.1.99-2) encoded by the alkJ gene, and particularly preferably an alcohol dehydrogenase derived from Pseudomonas putida GPol encoded by the alkJ gene.

[0042] The enzyme III mentioned above refers to the aminotransferase of the present invention, which is an enzyme that catalyzes the reaction of converting an oxoalkanoic acid ester into the corresponding aminoalkanoic acid ester.

[0043] In addition to the above polynucleotide, the vector of the present invention may contain, as enzyme IV, a polynucleotide encoding an esterase secreted by the cell. Specific examples of the enzyme IV include lipase LipA derived from Pseudomonas fluorescens HU380. By using a vector further comprising a polynucleotide encoding enzyme IV, it becomes possible to convert the aminoalkanoic acid esters produced in the transformant of the present invention, as described below, into aminoalkanoic acids.

[0044] The type of vector of the present invention is not particularly limited, and examples thereof include a plasmid vector, a viral vector, an artificial chromosome vector, etc. Methods for introducing the vector of the present invention into target cells include the competent cell method, the electroporation method, the lipofection method, the microinjection method, etc.

[0045] To enhance expression of a target polynucleotide (gene) in the transformant of the present invention, as described below, an episomal plasmid can be used. Many plasmid vectors are available for this purpose, including pUC, pET, pZ1 (Menkel et al., Applied and Environmental Microbiology, 64:549-554 (1989)), pEKEx1 (Eikmanns et al., Gene, 107:69-74 (1991)), pHS2-1 (Sonnen et al., Gene, 107:69-74 (1991)), the cryptic plasmid pHM1519, pBL1, or pGAI, as well as pCG4 (US Pat. No. 4,489,160), pNG2 (Serwold-Davis et al., FEMS Microbiology Letters, 66:119-124 (1990)), and pAG1 (US Pat. No. 5,158,891).

[0046] The vector can contain regulatory sequences such as a promoter, enhancer, ribosome binding sequence, terminator, polyadenylation site, etc. to enable expression of the gene of interest. Furthermore, if necessary, the vector can contain a selection marker sequence such as a drug resistance gene (e.g., a kanamycin resistance gene, an ampicillin resistance gene, a puromycin resistance gene, etc.), a thymidine kinase gene, a diphtheria toxin gene, etc., or a reporter gene sequence such as mCherry (red fluorescent protein), green fluorescent protein (GFP), β-glucuronidase (GUS), or FLAG.

[0047] <Transformant> The present invention also includes a transformant obtained by transforming a host cell with the vector of the present invention described above. That is, the present invention also includes a transformant having the vector of the present invention described above. The transformant of the present invention comprises the polynucleotide of the present invention and the aminotransferase of the present invention. Since the transformant of the present invention expresses the aminotransferase of the present invention intracellularly, the transamination reaction from a carbonyl compound to an amino compound can be efficiently carried out intracellularly by culturing the transformant in a culture medium containing an amino group acceptor (carbonyl compound) and an amino group donor or by dispersing the transformant in a reaction solution.

[0048] When the vector of the present invention used to transform a host cell is a vector containing the polynucleotides of the enzymes I to III described above, the transformant obtained by transformation can efficiently produce an aminoalkanoate ester from an alkanoate ester within the cell by adding an alkanoate ester and an amino group donor such as L-alanine to the culture medium or reaction solution. Similarly, when the enzyme III described above is introduced using the vector of the present invention and the enzymes I and II described above are introduced using separate vectors during the transformation of a host cell, the resulting transformant can efficiently produce an aminoalkanoate ester from an alkanoate ester within the cell by adding an alkanoate ester and an amino group donor such as L-alanine to the culture medium or reaction solution. When the vector of the present invention used to transform a host cell contains only the polynucleotide of the above-mentioned enzyme III (the aminotransferase of the present invention), the transformant obtained by transformation can efficiently produce an aminoalkanoate ester from an oxoalkanoate ester within the cell by adding an oxoalkanoate ester (formylalkanoate ester) as an amino group acceptor and L-alanine or the like as an amino group donor to the culture medium or reaction solution.

[0049] Specific examples of the alkanoate ester include methyl butanoate, ethyl butanoate, methyl pentanoate, ethyl pentanoate, methyl hexanoate, ethyl hexanoate, methyl heptanoate, ethyl heptanoate, methyl octanoate, ethyl octanoate, methyl nonanoate, ethyl nonanoate, methyl decanoate, ethyl decanoate, methyl undecanoate, ethyl undecanoate, methyl dodecanoate, ethyl dodecanoate, methyl tridecanoate, ethyl tridecanoate, methyl tetradecanoate, ethyl tetradecanoate, methyl pentadecanoate, ethyl pentadecanoate, methyl hexadecanoate, ethyl hexadecanoate, methyl heptadecanoate, ethyl heptadecanoate, methyl octadecanoate, and ethyl octadecanoate. Among these, from the viewpoint of the transamination activity of the aminotransferase of the present invention, methyl hexanoate, ethyl hexanoate, methyl octanoate, ethyl octanoate, methyl decanoate, ethyl decanoate, methyl undecanoate, ethyl undecanoate, methyl dodecanoate, ethyl dodecanoate, methyl tridecanoate, and ethyl tridecanoate are preferred, methyl hexanoate, ethyl hexanoate, methyl octanoate, ethyl octanoate, methyl decanoate, ethyl decanoate, methyl dodecanoate, and ethyl dodecanoate are more preferred, and methyl 6-hexanoate, methyl 8-octanoate, methyl 10-decanoate, and methyl 12-dodecanoate are even more preferred.

[0050] Examples of the amino group donor include amino acids and amine compounds. In the present invention, an ω-amino compound can serve as the amino group donor. Examples of the amino acid include L-alanine (aminopropionic acid), aminopropanoic acid (β-alanine), aminobutyric acid (tyrosine), aminopentanoic acid (norleucine), aminohexanoic acid (enosinic acid), aminoheptanoic acid (glycine), aminooctanoic acid (isoleucine), aminoundecanoic acid (arginine), aminododecanoic acid (ammonium dodecylsalicylic acid), aminoheptanoic acid (norvaline), aminoisobutyric acid (phenylalanine), methionine, tryptophan, asparagine, cysteine, glutamine, serine, threonine, glutamic acid, histidine, lysine, proline, and derivatives thereof. Examples of the amine compound include primary amines such as 1,4-diaminobutane, 1,5-diaminopentane, 1,6-diaminohexane, 6-aminohexan-1-ol, taurine, tyramine, cyclohexylamine, isopropylamine, 2-aminoindan, and derivatives thereof.

[0051] Specific examples of the aminoalkanoate ester include methyl aminobutanoate, ethyl aminobutanoate, methyl aminopentanoate, ethyl aminopentanoate, methyl aminohexanoate, ethyl aminohexanoate, methyl aminoheptanoate, ethyl aminoheptanoate, methyl aminooctanoate, ethyl aminooctanoate, methyl aminononanoate, ethyl aminononanoate, methyl aminodecanoate, ethyl aminodecanoate, methyl aminoundecanoate, ethyl aminoundecanoate, methyl aminododecanoate, ethyl aminododecanoate, methyl aminotridecanoate, ethyl aminotridecanoate, methyl aminotetradecanoate, ethyl aminotetradecanoate, methyl aminopentadecanoate, ethyl aminopentadecanoate, methyl aminohexadecanoate, ethyl aminohexadecanoate, methyl aminoheptadecanoate, ethyl aminoheptadecanoate, methyl aminooctadecanoate, and ethyl aminooctadecanoate. Among these, from the viewpoint of the transamination activity of the aminotransferase of the present invention, methyl aminohexanoate, ethyl aminohexanoate, methyl aminooctanoate, ethyl aminooctanoate, methyl aminodecanoate, ethyl aminodecanoate, methyl aminoundecanoate, ethyl aminoundecanoate, methyl aminododecanoate, ethyl aminododecanoate, methyl aminotridecanoate, and ethyl aminotridecanoate are preferred, methyl aminohexanoate, ethyl aminohexanoate, methyl aminooctanoate, ethyl aminooctanoate, methyl aminodecanoate, ethyl aminodecanoate, methyl aminododecanoate, and ethyl aminododecanoate are more preferred, and methyl 6-aminohexanoate, methyl 8-aminooctanoate, methyl 10-aminodecanoate, and methyl 12-aminododecanoate are even more preferred.

[0052] When the vector of the present invention used to transform a host cell further contains the above-mentioned polynucleotide of enzyme IV, the transformant obtained by transformation can convert the above-mentioned aminoalkanoic acid ester into an aminoalkanoic acid extracellularly.

[0053] The host cell may be a prokaryote or a eukaryote. Examples of the host cell include microorganisms such as fungi (e.g., bacteria, yeast, fungi, etc.), mammalian cells (e.g., human-derived cells), and plant cells. Among these, microorganisms such as fungi are preferred, and bacteria and yeast are more preferred.

[0054] Specific examples of the host cells include Corynebacterium, Brevibacterium, Bacillus, Lactobacillus, Lactococcus, Candida, Pichia, Kluveromyces, Saccharomyces, Escherichia coli, Zymomonas, Yarrowia, Methylobacterium, Ralstonia, Pseudomonas, Burkholderia, and Clostridium. Among these, Escherichia coli, Corynebacterium glutamicum, and Pseudomonas putida are preferred, with Escherichia coli being more preferred.

[0055] In the explanation of the transformant of the present invention, the explanations in the above respective sections regarding the vector, polynucleotide, and aminotransferase of the present invention are applicable as they are.

