阅读说明：本技术 重组微生物及使用该重组微生物的吡哆胺或其盐的制造方法 (Recombinant microorganism and method for producing pyridoxamine or a salt thereof using same ) 是由 馆野俊博 秀崎友则 安乐城正 进藤敦德 松本佳子 宫田敏男 本城胜 于 2018-05-11 设计创作，主要内容包括：重组微生物,其具有编码吡哆醇氧化酶的基因及编码吡哆胺合成酶的基因,所述吡哆胺合成酶具有从吡哆醛合成吡哆胺的酶活性,所述编码吡哆醇氧化酶的基因及所述编码吡哆胺合成酶的基因各自是从菌体外导入的基因,或者是内源于菌体但其表达得到增强的基因；以及,使用所述重组微生物从吡哆醇或其盐生产吡哆胺或其盐的方法。(A recombinant microorganism having a gene encoding pyridoxol oxidase having an enzyme activity of synthesizing pyridoxamine from pyridoxal and a gene encoding pyridoxamine synthase each of which is a gene introduced extracellularly or a gene endogenous to the bacterial cell and having enhanced expression; and a process for producing pyridoxamine or a salt thereof from pyridoxine or a salt thereof using the recombinant microorganism.)

1. A recombinant microorganism having a gene coding for pyridoxol oxidase and a gene coding for pyridoxamine synthase having an enzyme activity to synthesize pyridoxamine from pyridoxal,

the gene encoding pyridoxine oxidase and the gene encoding pyridoxamine synthase are each a gene introduced extracellularly or a gene endogenous to the cell but having enhanced expression.

2. The recombinant microorganism according to claim 1, wherein the pyridoxamine synthase is pyridoxamine-pyruvate transaminase, pyridoxamine-oxaloacetate transaminase, aspartate transaminase, or pyridoxamine phosphate transaminase.

3. The recombinant microorganism according to claim 1 or 2, wherein the pyridoxine oxidase is represented by the enzyme number EC1.1.3.12.

4. The recombinant microorganism according to any one of claims 1 to 3, wherein the gene encoding pyridoxol oxidase is derived from Microbacterium flavum.

5. The recombinant microorganism according to any one of claims 1 to 4, wherein the gene encoding pyridoxol oxidase

(a) A nucleotide sequence having sequence number 5; or

(b) A nucleotide sequence that hybridizes with a DNA having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO. 5 under stringent conditions and encodes a protein having pyridoxol oxidase activity; or

(c) A nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID NO. 1; or

(d) Has a nucleotide sequence encoding a protein having an amino acid sequence having 80% or more sequence identity to the amino acid sequence of SEQ ID NO. 1 and having pyridoxol oxidase activity.

6. The recombinant microorganism according to any one of claims 1 to 5, wherein the pyridoxamine synthase comprises at least one of a partial amino acid sequence (c), a partial amino acid sequence (d), a partial amino acid sequence (e), a partial amino acid sequence (f), a partial amino acid sequence (g), and a partial amino acid sequence (h), and has an enzymatic activity for synthesizing pyridoxamine from pyridoxal,

(c)X₈X₉X₁₀X₁₁X₁₂X₁₃(SEQ ID NO: 39)

X₈The representation of L, M, I or V indicates that,

X₉represents a group of a compound represented by H or Q,

X₁₀the representation G, C or A is shown as,

X₁₁represents a group of a compound represented by E or D,

X₁₂the expression P or A is shown in the specification,

X₁₃represents V, I, L or A;

(d)X₁₄X₁₅TPSGTX₁₆X₁₇(SEQ ID NO: 40)

X₁₄Represents a group of a compound represented by H or S,

X₁₅the expression D or E is shown in the specification,

X₁₆the designation I, V or L is shown as,

X₁₇represents N or T;

(e)X₁₈DX₁₉VSX₂₀X₂₁(SEQ ID NO. 41)

X₁₈The representation V, I or A is shown as,

X₁₉the representation A, T or the representation S,

X₂₀the representation of S, A or G is,

X₂₁represents F, W or V;

(f)X₂₂X₂₃X₂₄KCX₂₅GX₂₆X₂₇p (SEQ ID NO: 42)

X₂₂The expression G or S is used for expressing,

X₂₃the representation P, S or A is shown as,

X₂₄the representation of N, G, S, A or Q,

X₂₅represents L or M, and is represented by,

X₂₆the representation of A, S, C or G is,

X₂₇represents P, T, S or A;

(g)X₂₈X₂₉X₃₀X₃₁SX₃₂GX₃₃X₃₄(SEQ ID NO. 43)

X₂₈The expression G or D is shown in the specification,

X₂₉the expression V or I is shown in the specification,

X₃₀the designation V, T, A, S, M, I or L is shown as,

X₃₁the representation of F, M, L, I or V indicates that,

X₃₂the designation S, G, A, T, I, L or H is shown in,

X₃₃the representation of R, M or Q,

X₃₄represents G, R, A, D, H or K;

(h)X₃₅X₃₆RX₃₇X₃₈HMGX₃₉X₄₀a (SEQ ID NO: 44)

X₃₅Represents a group of a compound represented by L or V,

X₃₆the designation T, I, V or L is shown as,

X₃₇the designation I, V or L is shown as,

X₃₈the expression G or S is used for expressing,

X₃₉the expression P, A or R is used for,

X₄₀indicating T, V or S.

7. The recombinant microorganism according to any one of claims 1 to 6, wherein the pyridoxamine synthase is represented by enzyme number EC2.6.1.30.

8. The recombinant microorganism according to any one of claims 1 to 7, wherein the gene encoding pyridoxamine synthase is derived from Mesorhizobium parviflorum.