[0056] <Method for producing amino compound> The method for producing an amino compound of the present invention is characterized by contacting a carbonyl compound represented by the following formula (1) with an amino group donor in the presence of the aminotransferase of the present invention.

[0057] In the formulas (1) and (2), R represents a methyl group or an ethyl group, and n represents an integer of 2 to 16.

[0058] In the method for producing an amino compound of the present invention, the novel aminotransferase of the present invention is allowed to act on the alkanoic acid ester (carbonyl compound) represented by the above formula (1) as the amino group acceptor and L-alanine or the like as the amino donor, thereby producing an aminoalkanoic acid ester (amino compound) represented by the above formula (2) through a transamination reaction.

[0059] The conditions for the transamination reaction are as follows: pH: 6 to 9, preferably 7.0 to 8.5, and more preferably 8.0 Temperature: 20 to 40°C, preferably 25 to 37°C, and more preferably 30°C.

[0060] In the method for producing an amino compound of the present invention, the carbonyl compound used as a substrate by the aminotransferase is a compound represented by the following formula (1) which has an aldehyde group at the alkyl group terminal.

[0061] In the above formula (1), R represents a methyl group or an ethyl group, and n is an integer of 2 to 16.

[0062] The R may be either a methyl group or an ethyl group, but is preferably a methyl group. The n may be an integer of 2 to 16, but is preferably an integer of 5 to 14, more preferably an integer of 8 to 12, even more preferably 10 or 11, and particularly preferably 10.

[0063] The carbonyl compound is a methyl oxoalkanoate or an ethyl oxoalkanoate represented by the above formula (1), and specifically, methyl oxobutanoate, ethyl oxobutanoate, methyl oxopentanoate, ethyl oxopentanoate, methyl oxohexanoate, ethyl oxohexanoate, methyl oxoheptanoate, ethyl oxoheptanoate, methyl oxooctanoate, ethyl oxooctanoate, methyl oxononanoate, ethyl oxononanoate, methyl oxodecanoate, oxodecane, methyl oxobutanoate, ethyl oxopentanoate, methyl oxohexanoate, ethyl oxohexanoate, methyl oxoheptanoate, ethyl oxoheptanoate, methyl oxooctanoate, methyl oxononanoate, ethyl oxononanoate, methyl oxodecanoate, methyl oxodecanoate, methyl oxobutanoate, ethyl oxobutanoate, methyl oxopentanoate, ethyl oxopentanoate, methyl oxohexanoate, ethyl oxohexanoate, methyl oxohexanoate, methyl oxohexanoate, methyl oxononanoate, ethyl oxononanoate, methyl oxodecanoate, methyl oxodecanoate, methyl oxodecanoate, methyl oxobutanoate, ethyl oxobutanoate, methyl oxopentanoate, ethyl oxopentanoate, methyl oxohexanoate, ethyl oxohexanoate, methyl oxohexanoate, methyl oxohexanoate, methyl oxononanoate, ethyl oxodecanoate, methyl oxodecanoate, methyl oxodecanoate, methyl oxodecanoate, methyl oxodecanoate, methyl oxodecanoate, methyl methyl oxoundecanoate, ethyl oxoundecanoate, methyl oxododecanoate, ethyl oxododecanoate, methyl oxotridecanoate, ethyl oxotridecanoate, methyl oxotetradecanoate, ethyl oxotetradecanoate, methyl oxopentadecanoate, ethyl oxopentadecanoate, methyl oxohexadecanoate, ethyl oxohexadecanoate, methyl oxoheptadecanoate, ethyl oxoheptadecanoate, methyl oxooctadecanoate, or ethyl oxooctadecanoate. Among these, from the viewpoint of the transamination activity of the aminotransferase of the present invention, methyl oxohexanoate, ethyl oxohexanoate, methyl oxooctanoate, ethyl oxooctanoate, methyl oxodecanoate, ethyl oxodecanoate, methyl oxoundecanoate, ethyl oxoundecanoate, methyl oxododecanoate, ethyl oxododecanoate, methyl oxotridecanoate, and ethyl oxotridecanoate are preferred, methyl oxohexanoate, ethyl oxohexanoate, methyl oxooctanoate, ethyl oxooctanoate, methyl oxodecanoate, ethyl oxodecanoate, methyl oxododecanoate, and ethyl oxododecanoate are more preferred, and methyl 6-oxohexanoate, methyl 8-oxooctanoate, methyl 10-oxodecanoate, and methyl 12-oxododecanoate are even more preferred.

[0064] In the method for producing an amino compound of the present invention, the amino group donor may be an amino acid or an amine compound. Examples of the amino acid include L-alanine (aminopropionic acid), aminopropanoic acid (β-alanine), aminobutyric acid (tyrosine), aminopentanoic acid (norleucine), aminohexanoic acid (enosinic acid), aminoheptanoic acid (glycine), aminooctanoic acid (isoleucine), aminoundecanoic acid (arginine), aminododecanoic acid (ammonium dodecylsalicylic acid), aminoheptanoic acid (norvaline), aminoisobutyric acid (phenylalanine), methionine, tryptophan, asparagine, cysteine, glutamine, serine, threonine, glutamic acid, histidine, lysine, proline, and derivatives thereof.

[0065] Examples of the amine compound include primary amines such as 1,4-diaminobutane, 1,5-diaminopentane, 1,6-diaminohexane, 6-aminohexan-1-ol, taurine, tyramine, cyclohexylamine, isopropylamine, 2-aminoindan, and derivatives thereof.

[0066] The amino compound obtainable by the method of the present invention is an aminoalkanoate ester, specifically methyl aminobutanoate, ethyl aminobutanoate, methyl aminopentanoate, ethyl aminopentanoate, methyl aminohexanoate, ethyl aminohexanoate, methyl aminoheptanoate, ethyl aminoheptanoate, methyl aminooctanoate, ethyl aminooctanoate, methyl aminononanoate, ethyl aminononanoate, methyl aminodecanoate, ethyl aminodecanoate, methyl aminoundecanoate, ethyl aminoundecanoate, methyl aminododecanoate, ethyl aminododecanoate, methyl aminotridecanoate, ethyl aminotridecanoate, methyl aminotetradecanoate, ethyl aminotetradecanoate, methyl aminopentadecanoate, ethyl aminopentadecanoate, methyl aminohexadecanoate, ethyl aminohexadecanoate, methyl aminoheptadecanoate, ethyl aminoheptadecanoate, methyl aminooctadecanoate, or ethyl aminooctadecanoate. Among these, from the viewpoint of the transamination activity of the aminotransferase of the present invention, methyl aminohexanoate, ethyl aminohexanoate, methyl aminooctanoate, ethyl aminooctanoate, methyl aminodecanoate, ethyl aminodecanoate, methyl aminoundecanoate, ethyl aminoundecanoate, methyl aminododecanoate, ethyl aminododecanoate, methyl aminotridecanoate, and ethyl aminotridecanoate are preferred, methyl aminohexanoate, ethyl aminohexanoate, methyl aminooctanoate, ethyl aminooctanoate, methyl aminodecanoate, ethyl aminodecanoate, methyl aminododecanoate, and ethyl aminododecanoate are more preferred, and methyl 6-aminohexanoate, methyl 8-aminooctanoate, methyl 10-aminodecanoate, and methyl 12-aminododecanoate are even more preferred.

[0067] Another embodiment of the method for producing an amino compound of the present invention is a method for producing an amino compound represented by the following formula (2), which comprises contacting a transformant characterized by having the above-mentioned aminotransferase of the present invention or the above-mentioned polynucleotide of the present invention, with a carbonyl compound represented by the following formula (1) and an amino group donor. Another embodiment of the method for producing an amino compound of the present invention may be a method for producing an amino compound represented by the following formula (2), which comprises contacting a transformant characterized by having an aminotransferase that uses a carbonyl compound represented by the following formula (1) as a substrate, the aminotransferase comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 13, or a polynucleotide comprising a nucleotide sequence encoding the aminotransferase, with a carbonyl compound represented by the following formula (1) and an amino group donor. Another embodiment of the method for producing an amino compound of the present invention may be a method for producing an amino compound represented by formula (2) below, comprising contacting a transformant having an aminotransferase which uses a carbonyl compound represented by formula (1) below as a substrate, the aminotransferase comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 17, or a polynucleotide comprising a nucleotide sequence encoding the aminotransferase, with the carbonyl compound represented by formula (1) below and an amino group donor. Another embodiment of the method for producing an amino compound of the present invention may be a method for producing an amino compound represented by formula (2) below, comprising contacting a transformant having an aminotransferase which uses a carbonyl compound represented by formula (1) below as a substrate, the aminotransferase comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 21, or a polynucleotide comprising a nucleotide sequence encoding the aminotransferase, with the carbonyl compound represented by formula (1) below and an amino group donor.Another embodiment of the method for producing an amino compound of the present invention may be a method for producing an amino compound represented by formula (2) below, comprising contacting a transformant having an aminotransferase which uses a carbonyl compound represented by formula (1) below as a substrate, the aminotransferase comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 25, or a polynucleotide comprising a nucleotide sequence encoding the aminotransferase, with the carbonyl compound represented by formula (1) below and an amino group donor. Another embodiment of the method for producing an amino compound of the present invention may be a method for producing an amino compound represented by formula (2) below, comprising contacting a transformant having an aminotransferase which uses a carbonyl compound represented by formula (1) below as a substrate, the aminotransferase comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 29, or a polynucleotide comprising a nucleotide sequence encoding the aminotransferase, with the carbonyl compound represented by formula (1) below and an amino group donor. Among these, the method for producing an amino compound of the present invention is preferably a method for producing an amino compound represented by formula (2) below, which method comprises contacting a transformant having an aminotransferase which uses a carbonyl compound represented by formula (1) below as a substrate, the aminotransferase comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 13 or 25, or a polynucleotide comprising a nucleotide sequence encoding the aminotransferase, with a carbonyl compound represented by formula (1) below and an amino group donor.