9. The recombinant microorganism according to any one of claims 1 to 8, wherein the gene encoding pyridoxamine synthase

(a) Having the nucleotide sequence of any one of SEQ ID NO. 6 and SEQ ID NO. 25 to SEQ ID NO. 31, or

Has a region from the 18 th nucleotide to the 3 'end in the nucleotide sequence of SEQ ID NO. 10 or a region from the 18 th nucleotide to the 3' end in any of the nucleotide sequences of SEQ ID NO. 32 to SEQ ID NO. 38; or

(b) A nucleotide sequence that hybridizes under stringent conditions to a DNA having a nucleotide sequence complementary to the nucleotide sequence of any one of SEQ ID NO. 6 and SEQ ID NO. 25 to SEQ ID NO. 31, or a nucleotide sequence complementary to a region from the 18 th nucleotide to the 3 'end in the nucleotide sequence of SEQ ID NO. 10 or a region from the 18 th nucleotide to the 3' end in the nucleotide sequence of any one of SEQ ID NO. 32 to SEQ ID NO. 38, and encodes a protein having an enzymatic activity of synthesizing pyridoxamine from pyridoxal; or

(c) A nucleotide sequence encoding a protein having the amino acid sequence of any one of SEQ ID NO.2 and SEQ ID NO. 18 to SEQ ID NO. 24; or

(d) Has a nucleotide sequence encoding a protein having an amino acid sequence having 80% or more sequence identity to at least one of the amino acid sequences of SEQ ID NO.2 and SEQ ID NO. 18 to SEQ ID NO. 24 and having an enzymatic activity of synthesizing pyridoxamine from pyridoxal.

10. The recombinant microorganism according to any one of claims 1 to 5, wherein the pyridoxamine synthase is represented by enzyme No. EC2.6.1.31 or EC2.6.1.1.

11. The recombinant microorganism according to any one of claims 1 to 5 and 10, wherein the gene encoding pyridoxamine synthase is derived from Escherichia coli.

12. The recombinant microorganism according to any one of claims 1 to 5 and 10 to 11, wherein the gene encoding pyridoxamine synthase

(a) A nucleotide sequence having sequence number 8; or

(b) A nucleotide sequence that hybridizes under stringent conditions to a DNA having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO. 8 and encodes a protein having an enzymatic activity of synthesizing pyridoxamine from pyridoxal; or

(c) A nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID NO. 4; or

(d) Has a nucleotide sequence encoding a protein having an amino acid sequence having 80% or more sequence identity to the amino acid sequence of SEQ ID NO. 4 and having an enzymatic activity of synthesizing pyridoxamine from pyridoxal.

13. The recombinant microorganism as claimed in any one of claims 1 to 11, further having a gene encoding a hydrogen peroxide catabolic enzyme having an enzymatic activity of generating oxygen from hydrogen peroxide.

14. The recombinant microorganism according to claim 13, wherein the gene encoding a hydrogen peroxide catabolic enzyme is introduced extracellularly or is endogenous to the bacterial cell and has enhanced expression.

15. The recombinant microorganism as claimed in claim 13 or 14, wherein the hydrogen peroxide catabolic enzyme is represented by the enzyme number ec 1.11.1.6.

16. The recombinant microorganism according to any one of claims 13 to 15, wherein the gene encoding a hydrogen peroxide catabolic enzyme

(a) A nucleotide sequence having sequence number 7; or

(b) Hybridizing with a DNA having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO. 7 under stringent conditions and having an enzyme activity of generating oxygen from hydrogen peroxide; or

(c) A nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID NO. 3; or

(d) Has a nucleotide sequence encoding a protein having an amino acid sequence having 80% or more sequence identity to the amino acid sequence of SEQ ID NO. 3 and having an enzymatic activity of generating oxygen from hydrogen peroxide.

17. The recombinant microorganism of any one of claims 1-16, which is a recombinant escherichia coli.

18. A process for producing pyridoxamine or a salt thereof, wherein the recombinant microorganism or the culture of the recombinant microorganism or the treated product of the recombinant microorganism or the culture according to any one of claims 1 to 17 is contacted with pyridoxine or a salt thereof to produce pyridoxamine or a salt thereof in the presence of oxygen.

19. The production process according to claim 18, wherein the recombinant microorganism or a culture of the recombinant microorganism, or a treated matter of the recombinant microorganism or the culture contains the pyridoxol oxidase and the pyridoxamine synthase.

20. The production method according to claim 19, wherein the recombinant microorganism or the culture of the recombinant microorganism, or the treated product of the recombinant microorganism or the culture further comprises a hydrogen peroxide catabolic enzyme.

21. The production method according to any one of claims 18 to 20, wherein the treated product of the recombinant microorganism or the culture is a treated product obtained by 1 or more treatments selected from the group consisting of a heating treatment, a cooling treatment, mechanical destruction of cells, an ultrasonic treatment, a freeze-thaw treatment, a drying treatment, a pressure or reduced pressure treatment, an osmotic pressure treatment, autodigestion of cells, a surfactant treatment, an enzyme treatment, a cell separation treatment, a purification treatment, and an extraction treatment.

22. The production method according to any one of claims 18 to 21, which comprises either or both of the following (A) and (B):

(A) continuously adding or adding in divided portions pyridoxine or a salt thereof to a solution containing the recombinant microorganism or a culture of the recombinant microorganism, or a treatment of the recombinant microorganism or the culture;

(B) in a solution containing the recombinant microorganism or a culture of the recombinant microorganism, or a treated matter of the recombinant microorganism or the culture, the molar concentration of amino acids consumed by the pyridoxamine synthase is controlled to be 1-fold or more relative to the molar concentration of pyridoxine or a salt thereof.

23. The process according to claim 22, wherein the amino acid consumed by the pyridoxamine synthase is L-alanine, D-alanine, L-glutamic acid or D-glutamic acid.

Technical Field

The present disclosure relates to a recombinant microorganism capable of producing pyridoxamine or a salt thereof, and a process for producing pyridoxamine or a salt thereof using the recombinant microorganism.

Background

Pyridoxamine and its salt is vitamin B₆Has glycosylation reaction inhibiting effect, and is known. Pyridoxamine and its salts inhibit the accumulation of, for example, glycosylation end products (AGEs) associated with various aging reactions into the body. AGE is a generic term for substances produced by glycosylation of proteins, and accumulation of AGE is considered to worsen diseases such as diabetes, atherosclerosis, chronic renal failure, and alzheimer-type cognitive impairment. Accordingly, pyridoxamine and salts thereof are promising substances expected to be able to prevent and treat the above-mentioned diseases by preventing the accumulation of AGEs.

Pyridoxamine and its salts are known to have activity as a drug for schizophrenia, and various studies have been made on the practical use thereof. In addition, development of health foods and cosmetics utilizing various physiological activities of pyridoxamine and salts thereof has been advanced.

Pyridoxamine and salts thereof can be chemically synthesized. For example, international publication No. 2006/066806 discloses a method for chemically synthesizing pyridoxamine dihydrochloride using alanine and formic acid as starting materials. Further, international publication No. 2005/077902 discloses a method for chemically synthesizing pyridoxamine from pyridoxine.

On the other hand, biosynthesis of pyridoxamine has also been studied. International publication No. 2007/142222 discloses a method for obtaining pyridoxamine from pyridoxal using a specific microorganism belonging to the genus Achromobacter or the like. In addition, international publication No. 2007/142222 discloses an experiment in which an amount of pyridoxine is converted into pyridoxal by culturing Acremonium fusiformis in the presence of the pyridoxine.

Japanese unexamined patent publication No. 9-107985 discloses vitamin B₆The method for producing a microorganism having vitamin B belonging to the genus Rhizobium in a medium under aerobic conditions₆Culturing microorganism with production ability, and obtaining the vitamin B from the culture solution₆。

As related to vitamin B₆To investigate the synthesis, International publication No. 2004/035010 discloses production of vitamin B by culturing an organism in which the activity of at least one of yaaD and yaaE of Bacillus subtilis is enhanced as compared with that of a parent organism₆The method of (1). Journal of Molecular Catalysis B: enzymic, 2010, vol.67, p.104-110 disclose pyridoxamine-pyruvate transaminase (PPAT) engineered to be able to utilize L-glutamate by sequence modification. It is described that pyridoxamine can be produced by incubating Escherichia coli expressing PPAT having the modified sequence in the presence of a saturating amount of pyridoxal.

Disclosure of Invention

Problems to be solved by the invention

However, high reaction yields are not obtained by pyridoxamine synthesis based on chemical synthesis. For example, in the method described in international publication No. 2006/066806, pyridoxamine dihydrochloride is synthesized through a plurality of chemical reactions, and thus the reaction yield is lowered. Further, the method described in International publication No. 2005/077902 produces a dimer or trimer of pyridoxamine, and the reaction yield is not high.

On the other hand, in the method described in international publication No. 2007/142222, which is a method using a specific type of microorganism, the efficiency of conversion from pyridoxal to pyridoxamine varies greatly depending on the type of microorganism, and is not high as a whole. In addition, in the experiment for culturing Acremonium fusiforme in the presence of pyridoxine described in International publication No. 2007/142222, most of the products were pyridoxal rather than pyridoxamine. Further, vitamin B is produced as described in Japanese patent application laid-open No. 9-107985₆Almost all are pyridoxine (pyridoxine), and pyridoxamine is rarely produced.

International publication No. 2004/035010 describes that vitamin B is obtained by culturing a microorganism in which yaaD and yaaE genes are expressed at a high level in a medium₆However, the product is a mixture of pyridoxine, pyridoxal, pyridoxamine phosphate, etc., and pyridoxamine cannot be selectively obtained.

As described above, chemical synthesis of pyridoxamine or a salt thereof (for example, international publication nos. 2006/066806 and 2005/077902) has not achieved a high yield through a plurality of reaction and purification steps. Further, there are microorganisms having pyridoxamine-synthesizing ability (for example, International publication No. 2007/142222 and Japanese patent application laid-open No. 9-107985), but it is newly found that such microorganisms are labor-consuming and that vitamin B has not yet been found₆Also, pyridoxamine or a salt thereof is selectively synthesized in the family, and high production efficiency of pyridoxamine is not obtained. In addition, by screening a specific microorganism species from naturally occurring microorganism species in this way, it is not possible to obtain any enzyme or gene for efficiently producing vitamin B₆Is important such molecular biological information.

In addition, it is also effective against vitamin B₆Production of (2) is carried out using a recombinant microorganism prepared by a gene recombination technique (International publication No. 2004/035010 and Journal of Molecular Catalysis B: Enzymatic, 2010, vol.67, p.104-110), but in the production of a substance, vitamin B is obtained by using a usual medium without selection₆(International publication No. 2004/035010), or pyridoxamine (Journal of Molecular Catalysis B: Enzymatic, 2010, vol.67, p.104-110) obtained by using an expensive raw material pyridoxal in a saturated amount, has not been able to produce pyridoxamine or a salt thereof at low cost and with good selectivity.

In view of the above-described circumstances, the present disclosure provides a recombinant microorganism capable of producing pyridoxamine or a salt thereof from pyridoxine or a salt thereof with high production efficiency at low cost, and a method for producing pyridoxamine or a salt thereof from pyridoxine or a salt thereof with high production efficiency using the recombinant microorganism at low cost.

Means for solving the problems

The present disclosure includes the following aspects.

<1>

A recombinant microorganism having a gene coding for pyridoxol oxidase and a gene coding for pyridoxamine synthase having an enzyme activity to synthesize pyridoxamine from pyridoxal,

the gene encoding pyridoxine oxidase and the gene encoding pyridoxamine synthase are each a gene introduced extracellularly or a gene endogenous to the cell but having enhanced expression.

<2>

The recombinant microorganism according to <1>, wherein the pyridoxamine synthase is pyridoxamine-pyruvate transaminase, pyridoxamine-oxaloacetate transaminase, aspartate transaminase or pyridoxamine phosphate transaminase.

<3>

The recombinant microorganism according to <1> or <2>, wherein the pyridoxol oxidase is represented by the enzyme number EC1.1.3.12.

<4>

The recombinant microorganism according to any one of <1> to <3>, wherein the gene encoding pyridoxol oxidase is derived from Microbacterium flavum.

<5>

The recombinant microorganism according to any one of <1> to <4>, wherein the gene encoding pyridoxol oxidase is

(a) A nucleotide sequence having sequence number 5; or

(b) A nucleotide sequence that hybridizes with a DNA having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO. 5 under stringent conditions and encodes a protein having pyridoxol oxidase activity; or

(c) A nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID NO. 1; or

(d) Has a nucleotide sequence encoding a protein having an amino acid sequence having 80% or more sequence identity to the amino acid sequence of SEQ ID NO. 1 and having pyridoxol oxidase activity.