[0068]

[0069] In the formulas (1) and (2), R represents a hydrogen atom, a methyl group, or an ethyl group, and n represents an integer of 2 to 16.

[0070] According to the method for producing an amino compound of the present invention, the target amino compound can be efficiently produced by culturing the transformant or dispersing it in a reaction solution in the presence of a carbonyl compound as an amino group acceptor and L-alanine or the like as an amino group donor. Examples of amino group donors can be found above.

[0071] In the method for producing an amino compound of the present invention, the transformant is contacted with a medium or reaction solution containing an alkanoate ester, or with a medium or reaction solution adjacent to an organic phase containing an alkanoate ester, to carry out the hydroxylation, aldehyde formation, and amination of the alkanoate ester. That is, the transformant is cultured in a medium containing an alkanoate ester or dispersed in a reaction solution. Culture conditions can be appropriately adjusted depending on the type of host cell (e.g., type of strain) of the transformant. The medium to be used is selected to be suitable for each host cell. The reaction solution can be appropriately adjusted depending on the type of host cell (e.g., type of strain) of the transformant.

[0072] When the polynucleotides of the enzymes I to III described above have been introduced into the transformant, aminoalkanoate esters can be efficiently produced from alkanoate esters within the cells by adding an alkanoate ester and an amino group donor such as L-alanine to the culture medium or reaction solution. When the transformant contains only the polynucleotide of the enzyme III (the aminotransferase of the present invention), aminoalkanoate esters can be efficiently produced from oxoalkanoate esters within the cells by adding an oxoalkanoate ester (formylalkanoate ester) as an amino group acceptor and an amino group donor such as L-alanine to the culture medium or reaction solution.

[0073] The medium may contain, as a carbon source, hydrocarbons such as glucose, saccharose, lactose, fructose, maltose, molasses, starch, and cellulose; oils and fats such as soybean oil, sunflower oil, peanut oil, and coconut oil; fatty acids such as palmitic acid, stearic acid, and linoleic acid; alcohols such as glycerin and methanol; hydrocarbons such as methane; amino acids such as L-glutamate and L-valine; or organic acids such as acetic acid. These may be added individually or as a mixture. It is preferable to use a medium containing carbohydrates, monosaccharides, oligosaccharides, polysaccharides, glycerin, etc.

[0074] The medium may contain nitrogen sources such as nitrogen-containing organic compounds, e.g., peptone, yeast extract, meat extract, malt extract, corn swelling water, soybean flour, and urea, or inorganic compounds, e.g., ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate. The nitrogen sources may be added individually or as a mixture.

[0075] The medium may contain phosphoric acid, potassium phosphate, or dipotassium hydrogen phosphate or the corresponding sodium-containing salts as a phosphorus source. Preferably, the medium also contains metal salts necessary for growth, such as magnesium sulfate or ferrous sulfate. Finally, important growth materials, such as amino acids and vitamins, can be used in addition to the above materials.

[0076] To control the pH of the medium during cultivation, basic compounds such as sodium hydroxide, potassium hydroxide, ammonia, or aqueous ammonia, or acidic compounds such as phosphoric acid or sulfuric acid can be used in an appropriate manner. To maintain the stability of the plasmid in the transformant, antibiotics or the like may be added to the medium. Furthermore, to maintain aerobic conditions, oxygen or an oxygen-containing gas mixture, such as air, may be used during cultivation. The cultivation temperature is usually 20°C to 45°C, preferably 25°C to 40°C.

[0077] When recombinant cells derived from Escherichia coli are used as the transformant, it is preferable to use a mineral salt medium such as LB medium supplemented with carbenicillin, ampicillin, chloramphenicol, kanamycin, etc. as the nutrient medium.

[0078] <Aminotransferase composition> The aminotransferase composition of the present invention is characterized by containing the above-mentioned aminotransferase of the present invention. By containing the above-mentioned aminotransferase of the present invention, the aminotransferase composition of the present invention can improve the efficiency of amino compound production compared to conventionally known aminotransferases. The aminotransferase of the present invention is an aminotransferase that uses a carbonyl compound as a substrate, characterized by containing an amino acid sequence that has 90% or more identity to any of the amino acid sequences of SEQ ID NOs: 13, 17, 21, 25, and 29.

[0079] The aminotransferase composition of the present invention may further contain, in addition to the aminotransferase of the present invention, an alkane monooxygenase (the above-mentioned enzyme I), and an alkane monooxidase, alcohol dehydrogenase, or alcohol oxidase (the above-mentioned enzyme II). The above-mentioned enzyme I is an enzyme that catalyzes a reaction that converts an alkanoate ester into the corresponding hydroxyalkanoate ester. As the above-mentioned enzyme I, an alkane monooxygenase is preferred, and specific examples include an alkane monooxygenase encoded by the alkBGT gene derived from Pseudomonas putida GPO1, a cytochrome P450 monooxygenase derived from Candida (e.g., Candida tropicalis), and a cytochrome P450 monooxygenase derived from a plant (e.g., chickpea (Cicer arietinum L.)). The above-mentioned enzyme II is an enzyme that catalyzes a reaction for converting a hydroxyalkanoate ester into the corresponding oxoalkanoate ester. The enzyme II is preferably an alcohol dehydrogenase, specifically, for example, an alcohol dehydrogenase (EC. 1.1.99-2) encoded by the alkJ gene, and particularly, an alcohol dehydrogenase derived from Pseudomonas putida GPol encoded by the alkJ gene is preferred.

[0080] The aminotransferase composition of the present invention may further contain, in addition to the three enzymes described above, a compound that serves as an amino group acceptor and a compound that serves as an amino group donor. The amino group acceptor and amino group donor compounds may be added during the transamination reaction, and are stored in a container separate from the enzymes before the reaction. By containing the three enzymes described above, as well as an amino group acceptor and an amino group donor, the aminotransferase composition of the present invention can efficiently produce aminoalkanoate esters from alkanoate esters that serve as amino group acceptors.

[0081] The aminotransferase, enzyme I, enzyme II, amino group acceptor, and amino group donor contained in the aminotransferase composition of the present invention can be specifically described in the above sections "Aminotransferase," "Vector," "Transformant," and "Method for producing amino compounds."

[0082] The present invention will be described in more detail below with reference to examples. However, these examples are merely examples shown for the convenience of explanation, and the present invention is not limited to these examples in any sense.

[0083] <Gene nucleotide sequence and protein amino acid sequence> The gene nucleotide sequence and protein amino acid sequence were obtained from the National Center for Biotechnology Information (NCBI, https: / / www.ncbi.nlm.nih.gov / ) and can be identified by their accession numbers.

[0084] <Genetic Engineering Software> Molecular biology software SnapGene (registered trademark) 5.3.3 (GSL Biotech, LLC) was used for cloning foreign genes, designing primers, designing target plasmid DNA, and confirming DNA sequences.

[0085] <Synthesis of Artificial Genes and Oligonucleotides> Artificial genes and oligonucleotides were synthesized based on base sequence information in a database. For sequences that are difficult to synthesize (homopolymers or repeat sequences longer than a certain length), some codons were modified to avoid changing the amino acids. Current technology limits the length of artificial genes that can be synthesized, making it difficult to synthesize a group of genes to confer the ability to produce a target product in E. coli as a single artificial gene sequence. Therefore, the artificial gene was synthesized in multiple parts, each less than 3 kb in length. The artificial genes, primers for PCR reactions and short double-stranded DNA, and oligonucleotides (open column purification grade) were synthesized by Eurofins Genomics.

[0086] <PCR reaction> PCR reaction was carried out using DNA polymerase Q5 DNA polymerase (New England Biolabs). Amplification of DNA fragments was carried out using a thermal cycler SimpliAmp (Thermo Fisher Scientific) according to the specified protocol (thermal annealing at 98°C for 10 seconds, annealing temperature depending on the primers, extension reaction at 72°C for 35 cycles, reaction time at each temperature depending on the base length of the DNA fragment to be amplified: 30 seconds / 1 kb).

[0087] Comparison of Amino Compound Production Efficiency in Transformants Containing Various Aminotransferases 1. Preparation and Cultivation of Transformants (1) Construction and Cultivation of Recombinant E. coli (865838B) Carrying the Aminotransferase Gene from Chromobacterium violaceum ATCC 12472 (Comparative Example 1) A DNA solution containing the plasmid vector pKEF865 (Figure 1) containing artificially synthesized DNA sequences [alkBFG (SEQ ID NO: 1), alkJ (SEQ ID NO: 2), alkL (SEQ ID NO: 3), and alkT (SEQ ID NO: 4)] based on information from the alkane degradation gene cluster from Pseudomonas putida GPo01 (Accession No. AJ245436) was prepared. alkBFG (SEQ ID NO: 1) contains the sequences for alkane-1 monooxygenase (alkB), rubredoxin 1 (alkF), and rubredoxin 2 (alkG). alkJ (SEQ ID NO: 2) is an alcohol dehydrogenase, alkL (SEQ ID NO: 3) is an outer membrane protein, and alkT (SEQ ID NO: 4) is a rubredoxin reductase. A DNA solution containing the plasmid vector pKEF838 (Figure 2) was prepared. The DNA contained the nucleotide sequence (SEQ ID NO: 6) of a DNA artificially synthesized based on the amino acid sequence (SEQ ID NO: 5) translated from the cv2025 gene encoding an aminotransferase derived from Chromobacterium violaceum ATCC 12472 strain (accession number AAQ59697.1) and the nucleotide sequence (SEQ ID NO: 7) of a DNA artificially synthesized based on the alaD gene (accession number WP_003243280.1) encoding alanine synthase derived from Bacillus subtilis. 1 μL (0.1-0.2 μg) of a DNA solution of the plasmid vector pKEF838 (Figure 2), 1 μL (0.1-0.2 μg) of a DNA solution of the plasmid vector pKEF865 (Figure 1), and 50-100 μL of competent cells of the E. coli BL21(DE3) strain were mixed in a microtube and then heat-treated (42°C, 45 seconds).After cooling on ice, the mixture was diluted appropriately with SOC Outgrowth Medium (2% Vegetable Peptone, 0.5% Yeast Extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM Glucose) and plated on LB agar medium (1.0% Tryptone, 0.5% Yeast Extract, 1.0% NaCl, 1.5% agar) containing 100 μg / mL carbenicillin sodium and 50 μg / mL kanamycin sulfate. After static incubation overnight at 37°C, colonies formed on the agar medium were isolated to obtain recombinant E. coli (865838B) carrying the plasmid vectors pKEF865 and pKEF838.

[0088] The recombinant E. coli (865838B) was inoculated into 2 mL of LB medium (1.0% tryptone, 0.5% yeast extract, 1.0% NaCl) containing 50 μg / mL carbenicillin sodium, 50 μg / mL kanamycin sulfate, and 1.0% glucose in a test tube and cultured at 30°C and 180 rpm for 16 hours with shaking to prepare a preculture. 15 mL of LB medium containing 50 μg / mL carbenicillin sodium, 50 μg / mL kanamycin sulfate, and 1% glucose was prepared in a 125 mL baffled flask. 0.6 mL of the preculture was added, and main culture was carried out at 30°C and 180 rpm. When the turbidity (OD600) reached approximately 1.0, isopropyl-β-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM, and the main culture was continued for 4 hours. The resulting culture medium was centrifuged (3000 g, 5 minutes) to recover the bacterial cells, which were then used to evaluate the production reaction of aminoalkanoic acid esters.

[0089] (2) Preparation and Cultivation of Recombinant Escherichia coli (865913B) Carrying a Gene for a Protein of Unknown Function Derived from Stappia aggregata IAM 12614 Strain (Example 1) Using the above-mentioned plasmid vector pKEF838 (Figure 2) as template DNA and primers CP038 (SEQ ID NO: 8) and CP039 (SEQ ID NO: 9), a 5,400 bp DNA fragment was amplified by PCR. Additionally, a 1415-bp DNA fragment was amplified by PCR using primers NY231 (SEQ ID NO: 11) and NY232 (SEQ ID NO: 12) and artificially synthesized DNA sequence (SEQ ID NO: 10) based on information on a protein of unknown function derived from Stappia aggregata IAM 12614 (accession number EAV41574.1, https: / / www.ncbi.nlm.nih.gov / protein / EAV41574.1 / ), which is defined in the NCBI database as "hypothetical protein SIAM614_30726 [Stappia aggregata IAM 12614]." The two DNA fragments were purified using a QIAprep Gel Extraction Kit (Qiagen), and then ligated using NEBuilder HiFi DNA Assembly (New England Biolabs) according to the manufacturer's instructions. 1 μL of the reaction mixture was mixed with 50 μL of competent E. coli DH5α cells on ice, heat-treated (42°C, 45 seconds), cooled on ice, and diluted appropriately with SOC Outgrowth Medium (2% Vegetable Peptone, 0.5% Yeast Extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM Glucose). The mixture was then plated onto LB agar medium (1.0% Tryptone, 0.5% Yeast Extract, 1.0% NaCl, 1.5% agar) containing 50 μg / mL kanamycin sulfate. After static incubation overnight at 37°C, colonies formed on the agar medium were isolated to obtain recombinant E. coli carrying the plasmid vector pKEF913.

[0090] The plasmid vector was purified from the above recombinant E. coli using a QIAspin Miniprep Kit (Qiagen), and the base sequence was confirmed using a DNA sequencer. Then, the plasmid vector pKEF913 containing the gene for a protein of unknown function (sequence number 13) derived from Stappia aggregata IAM 12614 strain was obtained.

[0091] 1 μL (0.1-0.2 μg) of a DNA solution of the plasmid vector pKEF865 (Figure 1) containing the DNA base sequence [alkBFG (SEQ ID NO: 1), alkJ (SEQ ID NO: 2), alkL (SEQ ID NO: 3), alkT (SEQ ID NO: 4)] artificially synthesized based on the information of the alkane degradation gene cluster derived from the Pseudomonas putida GPo01 strain (accession number AJ245436), 1 μL (0.1-0.2 μg) of the DNA solution of the above-mentioned plasmid vector pKEF913, and 50-100 μL of competent cells of the Escherichia coli BL21(DE3) strain were mixed in a microtube and then heat-treated (42°C, 45 seconds). After cooling on ice, the mixture was diluted appropriately with SOC Outgrowth Medium (2% Vegetable Peptone, 0.5% Yeast Extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM Glucose) and plated on LB agar medium (1.0% Tryptone, 0.5% Yeast Extract, 1.0% NaCl, 1.5% agar) containing 100 μg / mL carbenicillin sodium and 50 μg / mL kanamycin sulfate. After static incubation overnight at 37°C, colonies formed on the agar medium were isolated to obtain recombinant E. coli (865913B) carrying the plasmid vectors pKEF865 and pKEF913.

[0092] The prepared recombinant Escherichia coli (865913B) was cultured in the same manner as described in Comparative Example 1, and the obtained cells were used to evaluate the production reaction of aminoalkanoic acid esters.

[0093] (3) Preparation and cultivation of recombinant Escherichia coli (865914B) carrying a gene encoding a protein of unknown function derived from Salmonella mucosus DSM 16094 strain (Example 2) Using the above-mentioned plasmid vector pKEF838 (Figure 2) as template DNA, a 5,400 bp DNA fragment was amplified by PCR using primers CP038 (SEQ ID NO: 8) and CP039 (SEQ ID NO: 9). Additionally, a 1448-bp DNA fragment was amplified by PCR using primers NY233 (SEQ ID NO: 15) and NY234 (SEQ ID NO: 16) and artificially synthesized DNA sequence (SEQ ID NO: 14) based on information from a protein of unknown function derived from Salipiger mucosus DSM 16094 (accession number EPX83375.1, https: / / www.ncbi.nlm.nih.gov / protein / EPX83375.1 / ), which is defined in the NCBI database as hypothetical protein Salmuc_01037 [Salipiger mucosus DSM 16094]. These two DNA fragments were purified using a QIAprep Gel Extraction Kit (Qiagen), and then ligated using NEBuilder HiFi DNA Assembly (New England Biolabs) according to the manufacturer's instructions. 1 μL of the reaction mixture was mixed with 50 μL of competent E. coli DH5α cells on ice, heat-treated (42°C, 45 seconds), cooled on ice, and diluted appropriately with SOC Outgrowth Medium (2% Vegetable Peptone, 0.5% Yeast Extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM Glucose). The mixture was then plated onto LB agar medium (1.0% Tryptone, 0.5% Yeast Extract, 1.0% NaCl, 1.5% agar) containing 50 μg / mL kanamycin sulfate. After static incubation overnight at 37°C, colonies formed on the agar medium were isolated to obtain recombinant E. coli carrying the plasmid vector pKEF914.

[0094] The plasmid vector was purified from the above recombinant E. coli using a QIAspin Miniprep Kit (Qiagen), and the base sequence was confirmed using a DNA sequencer.The plasmid vector pKEF914 containing the gene for a protein of unknown function (sequence number 17) derived from Salmonella mucosus DSM 16094 strain was obtained.

[0095] 1 μL (0.1–0.2 μg) of the DNA solution of the plasmid vector pKEF865 (Figure 1), 1 μL (0.1–0.2 μg) of the DNA solution of the plasmid vector pKEF914, and 50–100 μL of competent cells of E. coli BL21(DE3) were mixed in a microtube and heat-treated at 42°C for 45 seconds. After cooling on ice, the mixture was diluted appropriately with SOC Outgrowth Medium (2% Vegetable Peptone, 0.5% Yeast Extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM Glucose) and plated on LB agar medium (1.0% Tryptone, 0.5% Yeast Extract, 1.0% NaCl, 1.5% agar) containing 100 μg / mL carbenicillin sodium and 50 μg / mL kanamycin sulfate. After static cultivation overnight at 37°C, colonies formed on the agar medium were isolated to obtain recombinant E. coli (865914B) carrying the plasmid vectors pKEF865 and pKEF914.

[0096] The prepared recombinant Escherichia coli (865914B) was cultured in the same manner as described in Comparative Example 1, and the obtained cells were used to evaluate the production reaction of aminoalkanoic acid esters.