<6>

The recombinant microorganism according to any one of <1> to <5>, wherein the pyridoxamine synthase has at least one of a partial amino acid sequence (c), a partial amino acid sequence (d), a partial amino acid sequence (e), a partial amino acid sequence (f), a partial amino acid sequence (g), and a partial amino acid sequence (h), and has an enzymatic activity for synthesizing pyridoxamine from pyridoxal.

(c)X₈X₉X₁₀X₁₁X₁₂X₁₃(SEQ ID NO: 39)

(X₈The representation of L, M, I or V indicates that,

X₉represents a group of a compound represented by H or Q,

X₁₀the representation G, C or A is shown as,

X₁₁represents a group of a compound represented by E or D,

X₁₂the expression P or A is shown in the specification,

X₁₃representation V, I, L or A)

(d)X₁₄X₁₅TPSGTX₁₆X₁₇(SEQ ID NO: 40)

(X₁₄Represents a group of a compound represented by H or S,

X₁₅the expression D or E is shown in the specification,

X₁₆the designation I, V or L is shown as,

X₁₇represents N or T)

(e)X₁₈DX₁₉VSX₂₀X₂₁(SEQ ID NO. 41)

(X₁₈The representation V, I or A is shown as,

X₁₉the representation A, T or the representation S,

X₂₀the representation of S, A or G is,

X₂₁representation F, W or V)

(f)X₂₂X₂₃X₂₄KCX₂₅GX₂₆X₂₇P (SEQ ID NO: 42)

(X₂₂The expression G or S is used for expressing,

X₂₃the representation P, S or A is shown as,

X₂₄the representation of N, G, S, A or Q,

X₂₅represents L or M, and is represented by,

X₂₆the representation of A, S, C or G is,

X₂₇representation P, T, S or A)

(g)X₂₈X₂₉X₃₀X₃₁SX₃₂GX₃₃X₃₄(SEQ ID NO. 43)

(X₂₈The expression G or D is shown in the specification,

X₂₉the expression V or I is shown in the specification,

X₃₀the designation V, T, A, S, M, I or L is shown as,

X₃₁the representation of F, M, L, I or V indicates that,

X₃₂the designation S, G, A, T, I, L or H is shown in,

X₃₃the representation of R, M or Q,

X₃₄representation G, R, A, D, H or K)

(h)X₃₅X₃₆RX₃₇X₃₈HMGX₃₉X₄₀A (SEQ ID NO: 44)

(X₃₅Represents a group of a compound represented by L or V,

X₃₆the designation T, I, V or L is shown as,

X₃₇the designation I, V or L is shown as,

X₃₈the expression G or S is used for expressing,

X₃₉the expression P, A or R is used for,

X₄₀representation T, V or S)

<7>

The recombinant microorganism according to any one of <1> to <6>, wherein the pyridoxamine synthase is represented by enzyme number EC2.6.1.30.

<8>

The recombinant microorganism according to any one of <1> to <7>, wherein the gene encoding pyridoxamine synthase is derived from Mesorhizobium parviflorum.

<9>

The recombinant microorganism according to any one of <1> to <8>, wherein the gene encoding pyridoxamine synthase

(a) Having the nucleotide sequence of any one of SEQ ID NO. 6 and SEQ ID NO. 25 to SEQ ID NO. 31, or

Has a region from the 18 th nucleotide to the 3 'end in the nucleotide sequence of SEQ ID NO. 10 or a region from the 18 th nucleotide to the 3' end in any of the nucleotide sequences of SEQ ID NO. 32 to SEQ ID NO. 38; or

(b) A nucleotide sequence that hybridizes under stringent conditions to a DNA having a nucleotide sequence complementary to the nucleotide sequence of any one of SEQ ID NO. 6 and SEQ ID NO. 25 to SEQ ID NO. 31, or a nucleotide sequence complementary to a region from the 18 th nucleotide to the 3 'end in the nucleotide sequence of SEQ ID NO. 10 or a region from the 18 th nucleotide to the 3' end in the nucleotide sequence of any one of SEQ ID NO. 32 to SEQ ID NO. 38, and encodes a protein having an enzymatic activity of synthesizing pyridoxamine from pyridoxal; or

(c) A nucleotide sequence encoding a protein having the amino acid sequence of any one of SEQ ID NO.2 and SEQ ID NO. 18 to SEQ ID NO. 24; or

(d) Has a nucleotide sequence encoding a protein having an amino acid sequence having 80% or more sequence identity to at least one of the amino acid sequences of SEQ ID NO.2 and SEQ ID NO. 18 to SEQ ID NO. 24 and having an enzymatic activity of synthesizing pyridoxamine from pyridoxal.

<10>

The recombinant microorganism according to any one of <1> to <9>, wherein the gene encoding pyridoxamine synthase

(a) A nucleotide sequence having sequence number 6; or

(b) A nucleotide sequence that hybridizes under stringent conditions to a DNA having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO. 6 and encodes a protein having an enzymatic activity of synthesizing pyridoxamine from pyridoxal; or

(c) A nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID NO. 2; or

(d) Has a nucleotide sequence encoding a protein having an amino acid sequence having 80% or more sequence identity to the amino acid sequence of SEQ ID NO.2 and having an enzymatic activity of synthesizing pyridoxamine from pyridoxal.

<11>

The recombinant microorganism according to any one of <1> to <5>, wherein the pyridoxamine synthase is represented by enzyme No. EC2.6.1.31 or EC2.6.1.1.

<12>

The recombinant microorganism according to any one of <1> to <5> and <11>, wherein the gene encoding pyridoxamine synthase is derived from Escherichia coli.

<13>

The recombinant microorganism according to any one of <1> to <5> and <11> to <12>, wherein the gene encoding pyridoxamine synthase is

(a) A nucleotide sequence having sequence number 8; or

(b) A nucleotide sequence that hybridizes under stringent conditions to a DNA having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO. 8 and encodes a protein having an enzymatic activity of synthesizing pyridoxamine from pyridoxal; or

(c) A nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID NO. 4; or

(d) Has a nucleotide sequence encoding a protein having an amino acid sequence having 80% or more sequence identity to the amino acid sequence of SEQ ID NO. 4 and having an enzymatic activity of synthesizing pyridoxamine from pyridoxal.