[0097] (4) Preparation and Cultivation of Recombinant Escherichia coli (865915B) Carrying a Gene for a Protein of Unknown Function Derived from Ensifer sp. Root1252 Strain (Example 3) Using the above-mentioned plasmid vector pKEF838 (FIG. 2) as template DNA and primers CP038 (SEQ ID NO: 8) and CP039 (SEQ ID NO: 9), a 5,400 bp DNA fragment was amplified by PCR. In addition, a 1406-bp DNA fragment was amplified by PCR using primers NY235 (SEQ ID NO: 19) and NY236 (SEQ ID NO: 20) and artificially synthesized DNA sequence (SEQ ID NO: 18) based on information from an Ensifer sp. Root1252-derived protein of unknown function (accession number KQW54728.1, https: / / www.ncbi.nlm.nih.gov / protein / KQW54728.1 / ), which is defined in the NCBI database as hypothetical protein ASD02_30575 [Ensifer sp. Root1252]. These two DNA fragments were purified using a QIAprep Gel Extraction Kit (Qiagen), and then ligated using NEBuilder HiFi DNA Assembly (New England Biolabs) according to the product's instructions. 1 μL of the reaction mixture was mixed with 50 μL of competent E. coli DH5α cells on ice, heat-treated (42°C, 45 seconds), cooled on ice, and diluted appropriately with SOC Outgrowth Medium (2% Vegetable Peptone, 0.5% Yeast Extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM Glucose). The mixture was then plated onto LB agar medium (1.0% Tryptone, 0.5% Yeast Extract, 1.0% NaCl, 1.5% agar) containing 50 μg / mL kanamycin sulfate. After static incubation at 37°C overnight, colonies formed on the agar medium were isolated to obtain recombinant E. coli carrying the plasmid vector pKEF915.

[0098] The plasmid vector was purified from the above recombinant E. coli using a QIAspin Miniprep Kit (Qiagen), and the base sequence was confirmed using a DNA sequencer. Then, the plasmid vector pKEF915 containing the gene for a protein of unknown function (sequence number 21) derived from Ensifer sp. Root1252 strain was obtained.

[0099] 1 μL (0.1–0.2 μg) of the DNA solution of the plasmid vector pKEF865 and 1 μL (0.1–0.2 μg) of the DNA solution of the plasmid vector pKEF915 were mixed with 50–100 μL of competent cells of E. coli BL21(DE3) in a microtube and then heat-treated at 42°C for 45 seconds. After cooling on ice, the mixture was diluted appropriately with SOC Outgrowth Medium (2% Vegetable Peptone, 0.5% Yeast Extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM Glucose) and plated on LB agar medium (1.0% Tryptone, 0.5% Yeast Extract, 1.0% NaCl, 1.5% agar) containing 100 μg / mL carbenicillin sodium and 50 μg / mL kanamycin sulfate. After static cultivation overnight at 37°C, colonies formed on the agar medium were isolated to obtain recombinant E. coli (865915B) carrying the plasmid vectors pKEF865 and pKEF915.

[0100] The prepared recombinant Escherichia coli (865915B) was cultured in the same manner as described in Comparative Example 1, and the obtained cells were used to evaluate the production reaction of aminoalkanoic acid esters.

[0101] (5) Preparation and Cultivation of Recombinant Escherichia coli (865922B) Carrying a Gene for a Protein of Unknown Function Derived from Mameliella alba DSM 26384 Strain (Example 4) Using the above-mentioned plasmid vector pKEF838 (FIG. 2) as template DNA and primers CP038 (SEQ ID NO: 8) and CP039 (SEQ ID NO: 9), a 5,400 bp DNA fragment was amplified by PCR. Additionally, a 1403-bp DNA fragment was amplified by PCR using primers NY249 (SEQ ID NO: 23) and NY250 (SEQ ID NO: 24) and artificially synthesized DNA sequence (SEQ ID NO: 22) based on information on a protein of unknown function derived from Mameliella alba DSM26384 strain (accession number PTR37408.1, https: / / www.ncbi.nlm.nih.gov / protein / PTR37408.1 / ), which is defined in the NCBI database as hypothetical protein LX94_03747 [Mameliella alba]. These two DNA fragments were purified using a QIAprep Gel Extraction Kit (Qiagen), and then ligated using NEBuilder HiFi DNA Assembly (New England Biolabs) according to the product's instructions. 1 μL of the reaction mixture was mixed with 50 μL of competent E. coli DH5α cells on ice, heat-treated (42°C, 45 seconds), cooled on ice, and diluted appropriately with SOC Outgrowth Medium (2% Vegetable Peptone, 0.5% Yeast Extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM Glucose). The mixture was then plated onto LB agar medium (1.0% Tryptone, 0.5% Yeast Extract, 1.0% NaCl, 1.5% agar) containing 50 μg / mL kanamycin sulfate. After static incubation at 37°C overnight, colonies formed on the agar medium were isolated to obtain recombinant E. coli carrying the plasmid vector pKEF922.

[0102] The plasmid vector was purified from the above recombinant E. coli using a QIAspin Miniprep Kit (Qiagen), and the base sequence was confirmed using a DNA sequencer. Then, the plasmid vector pKEF922 containing the gene for a protein of unknown function (SEQ ID NO: 25) derived from the Mameliella alba DSM 26384 strain was obtained.

[0103] 1 μL (0.1–0.2 μg) of the DNA solution of the plasmid vector pKEF865 and 1 μL (0.1–0.2 μg) of the DNA solution of the plasmid vector pKEF922 were mixed with 50–100 μL of competent cells of E. coli BL21(DE3) in a microtube and then heat-treated at 42°C for 45 seconds. After cooling on ice, the mixture was diluted appropriately with SOC Outgrowth Medium (2% Vegetable Peptone, 0.5% Yeast Extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM Glucose) and plated on LB agar medium (1.0% Tryptone, 0.5% Yeast Extract, 1.0% NaCl, 1.5% agar) containing 100 μg / mL carbenicillin sodium and 50 μg / mL kanamycin sulfate. After static cultivation overnight at 37°C, colonies formed on the agar medium were isolated to obtain recombinant E. coli (865922B) carrying the plasmid vectors pKEF865 and pKEF922.

[0104] The prepared recombinant Escherichia coli (865922B) was cultured in the same manner as described in Comparative Example 1, and the obtained cells were used to evaluate the production reaction of aminoalkanoic acid esters.

[0105] (6) Preparation and Cultivation of Recombinant Escherichia coli (865923B) Carrying a Gene for a Protein of Unknown Function Derived from Mesorhizobium sp. Strain LSJC285A00 (Example 5) Using the above-mentioned plasmid vector pKEF838 (Figure 2) as template DNA and primers CP038 (SEQ ID NO: 8) and CP039 (SEQ ID NO: 9), a 5,400 bp DNA fragment was amplified by PCR. Additionally, a 1397-bp DNA fragment was amplified by PCR using primers NY251 (SEQ ID NO: 27) and NY252 (SEQ ID NO: 28) and artificially synthesized DNA based on information on a protein of unknown function derived from Mesorhizobium sp. LSJC285A00 (accession number ESW90402.1, https: / / www.ncbi.nlm.nih.gov / protein / ESW90402.1 / ), which is identified in the NCBI database as hypothetical protein X773_02165 [Mesorhizobium sp. LSJC285A00]. The two DNA fragments were purified using a QIAprep Gel Extraction Kit (Qiagen) and then ligated using NEBuilder HiFi DNA Assembly (New England Biolabs) according to the manufacturer's instructions. 1 μL of the reaction mixture was mixed with 50 μL of competent E. coli DH5α cells on ice, heat-treated (42°C, 45 seconds), cooled on ice, and diluted appropriately with SOC Outgrowth Medium (2% Vegetable Peptone, 0.5% Yeast Extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM Glucose). The mixture was then plated onto LB agar medium (1.0% Tryptone, 0.5% Yeast Extract, 1.0% NaCl, 1.5% agar) containing 50 μg / mL kanamycin sulfate. After static incubation at 37°C overnight, colonies formed on the agar medium were isolated to obtain recombinant E. coli carrying the plasmid vector pKEF923.

[0106] The plasmid vector was purified from the above recombinant E. coli using a QIAspin Miniprep Kit (Qiagen), and the base sequence was confirmed using a DNA sequencer. Then, the plasmid vector pKEF923 containing the gene for a protein of unknown function (sequence number 29) derived from Mesorhizobium sp. LSJC285A00 strain was obtained.

[0107] 1 μL (0.1–0.2 μg) of the DNA solution of the plasmid vector pKEF865 and 1 μL (0.1–0.2 μg) of the DNA solution of the plasmid vector pKEF923 were mixed with 50–100 μL of competent cells of E. coli BL21(DE3) in a microtube and then heat-treated at 42°C for 45 seconds. After cooling on ice, the mixture was diluted appropriately with SOC Outgrowth Medium (2% Vegetable Peptone, 0.5% Yeast Extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM Glucose) and plated on LB agar medium (1.0% Tryptone, 0.5% Yeast Extract, 1.0% NaCl, 1.5% agar) containing 100 μg / mL carbenicillin sodium and 50 μg / mL kanamycin sulfate. After static cultivation overnight at 37°C, colonies formed on the agar medium were isolated to obtain recombinant E. coli (865923B) carrying the plasmid vectors pKEF865 and pKEF923.

[0108] The prepared recombinant Escherichia coli (865923B) was cultured in the same manner as described in Comparative Example 1, and the obtained cells were used to evaluate the production reaction of aminoalkanoic acid esters.

[0109] 2. Comparison of aminoalkanoate ester production efficiency in each transformant. 0.2 mL of reaction solution (200 mM Tris-HCl buffer (pH 8.0) containing 5.0% methyl dodecanoate, 2.0% glucose, 50 mM L-alanine, and 0.1 mM pyridoxal phosphate) was prepared in a 2.0 mL microtube. The bacterial cells obtained in Comparative Example 1 and Examples 1-5 were dispersed to a turbidity (OD600) of 25, and the reaction was carried out by stirring at 30°C and 1400 rpm for 1 hour. The pH of the resulting reaction solution was adjusted to approximately 9.0 by adding 1 M NaOH. After solvent extraction, t-butyl methyl ether (TBME) containing an internal standard was added, and the organic phase containing the produced aminoalkanoate ester (methyl 12-aminododecanoate) was recovered. N,N-dimethylformamide dimethyl acetal was added to the recovered organic phase to derivatize methyl 12-aminododecanoate. The derivatized product was analyzed by GC-FID (column: DB-FATWAX Ultra Inert / inner diameter 0.32 mm, film thickness 0.25 μm, length 30 m), and the amount of methyl 12-aminododecanoate produced in the reaction was calculated.

[0110] The amount of aminoalkanoic acid ester produced by the transformant of Comparative Example 1 was set to 1, and the relative values ​​of the amounts of aminoalkanoic acid ester produced by the transformants of Examples 1 to 5 were calculated to compare the production efficiencies. The results are shown in Figure 3.

[0111] 3 , it was confirmed that the transformants of Examples 1 to 5 had significantly higher aminoalkanoate ester production efficiency than the transformant of Comparative Example 1. From these results, it was found that the polypeptides (proteins of unknown function) in the transformants of Examples 1 to 5 have transamination activity and are significantly superior in aminoalkanoate ester production efficiency compared to the conventionally known aminotransferase (Comparative Example 1).

[0112] The identity of the amino acid sequences of a conventionally known aminotransferase (Comparative Example 1) and polypeptides (Examples 1 to 5) having significantly superior transamination activity was examined, and the results are shown in Table 1 below.

[0113]

[0114] As shown in Table 1, the amino acid sequences of the polypeptides of Examples 1 to 5 (SEQ ID NOs: 13, 17, 21, 25, and 29) share very low identities of 33% to 54% with the amino acid sequence of Comparative Example 1 (SEQ ID NO: 5), a conventionally known aminotransferase. Therefore, a person skilled in the art would not have anticipated that the polypeptides of SEQ ID NOs: 13, 17, 21, 25, and 29 have transamination activity, and it would have been impossible to consider utilizing these proteins of unknown function in the biosynthesis of aminoalkanoic acid esters.

[0115] [Comparison of transamination reaction rates between aminotransferases and proteins of unknown function] 1. Transformant Preparation and Culturing, and Production of His-tagged Aminotransferase or His-tagged Protein of Unknown Function (1) Preparation and Culturing of Recombinant Escherichia coli (840B) Carrying the Gene for Chromobacterium violaceum ATCC 12472-Derived Aminotransferase, and Purification of His-tagged Aminotransferase (Comparative Example 2) 1 μL (0.1-0.2 μg) of a DNA solution of plasmid vector pKEF840 ( FIG. 4 ) containing the base sequence (SEQ ID NO: 6) of artificially synthesized DNA based on the amino acid sequence (SEQ ID NO: 5) translated from the cv2025 gene encoding the Chromobacterium violaceum ATCC 12472-derived aminotransferase (Accession No. AAQ59697.1) was mixed with 50-100 μL of competent cells of Escherichia coli BL21(DE3) in a microtube, followed by heat treatment (42°C, 45 seconds). After cooling on ice, the mixture was diluted appropriately with SOC Outgrowth Medium (2% Vegetable Peptone, 0.5% Yeast Extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM Glucose) and plated on LB agar medium (1.0% Tryptone, 0.5% Yeast Extract, 1.0% NaCl, 1.5% agar) containing 50 μg / mL kanamycin sulfate. After static cultivation overnight at 37°C, colonies formed on the agar medium were isolated to obtain recombinant E. coli (840B) carrying the plasmid vector pKEF840.

[0116] The recombinant E. coli (840B) was inoculated into 2 mL of LB medium (1.0% tryptone, 0.5% yeast extract, 1.0% NaCl) containing 50 μg / mL kanamycin sulfate and 0.1 mL of 20% glucose in a test tube. The resulting preculture was then cultured at 30°C and 180 rpm for 16 hours with shaking. 15 mL of LB medium containing 50 μg / mL kanamycin sulfate and 0.75 mL of 20% glucose were added to a 125 mL baffled flask. 0.3 mL of the preculture was added, and the main culture was carried out at 30°C and 180 rpm. When the turbidity (OD600) reached approximately 1.0, isopropyl-β-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM, and the main culture was continued for 4 hours. The resulting culture was centrifuged at 3000 × g for 5 minutes to harvest the bacterial cells.

[0117] To the collected cells, 9.0 mL of BugBuster® Protein Extraction Reagent (Merck), 90 μL of 4.0% lysozyme solution (Fujifilm Wako Pure Chemical Industries, Ltd., egg white lysozyme dissolved in purified water), and 9.0 μL of Benzonase® nuclease (Merck) were added and stirred to disperse the cells. The mixture was then left at room temperature for 10 minutes, frozen at -80°C for 20 minutes, and then returned to room temperature. The resulting solution was centrifuged (3000 × g, 10 minutes) to separate the supernatant and precipitate. The supernatant fraction was filtered through a membrane filter (material: cellulose acetate, pore size: 0.45 μm, ADVANTEC), and the filtrate (containing His-tagged aminotransferase with a histidine tag (His-tag) attached to the carboxyl terminal of SEQ ID NO: 5) was used as a crude extract.

[0118] The His-tagged aminotransferase was purified from the crude extract using the AKTA start™ protein purification chromatography system (Cytiva). The flow rate was set to 1.0 mL / min. The crude extract was then pumped onto a His-tagged protein purification column (HisTrap™ HP 1 mL, Cytiva) equilibrated with eluent A (20 mM sodium phosphate buffer, pH 7.4, 30 mM imidazole, 500 mM NaCl) to adsorb the His-tagged aminotransferase to the column. After removing unadsorbed components from the column with eluent A, the His-tagged aminotransferase was eluted with eluent B (20 mM sodium phosphate buffer, pH 7.4, 500 mM imidazole, 500 mM NaCl) in a linear gradient of imidazole from 30 mM to 300 mM over 25 min. The fraction containing the His-tagged aminotransferase was collected as the eluate.

[0119] The eluate was transferred to a centrifugal ultrafiltration filter unit (10,000 MWCO, AS ONE Corporation) and concentrated by centrifugation (4,000 × g). Ten volumes of 20 mM Tris-HCl buffer (pH 8.0) were added to the concentrate, followed by stirring and centrifuging (4,000 × g) to concentrate again (referred to as solvent substitution). After two more solvent substitutions, the resulting concentrate was designated as purified enzyme (840B-TA). The purified enzyme was subjected to polyacrylamide gel electrophoresis (SDS-PAGE). The gel was stained and destained with a staining reagent (Quick CBB Plus, Fujifilm Wako Pure Chemical Corporation) to confirm the absence of contaminating proteins. The enzyme concentration of the purified enzyme solution was quantified using Pierce™ BCA Protein Assay Kits (Thermo Fisher Scientific).

[0120] (2) Construction and cultivation of recombinant Escherichia coli (853B) carrying the gene encoding a protein of unknown function derived from Stappia aggregata IAM 12614 strain, and production of a His-tagged protein of unknown function (Example 6) Using pET26b(+) (Novagen / Merck Millipore) as template DNA, a 5190 bp DNA fragment was amplified by PCR using primers CP014 (SEQ ID NO: 30) and CP015 (SEQ ID NO: 31). Additionally, a 1495-bp DNA fragment was amplified by PCR using primers CP016 (SEQ ID NO: 32) and NY173 (SEQ ID NO: 33) and artificially synthesized DNA sequence (SEQ ID NO: 10) based on information on a protein of unknown function derived from Stappia aggregata IAM 12614 (accession number EAV41574.1, https: / / www.ncbi.nlm.nih.gov / protein / EAV41574.1 / ) defined in the NCBI database as "hypothetical protein SIAM614_30726 [Stappia aggregata IAM 12614]." These two DNA fragments were purified using a QIAprep Gel Extraction Kit (Qiagen) and then ligated using NEBuilder HiFi DNA Assembly (New England Biolabs) according to the manufacturer's instructions. 1 μL of the reaction mixture was mixed with 50 μL of competent E. coli DH5α cells on ice, heat-treated (42°C, 45 seconds), cooled on ice, and diluted appropriately with SOC Outgrowth Medium (2% Vegetable Peptone, 0.5% Yeast Extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM Glucose). The mixture was then plated onto LB agar medium (1.0% Tryptone, 0.5% Yeast Extract, 1.0% NaCl, 1.5% agar) containing 50 μg / mL kanamycin sulfate. After static incubation overnight at 37°C, colonies formed on the agar medium were isolated to obtain recombinant E. coli (853B) carrying the plasmid vector pKEF853.

[0121] The plasmid vector was purified from the above recombinant E. coli using a QIAspin Miniprep Kit (Qiagen), and the base sequence was confirmed using a DNA sequencer. Then, the plasmid vector pKEF853 containing the gene for a protein of unknown function (sequence number 13) derived from Stappia aggregata IAM 12614 strain was obtained.

[0122] Two test tubes were prepared by inoculating the recombinant E. coli (853B) into 2 mL of LB medium (1.0% tryptone, 0.5% yeast extract, 1.0% NaCl) containing 50 μg / mL kanamycin sulfate with 0.1 mL of 20% glucose. The resulting preculture was then cultured at 30°C and 180 rpm for 16 hours. A 1.0 L baffled flask was prepared by adding 4.0 mL of the preculture to 200 mL of LB medium containing 50 μg / mL kanamycin sulfate with 10 mL of 20% glucose. Main culture was then performed at 30°C and 160 rpm. When the turbidity (OD600) reached approximately 1.0, isopropyl-β-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM, and the main culture was continued for 4 hours. The resulting culture was then centrifuged (3000 × g, 5 minutes) to harvest the bacterial cells.