<14>

The recombinant microorganism according to any one of <1> to <13>, which further has a gene encoding a hydrogen peroxide catabolic enzyme having an enzyme activity of generating oxygen from hydrogen peroxide.

<15>

The recombinant microorganism according to <14>, wherein the gene encoding a hydrogen peroxide catabolic enzyme is introduced extracellularly or is endogenous to the bacterial cell and expression thereof is enhanced.

<16>

The recombinant microorganism according to <14> or <15>, wherein the hydrogen peroxide catabolic enzyme is represented by the enzyme number EC 1.11.1.6.

<17>

The recombinant microorganism according to any one of <14> to <16>, wherein the gene encoding a hydrogen peroxide catabolic enzyme

(a) A nucleotide sequence having sequence number 7; or

(b) Hybridizing with a DNA having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO. 7 under stringent conditions and having an enzyme activity of generating oxygen from hydrogen peroxide; or

(c) A nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID NO. 3; or

(d) Has a nucleotide sequence encoding a protein having an amino acid sequence having 80% or more sequence identity to the amino acid sequence of SEQ ID NO. 3 and having an enzymatic activity of generating oxygen from hydrogen peroxide.

<18>

The recombinant microorganism according to any one of <1> to <17>, which is a recombinant Escherichia coli.

<19>

A process for producing pyridoxamine or a salt thereof, wherein the recombinant microorganism or the culture of the recombinant microorganism or the treated product of the recombinant microorganism or the culture of any one of <1> to <18> is contacted with pyridoxine or a salt thereof to produce pyridoxamine or a salt thereof in the presence of oxygen.

<20>

The process according to <19>, wherein the recombinant microorganism or the culture of the recombinant microorganism, or the treated product of the recombinant microorganism or the culture contains the pyridoxol oxidase and the pyridoxamine synthase.

<21>

The production method according to <20>, wherein the recombinant microorganism or the culture of the recombinant microorganism, or the treated product of the recombinant microorganism or the culture further comprises a hydrogen peroxide catabolic enzyme.

<22>

The production method according to any one of <19> to <21>, wherein the treated product of the recombinant microorganism or the culture is a treated product obtained by at least one treatment selected from the group consisting of a heating treatment, a cooling treatment, mechanical disruption of cells, an ultrasonic treatment, a freeze-thaw treatment, a drying treatment, a pressurization or depressurization treatment, an osmotic pressure treatment, autodigestion of cells, a surfactant treatment, an enzyme treatment, a cell separation treatment, a purification treatment, and an extraction treatment.

<23>

The production method according to any one of <19> to <22>, which includes either one or both of the following (a) and (B):

(A) continuously adding or adding in divided portions pyridoxine or a salt thereof to a solution containing the recombinant microorganism or a culture of the recombinant microorganism, or a treatment of the recombinant microorganism or the culture;

(B) in a solution containing the recombinant microorganism or a culture of the recombinant microorganism, or a treated matter of the recombinant microorganism or the culture, the molar concentration of amino acids consumed by the pyridoxamine synthase is controlled to be 1-fold or more relative to the molar concentration of pyridoxine or a salt thereof.

<24>

The process according to <23>, wherein the amino acid consumed by the pyridoxamine synthase is L-alanine, D-alanine, L-glutamic acid or D-glutamic acid.

Effects of the invention

According to the present disclosure, a recombinant microorganism capable of producing pyridoxamine or a salt thereof from pyridoxine or a salt thereof with high production efficiency and at low cost, and a method for producing pyridoxamine or a salt thereof from pyridoxine or a salt thereof with high production efficiency and at low cost using the recombinant microorganism can be provided.

Drawings

[ FIG. 1] shows the results of determination of pyridoxine hydrochloride concentration, pyridoxal hydrochloride concentration, and pyridoxamine dihydrochloride concentration in test No. 7.

[ FIG. 2] shows the results of determination of pyridoxine hydrochloride concentration, pyridoxal hydrochloride concentration, and pyridoxamine dihydrochloride concentration in test 8.

[ FIG. 3] shows the measurement results of the pyridoxine hydrochloride concentration, the pyridoxal hydrochloride concentration, and the pyridoxamine dihydrochloride concentration in test 9.

FIG. 4-1 shows an alignment of SEQ ID NO.2 and SEQ ID NO. 18 to SEQ ID NO. 24.

FIG. 4-2 shows an alignment of SEQ ID NO.2 and SEQ ID NO. 18 to SEQ ID NO. 24.

Detailed Description

The present disclosure provides a recombinant microorganism (hereinafter referred to as a recombinant microorganism according to the present disclosure) having a gene encoding pyridoxol oxidase having an enzymatic activity of synthesizing pyridoxamine from pyridoxal and a gene encoding pyridoxamine synthase each of which is a gene introduced extracellularly or a gene endogenous to the cell and having enhanced expression. In the present disclosure, the term "introduced" means that the cells are introduced so as to be expressed in the cells.

Heretofore, a method for producing pyridoxamine or a salt thereof from pyridoxine or a salt thereof with high production efficiency and at low cost, by either chemical or biological methods, has not been known. The structure of pyridoxamine is shown below.

[ chemical formula 1]

However, the inventors of the present application have found that, surprisingly, pyridoxamine or a salt thereof can be produced from pyridoxine or a salt thereof with high production efficiency and at low cost by using a recombinant microorganism or a culture of the recombinant microorganism or a treated product of the recombinant microorganism or the culture having the above-described configuration. The reason for this is not necessarily clear, but it is presumed that the 2 enzymes are introduced from outside the cell body or are endogenous to the cell body but the expression thereof is enhanced, and thus the enzymes can be expressed at a high expression level based on the introduced or enhanced genes, whereby the balance of the respective reactions in the process of producing pyridoxamine or a salt thereof from pyridoxine or a salt thereof, by-products and the like produced in the process, the amount of raw materials consumed, and the like become favorable for producing pyridoxamine or a salt thereof from pyridoxine or a salt thereof due to the synergistic action of the 2 enzymes.