[0123] To the collected cells, 15 mL of BugBuster® Protein Extraction Reagent (Merck), 150 μL of 4.0% lysozyme solution (Fujifilm Wako Pure Chemical Industries, Ltd., egg white lysozyme dissolved in purified water), and 15 μL of Benzonase® nuclease (Merck) were added and stirred to disperse the cells. The mixture was then left at room temperature for 20 minutes, frozen at -80°C for 20 minutes, and then returned to room temperature. The resulting solution was centrifuged (3000 × g, 10 minutes) to separate the supernatant and precipitate. The supernatant fraction was filtered through a membrane filter (material: cellulose acetate, pore size: 0.45 μm, ADVANTEC), and the filtrate (containing a protein with unknown His-tag binding function, in which a histidine tag (His-tag) was attached to the carboxyl terminal of SEQ ID NO: 13) was used as a crude extract.

[0124] Purification of the His-tag binding protein with unknown function from the crude extract, concentration of the collected eluate, and solvent exchange were performed in the same manner as in Comparative Example 2. The resulting concentrate was used as the purified enzyme (853B-TA). SDS-PAGE analysis of the purified enzyme and quantification of the enzyme concentration in the purified enzyme solution were also performed in the same manner as in Comparative Example 2.

[0125] (3) Construction and cultivation of recombinant Escherichia coli (854B) carrying the gene encoding a protein of unknown function derived from Salmonella mucosus DSM 16094 strain, and production of a His-tagged protein of unknown function (Example 7) Using pET26b(+) (Novagen / Merck Millipore) as template DNA, a 5190 bp DNA fragment was amplified by PCR using primers CP014 (SEQ ID NO: 30) and CP015 (SEQ ID NO: 31). Additionally, a 1528-bp DNA fragment was amplified by PCR using primers CP016 (SEQ ID NO: 32) and NY174 (SEQ ID NO: 34) and artificially synthesized DNA sequence (SEQ ID NO: 14) based on information from a protein of unknown function derived from Salipiger mucosus DSM 16094 (accession number EPX83375.1, https: / / www.ncbi.nlm.nih.gov / protein / EPX83375.1 / ), which is defined in the NCBI database as hypothetical protein Salmuc_01037 [Salipiger mucosus DSM 16094]. These two DNA fragments were purified using a QIAprep Gel Extraction Kit (Qiagen) and then ligated using NEBuilder HiFi DNA Assembly (New England Biolabs) according to the manufacturer's instructions. 1 μL of the reaction mixture was mixed with 50 μL of competent E. coli DH5α cells on ice, heat-treated (42°C, 45 seconds), cooled on ice, and diluted appropriately with SOC Outgrowth Medium (2% Vegetable Peptone, 0.5% Yeast Extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM Glucose). The mixture was then plated onto LB agar medium (1.0% Tryptone, 0.5% Yeast Extract, 1.0% NaCl, 1.5% agar) containing 50 μg / mL kanamycin sulfate. After static incubation overnight at 37°C, colonies formed on the agar medium were isolated to obtain recombinant E. coli (854B) carrying the plasmid vector pKEF854.

[0126] The plasmid vector was purified from the above recombinant E. coli using a QIAspin Miniprep Kit (Qiagen), and the base sequence was confirmed using a DNA sequencer.The plasmid vector pKEF854 containing the gene for a protein of unknown function (sequence number 17) derived from Salmonella mucosus DSM 16094 strain was obtained.

[0127] The prepared recombinant E. coli (854B) was cultured in the same manner as described in Comparative Example 2, and the resulting culture medium was centrifuged (3000×g, 5 minutes) to recover the bacterial cells.

[0128] A crude extract was prepared from the recovered bacterial cells using the same method as in Comparative Example 2, and the resulting filtrate (containing a protein of unknown His-tag binding function in which a histidine tag (His-tag) was attached to the carboxyl terminal side of SEQ ID NO: 17) was used as the crude extract. Purification of the protein of unknown His-tag binding function from the crude extract, and concentration and solvent substitution of the recovered eluate were also performed using the same methods as in Comparative Example 2. The resulting concentrate was used as the purified enzyme (854B-TA). SDS-PAGE analysis of the purified enzyme and quantification of the enzyme concentration of the purified enzyme solution were also performed using the same methods as in Comparative Example 2.

[0129] (4) Construction and cultivation of recombinant Escherichia coli (855B) carrying the gene encoding a protein of unknown function derived from Ensifer sp. Root1252 strain, and production of a His-tagged protein of unknown function (Example 8) Using pET26b(+) (Novagen / Merck Millipore) as template DNA, a 5190 bp DNA fragment was amplified by PCR using primers CP014 (SEQ ID NO: 30) and CP015 (SEQ ID NO: 31). In addition, a 1486-bp DNA fragment was amplified by PCR using primers CP016 (SEQ ID NO: 32) and NY175 (SEQ ID NO: 35) and artificially synthesized DNA sequence (SEQ ID NO: 18) based on information from an Ensifer sp. Root1252-derived protein of unknown function (accession number KQW54728.1, https: / / www.ncbi.nlm.nih.gov / protein / KQW54728.1 / ), which is defined in the NCBI database as hypothetical protein ASD02_30575 [Ensifer sp. Root1252]. These two DNA fragments were purified using a QIAprep Gel Extraction Kit (Qiagen), and then ligated using NEBuilder HiFi DNA Assembly (New England Biolabs) according to the product's instructions. 1 μL of the reaction mixture was mixed with 50 μL of competent E. coli DH5α cells on ice, heat-treated (42°C, 45 seconds), cooled on ice, and diluted appropriately with SOC Outgrowth Medium (2% Vegetable Peptone, 0.5% Yeast Extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM Glucose). The mixture was then plated onto LB agar medium (1.0% Tryptone, 0.5% Yeast Extract, 1.0% NaCl, 1.5% agar) containing 50 μg / mL kanamycin sulfate. After static incubation overnight at 37°C, colonies formed on the agar medium were isolated to obtain recombinant E. coli (855B) carrying the plasmid vector pKEF855.

[0130] The plasmid vector was purified from the above recombinant E. coli using a QIAspin Miniprep Kit (Qiagen), and the base sequence was confirmed using a DNA sequencer. Then, the plasmid vector pKEF855 containing the gene for a protein of unknown function (sequence number 21) derived from Ensifer sp. Root1252 strain was obtained.

[0131] The prepared recombinant E. coli (855B) was cultured in the same manner as described in Comparative Example 2, and the resulting culture medium was centrifuged (3000×g, 5 minutes) to recover the bacterial cells.

[0132] A crude extract was prepared from the recovered bacterial cells using the same method as in Comparative Example 2, and the resulting filtrate (containing a protein of unknown His-tag binding function in which a histidine tag (His-tag) was attached to the carboxyl terminal side of SEQ ID NO: 21) was used as the crude extract. Purification of the protein of unknown His-tag binding function from the crude extract, and concentration and solvent substitution of the recovered eluate were also performed using the same methods as in Comparative Example 2. The resulting concentrate was used as the purified enzyme (855B-TA). SDS-PAGE analysis of the purified enzyme and quantification of the enzyme concentration of the purified enzyme solution were also performed using the same methods as in Comparative Example 2.

[0133] (5) Construction and cultivation of recombinant Escherichia coli (862B) carrying the gene encoding a protein of unknown function derived from Mameliella alba DSM 26384 strain, and production of a His-tagged protein of unknown function (Example 9) Using pET26b(+) (Novagen / Merck Millipore) as template DNA, a 5190 bp DNA fragment was amplified by PCR using primers CP014 (SEQ ID NO: 30) and CP015 (SEQ ID NO: 31). Additionally, a 1478-bp DNA fragment was amplified by PCR using primers CP016 (SEQ ID NO: 32) and NY192 (SEQ ID NO: 36) and artificially synthesized DNA sequence (SEQ ID NO: 22) based on information on a protein of unknown function derived from Mameliella alba DSM26384 strain (accession number PTR37408.1, https: / / www.ncbi.nlm.nih.gov / protein / PTR37408.1 / ), which is defined in the NCBI database as hypothetical protein LX94_03747 [Mameliella alba]. These two DNA fragments were purified using a QIAprep Gel Extraction Kit (Qiagen), and then ligated using NEBuilder HiFi DNA Assembly (New England Biolabs) according to the product's instructions. 1 μL of the reaction mixture was mixed with 50 μL of competent E. coli DH5α cells on ice, heat-treated (42°C, 45 seconds), cooled on ice, and diluted appropriately with SOC Outgrowth Medium (2% Vegetable Peptone, 0.5% Yeast Extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM Glucose). The mixture was then plated onto LB agar (1.0% Tryptone, 0.5% Yeast Extract, 1.0% NaCl, 1.5% agar) containing 50 μg / mL kanamycin sulfate. After overnight static incubation at 37°C, colonies formed on the agar were isolated to obtain recombinant E. coli (862B) harboring the plasmid vector pKEF862.

[0134] The plasmid vector was purified from the above recombinant E. coli using a QIAspin Miniprep Kit (Qiagen), and the base sequence was confirmed using a DNA sequencer. Then, the plasmid vector pKEF862 containing the gene for a protein of unknown function (SEQ ID NO: 25) derived from the Mameliella alba DSM 26384 strain was obtained.

[0135] The prepared recombinant E. coli (862B) was cultured in the same manner as described in Comparative Example 2, and the resulting culture medium was centrifuged (3000×g, 5 minutes) to recover the bacterial cells.

[0136] A crude extract was prepared from the recovered bacterial cells using the same method as in Comparative Example 2, and the resulting filtrate (containing a protein of unknown His-tag binding function in which a histidine tag (His-tag) was attached to the carboxyl terminal side of SEQ ID NO: 25) was used as the crude extract. Purification of the protein of unknown His-tag binding function from the crude extract, and concentration and solvent substitution of the recovered eluate were also performed using the same methods as in Comparative Example 2. The resulting concentrate was used as the purified enzyme (862B-TA). SDS-PAGE analysis of the purified enzyme and quantification of the enzyme concentration of the purified enzyme solution were also performed using the same methods as in Comparative Example 2.

[0137] (6) Construction and cultivation of recombinant Escherichia coli (863B) carrying the gene encoding a protein of unknown function derived from Mesorhizobium sp. strain LSJC285A00, and production of a His-tagged protein of unknown function (Example 10) Using pET26b(+) (Novagen / Merck Millipore) as template DNA, a 5190 bp DNA fragment was amplified by PCR using primers CP014 (SEQ ID NO: 30) and CP015 (SEQ ID NO: 31). Additionally, a 1472-bp DNA fragment was amplified by PCR using primers CP016 (SEQ ID NO: 32) and NY193 (SEQ ID NO: 37) and artificially synthesized DNA sequence (SEQ ID NO: 26) based on information from a protein of unknown function derived from Mesorhizobium sp. LSJC285A00 (accession number ESW90402.1, https: / / www.ncbi.nlm.nih.gov / protein / ESW90402.1 / ), which is identified in the NCBI database as hypothetical protein X773_02165 [Mesorhizobium sp. LSJC285A00]. These two DNA fragments were purified using a QIAprep Gel Extraction Kit (Qiagen) and then ligated using NEBuilder HiFi DNA Assembly (New England Biolabs) according to the manufacturer's instructions. 1 μL of the reaction mixture was mixed with 50 μL of competent E. coli DH5α cells on ice, heat-treated (42°C, 45 seconds), cooled on ice, and diluted appropriately with SOC Outgrowth Medium (2% Vegetable Peptone, 0.5% Yeast Extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM Glucose). The mixture was then plated onto LB agar (1.0% Tryptone, 0.5% Yeast Extract, 1.0% NaCl, 1.5% agar) containing 50 μg / mL kanamycin sulfate. After overnight static incubation at 37°C, colonies formed on the agar were isolated to obtain recombinant E. coli (863B) carrying the plasmid vector pKEF863.

[0138] The plasmid vector was purified from the above recombinant E. coli using a QIAspin Miniprep Kit (Qiagen), and the base sequence was confirmed using a DNA sequencer. Then, the plasmid vector pKEF863 containing the gene for a protein of unknown function (sequence number 29) derived from Mesorhizobium sp. LSJC285A00 strain was obtained.

[0139] The prepared recombinant E. coli (863B) was cultured in the same manner as described in Comparative Example 2, and the resulting culture medium was centrifuged (3000×g, 5 minutes) to recover the bacterial cells.

[0140] A crude extract was prepared from the recovered bacterial cells using the same method as in Comparative Example 2, and the resulting filtrate (containing a protein of unknown His-tag binding function in which a histidine tag (His-tag) was attached to the carboxyl terminal side of SEQ ID NO: 29) was used as the crude extract. Purification of the protein of unknown His-tag binding function from the crude extract, and concentration and solvent substitution of the recovered eluate were also performed using the same methods as in Comparative Example 2. The resulting concentrate was used as the purified enzyme (863B-TA). SDS-PAGE analysis of the purified enzyme and quantification of the enzyme concentration of the purified enzyme solution were also performed using the same methods as in Comparative Example 2.

[0141] 2. Comparison of the transamination reaction rates of His-tagged aminotransferase and His-tagged proteins of unknown function The enzyme activity was measured using a modified version of the method described in Non-Patent Document A1 (J. Biol. Chem. 1993, 268, 17687-17694), and the transamination reaction rates of His-tagged aminotransferase and each His-tagged protein of unknown function were calculated.

[0142] Specifically, 0.2 mL of reaction solution (100 mM Tris-HCl buffer (pH 8.0) containing 50 mM L-alanine, 0.1 mM pyridoxal phosphate, 0.2 mM nicotinamide adenine dinucleotide (NADH), 10 U / mL lactate dehydrogenase (LDH), and 0.1% (v / v) methyl oxoalkanoate (methyl 6-oxohexanoate, methyl 8-oxooctanoate, or methyl 10-oxodecanoate)) was prepared in each well of a 96-well microplate, and then 20 μL of an enzyme solution (20 mM Tris-HCl buffer (pH 8.0) containing 20 μg / mL enzyme) containing the purified His-tag binding aminotransferase (Comparative Example 2) or a protein of unknown His-tag binding function (Examples 6 to 10) was added. The 96-well microplate containing the sample was then read using an absorbance microplate reader (Tecan Infinite M). The mixture was set on a 500-μL chromatograph (STEM) in a 500-μL Nano tube. After stirring for 10 seconds, the change in absorbance at 340 nm at 25°C was measured over 10 minutes. The NADH consumption rate was calculated from the absorbance change, and this was converted into the transamination reaction rate, allowing the transamination activity of various aminotransferases to be evaluated.

[0143] The transamination reaction rate of the His-tagged aminotransferase for methyl 6-oxohexanoate in Comparative Example 2 was set to 1, and the relative transamination reaction rates for the His-tagged proteins of unknown function in Examples 6 to 10 were calculated to compare the enzyme activities. The results are shown in Figure 5.

[0144] The transamination reaction rate of the His-tagged aminotransferase for methyl 8-oxooctanoate in Comparative Example 2 was set to 1, and the relative values ​​of the transamination reaction rates for the His-tagged proteins of unknown function in Examples 6 to 10 were calculated to compare the enzyme activities. The results are shown in Figure 6.

[0145] The transamination reaction rate of the His-tagged aminotransferase for methyl 10-oxodecanoate in Comparative Example 2 was set to 1, and the relative values ​​of the transamination reaction rates for the His-tagged proteins of unknown function in Examples 6 to 10 were calculated to compare the enzyme activities. The results are shown in Figure 7.

[0146] 5 to 7 , the transamination reaction rates of the His-tagged proteins of unknown function in Examples 6 to 10 using methyl 6-oxohexanoate, methyl 8-oxooctanoate, and methyl 10-oxodecanoate as substrates were all higher than the transamination reaction rate of the His-tagged aminotransferase of Comparative Example 2. In particular, the His-tagged protein of unknown function in Example 9 (SEQ ID NO: 29) showed significantly higher transamination reaction rates for the three carbonyl compounds, 53.7-fold, 47.9-fold, and 88.4-fold higher than the transamination reaction rate of the His-tagged aminotransferase of Comparative Example 2.

[0147] From the above results, it was found that all of the proteins with unknown His-tag binding function in Examples 6 to 10 have transamination activity and have superior transamination reaction activity compared to conventionally known aminotransferases, with Examples 8 to 10 being particularly superior.

[0148] The amino acid sequences or nucleic acid sequences of each SEQ ID NO: in the sequence listing are shown below.

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159] The present invention provides a novel aminotransferase with improved amino compound production efficiency and a transformant containing the same. Furthermore, the present invention enables the supply of bio-derived products and carbon recycling, thereby contributing to the achievement of Goal 12 of the SDGs.

Claims

1. An amino acid transferase that uses a carbonyl compound as a substrate, characterized in that it contains an amino acid sequence that has 90% or more identity to any one of the amino acid sequences of SEQ ID NO: 13, 17, 21, 25, or 29.

2. The aminotransferase according to claim 1, wherein the carbonyl compound is a compound represented by the following formula (1): (In formula (1), R represents a methyl group or an ethyl group, and n is an integer from 2 to 16.) 3. A polynucleotide comprising an amino acid sequence having 90% or more identity to any one of the amino acid sequences of SEQ ID NO: 13, 17, 21, 25, and 29, and comprising a base sequence encoding a protein having transamination activity using a carbonyl compound as a substrate.

4. A vector comprising the polynucleotide of claim 3.

5. A transformant having the vector according to claim 4.

6. A transformant for producing an aminoalkanoic acid ester from an alkanoic acid ester, comprising the aminotransferase according to claim 1 or 2.

7. A transformant for producing an aminoalkanoic acid ester from an alkanoic acid ester, comprising the polynucleotide according to claim 3.

8. A method for producing an amino compound represented by the following formula (2), which comprises contacting a carbonyl compound represented by the following formula (1) with an amino group donor in the presence of the aminotransferase described in claim 1 or 2. (In the formulas (1) and (2), R represents a methyl group or an ethyl group, and n is an integer from 2 to 16.) 9. A method for producing an amino compound represented by the following formula (2), comprising contacting a transformant having the aminotransferase described in claim 1 or the polynucleotide described in claim 3 with a carbonyl compound represented by the following formula (1) and an amino group donor. (In the formulas (1) and (2), R represents a hydrogen atom, a methyl group, or an ethyl group, and n is an integer from 2 to 16.) 10. A method for producing an aminotransferase, comprising the step of culturing a transformant having the aminotransferase according to claim 1 or the polynucleotide according to claim 3.

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