Furthermore, it is presumed that pyridoxal is temporarily produced as an intermediate product when the recombinant microorganism or the culture of the recombinant microorganism or the treated product of the recombinant microorganism or the culture according to the present disclosure is used. However, pyridoxal has high reactivity due to being an aldehyde, and therefore reacts spontaneously with an amino donor such as alanine under a pH condition near neutral without a catalyst such as an enzyme to produce pyridoxamine and a by-product. Thus, it is considered that the production efficiency of pyridoxamine is not increased because pyridoxal is produced as a by-product in a spontaneous reaction and pyridoxamine is not produced at a high selectivity. On the other hand, when the recombinant microorganism or the culture of the recombinant microorganism or the treated product of the recombinant microorganism or the culture according to the present disclosure is used, pyridoxal as an intermediate is rapidly converted to pyridoxamine by pyridoxamine synthase, accumulation of pyridoxal is suppressed, and production of byproducts is also suppressed. This enables high pyridoxamine production efficiency to be achieved.

Such synergistic effects of the 2 enzymes have not been found so far, and thus pyridoxamine or a salt thereof can be produced with high production efficiency by using pyridoxine or a salt thereof without performing a reaction of multiple steps one by one as in the case of a chemical synthesis method. Moreover, by using the recombinant microorganism to which the present disclosure relates, it is possible to avoid the production of vitamin B in a large amount as a by-product₆Family of other substances. Pyridoxine is a raw material that is industrially available at a lower cost than pyridoxal, and pyridoxamine or a salt thereof can be produced at a lower cost because pyridoxine can be used as a starting material.

< pyridoxine oxidase >

Pyridoxine oxidase is an enzyme also known as pyridoxine-4-oxidase. The pyridoxine oxidase may be the enzyme represented by enzyme number EC1.1.3.12. Pyridoxine oxidase is an enzyme having an enzymatic activity that catalyzes a reaction of oxidizing pyridoxine with oxygen to convert it into pyridoxal. It should be noted that pyridoxal and pyridoxine may exist as salts depending on the surrounding environment, but for the sake of simplicity, the description of the activity of the enzyme in the present disclosure is omitted and the description is omitted. Pyridoxine oxidase consumes oxygen to produce hydrogen peroxide when it oxidizes pyridoxine. Since hydrogen peroxide harmful to living organisms is produced, it has been considered that high yield cannot be achieved even when pyridoxine oxidase is used for producing other substances from pyridoxine. However, when the recombinant microorganism according to the present disclosure is used, an unexpected effect is obtained that production of pyridoxamine or a salt thereof using pyridoxine or a salt thereof as a raw material can be achieved with high production efficiency.

The pyridoxol oxidase of EC1.1.3.12 can be, for example, a pyridoxol oxidase derived from Enterobacter cloacae (Enterobacter cloacae), Mesorhizobium loti (Mesorhizobium loti), Microbacterium luteum (Microbacterium luteolus), Microbacterium anthropi (Ochrobactrum anthropi), Pseudomonas (Pseudomonas) strain MA-1, and the like. The pyridoxol oxidase of Microbacterium flavum has the amino acid sequence of SEQ ID No. 1.

< pyridoxamine synthase >

The pyridoxamine synthase used in the present disclosure refers to any enzyme having an enzymatic activity of synthesizing pyridoxamine from pyridoxal. It should be noted that pyridoxal and pyridoxamine may exist as salts depending on the surrounding environment, but for the sake of simplicity, the description of the activity of the enzyme in the present disclosure is omitted and the description is omitted. Examples of the pyridoxamine synthase include pyridoxamine-pyruvate transaminase represented by the enzyme number EC2.6.1.30, pyridoxamine-oxaloacetate transaminase represented by EC2.6.1.31, aspartate transaminase represented by EC2.6.1.1, and pyridoxamine phosphate transaminase represented by EC2.6.1.54.

The aspartate aminotransferase represented by EC2.6.1.1 is a holoenzyme having pyridoxal phosphate as a coenzyme, and has an enzymatic activity of transferring an amino group of aspartate to 2-oxoglutarate to produce glutamate and oxaloacetate. It is known that this aspartate aminotransferase transfers an amino group OF glutamic acid or aspartic acid to pyridoxal in the state OF apoenzyme (apoenzyme) which is not bound to pyridoxal phosphate to synthesize pyridoxamine (JOURNAL OF BIOLOGICAL CHEMISTRY, 1/1962, Vol.237, No.l, p.127-132). That is, the apoenzyme form of aspartate aminotransferase represented by EC2.6.1.1 is pyridoxamine-oxaloacetate aminotransferase of EC2.6.1.31. Therefore, aspartate aminotransferase exists as apoenzyme in the absence or in a small amount of pyridoxal phosphate as a coenzyme to synthesize pyridoxamine.

Pyridoxamine synthase has an enzymatic activity of partially oxidizing (═ O) an amino group of a specific amino acid and transferring the amino group to produce pyridoxamine or a salt thereof when pyridoxamine or a salt thereof is synthesized from pyridoxal or a salt thereof. For example, pyridoxamine-pyruvate transaminase can utilize any of L-alanine and D-alanine, pyridoxamine-oxaloacetate transaminase and apoenzyme states aspartate transaminase can utilize any of D-aspartate, L-aspartate, D-glutamate, and L-glutamate, and pyridoxamine phosphate transaminase can utilize D-glutamate.

The aforementioned pyridoxamine-pyruvate transaminase can also be derived, for example, from a microorganism belonging to the phylum Proteobacteria, Actinomycetes, Spirochaeta or Mycoplasma. The pyridoxamine-pyruvate transaminase may be a pyridoxamine-pyruvate transaminase derived from, for example, Leptosphaeria maculans (Mesorhizobium loti), Ochrobactrum anthropi (Ochrobactrum anthropi), Pseudomonas (Pseudomonas) such as Pseudomonas strain MA-1, and the like. For example, pyridoxamine-pyruvate transaminase of bradyrhizobium in crowtoe has the amino acid sequence of SEQ ID No. 2.

Furthermore, the pyridoxamine-pyruvate transaminase may also be, for example, a pyridoxamine-pyruvate transaminase (having an amino acid sequence of seq id No. 18) derived from bradyrhizobium strain YR577(Mesorhizobium sp.yr577), a pyridoxamine-pyruvate transaminase (having an amino acid sequence of seq id No. 19) derived from pseudoamino acid salicylate oxydans (pseudosalicylates), a pyridoxamine-pyruvate transaminase (having an amino acid sequence of seq id No. 20) derived from bacillus shore (Bauldia littoralis), a pyridoxamine-pyruvate transaminase (having an amino acid sequence of seq id No. 21) derived from staphylinicoccus antimoniatus (skermanasetibiiisistens), a pyridoxamine-pyruvate transaminase (having an amino acid sequence of seq id No. 22) derived from Rhizobium strain AC44/96(Rhizobium sp.ac44/96), a pyridoxamine-pyruvate transaminase (having an amino acid sequence of seq id No. 23) derived from Erwinia Lepidium), pyridoxamine-pyruvate transaminase (having the amino acid sequence of SEQ ID NO: 24) derived from Raffinium Ginseng (Herbicoux ginsengi).

The pyridoxamine-oxaloacetate transaminase can be a pyridoxamine-oxaloacetate transaminase derived from, for example, Escherichia coli (Escherichia coli), Oryctolagus cuniculus, Rattus norvegicus (Rattus norvegicus), or the like. For example, the pyridoxamine-oxaloacetate transaminase of Escherichia coli has the amino acid sequence of SEQ ID NO. 4. The above-mentioned aspartate aminotransferase may be an aspartate aminotransferase derived from, for example, Escherichia coli (Escherichia coli), Trichoderma viride (Trichoderma viride), or the like. The pyridoxamine phosphate transaminase may be derived from, for example, Clostridium butyricum (Clostridium butyricum).

< Hydrogen peroxide catabolic enzyme >

The recombinant microorganism to which the present disclosure relates may further have a gene encoding a hydrogen peroxide catabolic enzyme having an enzymatic activity of decomposing hydrogen peroxide. The hydrogen peroxide catabolic enzyme used in the present disclosure is any enzyme having an enzymatic activity of decomposing hydrogen peroxide generated when pyridoxine is oxidized by the pyridoxine oxidase. By further including a gene encoding a hydrogen peroxide catabolic enzyme, accumulation of hydrogen peroxide harmful to living organisms and enzyme activity can be further reduced, which is advantageous in further improving production efficiency of pyridoxamine or a salt thereof and prolonging the reaction duration. Further, the hydrogen peroxide catabolic enzyme may have an enzyme activity of regenerating oxygen. The presence of the enzyme activity for regenerating oxygen is advantageous in that the concentration of oxygen in the reaction system can be increased particularly when producing pyridoxamine or a salt thereof in a low-oxygen environment, and the production efficiency of pyridoxamine or a salt thereof can be further improved.

Examples of such a hydrogen peroxide catabolic enzyme include enzymes represented by enzyme numbers (EC)1.11.1.1, 1.11.1.2, 1.11.1.3, 1.11.1.5, 1.11.1.6, 1.11.1.7, 1.11.1.8, 1.11.1.9, 1.11.1.10, 1.11.1.11, 1.11.1.13, 1.11.1.14, 1.11.1.16, 1.11.1.17, 1.11.1.18, 1.11.1.19, 1.11.1.21, or 1.11.1.23. Among them, in view of oxygen regeneration ability, catalase represented by the enzyme number ec1.11.1.6 and catalase-peroxidase represented by EC1.11.1.21 are preferable, and catalase represented by the enzyme number ec1.11.1.6 is more preferable.

The hydrogen peroxide catabolic enzyme may be a catalase derived from, for example, Listeria seelii (Listeria seeligeri), Escherichia coli (Escherichia coli), Saccharomyces cerevisiae (Saccharomyces cerevisiae), and the like. Alternatively, the hydrogen peroxide catabolic enzyme may be a catalase-peroxidase derived from, for example, Escherichia coli (Escherichia coli). For example, Listeria seelerii catalase has the amino acid sequence of SEQ ID No. 3.

The pyridoxine oxidase, the pyridoxamine synthase, and the hydrogen peroxide catabolic enzyme may be proteins that have known amino acid sequences having the enzymatic activity (e.g., amino acid sequences encoded by genes naturally occurring in the organism, such as those of the above-exemplified microorganisms) without modification, or may be proteins having amino acid sequences obtained by sequence modification of such amino acid sequences within a range that does not lose the enzymatic activity (the above-mentioned enzymatic activity). Examples of such modifications include insertion, deletion, substitution of amino acid residues, and addition of an additional amino acid residue to the N-terminus or C-terminus or both of the amino acid sequence. When one or more of insertion, deletion and substitution of amino acid residues are present, the insertion, deletion and substitution may be, for example, 1 to 30 amino acid residues, or 1 to 20 amino acid residues, or 1 to 10 amino acid residues, or 1 to 5 amino acid residues, respectively, when present, and the total number of insertion, deletion and substitution of amino acid residues may be, for example, 1 to 50 amino acid residues, or 1 to 30 amino acid residues, or 1 to 10 amino acid residues, or 1 to 5 amino acid residues. In addition, as the number of amino acid residues added to the terminal, each terminal may be, for example, 1 to 50 amino acid residues, or 1 to 30 amino acid residues, or 1 to 10 amino acid residues, or 1 to 5 amino acid residues, when present. Such additional amino acid residues may form a signal sequence for secretion to the outside of the cell, etc. Examples of the signal sequence include an OmpA signal sequence of Escherichia coli.

Alternatively, each enzyme may be a protein having an amino acid sequence itself known to have the enzyme activity (for example, an amino acid sequence encoded by a gene naturally occurring in an organism); or a protein having an amino acid sequence having 80% or more, or 85% or more, or 90% or more, or 95% or more sequence identity to a known amino acid sequence having the enzyme activity (e.g., an amino acid sequence encoded by a gene naturally present in an organism), and having a desired enzyme activity (the above-mentioned enzyme activity). Here, sequence identity can be evaluated based on default parameters using, for example, BLAST (registered trademark) program.

For example, the pyridoxol oxidase may be a protein having the amino acid sequence of SEQ ID NO. 1, or a protein having an amino acid sequence obtained by substituting, deleting, inserting, or adding at least one of an amino acid residue to the amino acid sequence of SEQ ID NO. 1, or an additional amino acid residue to the N-terminus or the C-terminus or both of the amino acid sequence. Examples of the degree of substitution, deletion, insertion, and addition of an additional amino acid residue to the N-terminus, the C-terminus, or both of the amino acid sequences are as described above.

Alternatively, the pyridoxol oxidase may be a protein having the amino acid sequence of SEQ ID NO. 1; or a protein having an amino acid sequence having, for example, 80% or more, or 85% or more, or 90% or more, or 95% or more sequence identity with the amino acid sequence of SEQ ID NO. 1.

When a protein having an amino acid sequence similar to that of SEQ ID NO. 1 is used, the protein should have an activity as pyridoxol oxidase. Pyridoxine oxidase activity can be measured, for example, by the following method: adding a protein to be tested to an aqueous solution of pyridoxine as a substrate in the presence of oxygen, and quantifying the produced pyridoxal by high performance liquid chromatography; alternatively, Schiff bases are formed between the produced pyridoxal and an amine such as trishydroxymethylaminomethane, and the Schiff bases are quantified by measuring the absorbance at 415nm or the like.

Alternatively, the pyridoxamine synthase may be a protein having the amino acid sequence of any one of SEQ ID NO.2 and SEQ ID NO. 18 to SEQ ID NO. 24; or a protein having an amino acid sequence having, for example, 80% or more, or 85% or more, or 90% or more, or 95% or more sequence identity to at least one of the amino acid sequence of SEQ ID NO.2, the amino acid sequence of SEQ ID NO. 18, the amino acid sequence of SEQ ID NO. 19, the amino acid sequence of SEQ ID NO. 20, the amino acid sequence of SEQ ID NO. 21, the amino acid sequence of SEQ ID NO. 22, the amino acid sequence of SEQ ID NO. 23, and the amino acid sequence of SEQ ID NO. 24. The protein should also have activity as a pyridoxamine synthase. In this case, the enzymatic activity for synthesizing pyridoxamine from pyridoxal can be measured, for example, by the following method: a protein to be tested is added to an aqueous solution containing pyridoxal and L-alanine as substrates, and the amount of pyridoxamine produced is quantified by high performance liquid chromatography or the like.

Alternatively, the pyridoxamine synthase may be a pyridoxamine synthase having an enzymatic activity for synthesizing pyridoxamine from pyridoxal, which pyridoxamine synthase contains at least one of the following partial amino acid sequence (c), partial amino acid sequence (d), partial amino acid sequence (e), partial amino acid sequence (f), partial amino acid sequence (g) and partial amino acid sequence (h). In this case, the enzymatic activity for synthesizing pyridoxamine from pyridoxal can be measured, for example, by the following method: a protein to be tested is added to an aqueous solution containing pyridoxal and L-alanine as substrates, and the amount of pyridoxamine produced is quantified by high performance liquid chromatography or the like.

(c)X₈X₉X₁₀X₁₁X₁₂X₁₃(SEQ ID NO: 39)

(X₈The representation of L, M, I or V indicates that,

X₉represents a group of a compound represented by H or Q,

X₁₀the representation G, C or A is shown as,

X₁₁represents a group of a compound represented by E or D,

X₁₂the expression P or A is shown in the specification,

X₁₃representation V, I, L or A)

(d)X₁₄X₁₅TPSGTX₁₆X₁₇(SEQ ID NO: 40)

(X₁₄Represents a group of a compound represented by H or S,

X₁₅the expression D or E is shown in the specification,

X₁₆the designation I, V or L is shown as,

X₁₇represents N or T)

(e)X₁₈DX₁₉VSX₂₀X₂₁(SEQ ID NO. 41)

(X₁₈The representation V, I or A is shown as,

X₁₉the representation A, T or the representation S,

X₂₀the representation of S, A or G is,

X₂₁representation F, W or V)

(f)X₂₂X₂₃X₂₄KCX₂₅GX₂₆X₂₇P (SEQ ID NO: 42)

(X₂₂The expression G or S is used for expressing,

X₂₃the representation P, S or A is shown as,

X₂₄the representation of N, G, S, A or Q,

X₂₅represents L or M, and is represented by,

X₂₆the representation of A, S, C or G is,

X₂₇representation P, T, S or A)

(g)X₂₈X₂₉X₃₀X₃₁SX₃₂GX₃₃X₃₄(SEQ ID NO. 43)

(X₂₈The expression G or D is shown in the specification,

X₂₉the expression V or I is shown in the specification,

X₃₀the designation V, T, A, S, M, I or L is shown as,

X₃₁the representation of F, M, L, I or V indicates that,

X₃₂the designation S, G, A, T, I, L or H is shown in,

X₃₃the representation of R, M or Q,

X₃₄representation G, R, A, D, H or K)

(h)X₃₅X₃₆RX₃₇X₃₈HMGX₃₉X₄₀A (SEQ ID NO: 44)

(X₃₅Represents a group of a compound represented by L or V,

X₃₆the designation T, I, V or L is shown as,

X₃₇the designation I, V or L is shown as,

X₃₈the expression G or S is used for expressing,

X₃₉the expression P, A or R is used for,

X₄₀representation T, V or S)

FIGS. 4-1 and 4-2 show alignments of the sequences of SEQ ID NO.2 and SEQ ID NO. 18 to SEQ ID NO. 24. In FIGS. 4-1 and 4-2, MlPPAT represents pyridoxamine-pyruvate transaminase of bradyrhizobium giganteum, MsPPAT represents pyridoxamine-pyruvate transaminase of bradyrhizobium giganteum strain YR577, PsPPAT represents pyridoxamine-pyruvate transaminase of Pseudoaminobacter oxysalicylate, BlPPAT represents pyridoxamine-pyruvate transaminase of Bacillus lithamii, SsPT represents pyridoxamine-pyruvate transaminase against Pseudomonas antimonicola, RsP represents pyridoxamine-pyruvate transaminase of Rhizobium strain AC44/96, EtPPAT represents pyridoxamine-pyruvate transaminase of Salmonella torpedo, and HgPPAT represents pyridoxamine-pyruvate transaminase of Ramopsis. The partial amino acid sequence (c) corresponds to amino acid residues that correspond in alignment to the amino acid residues at positions 65 to 70 from the N-terminus of pyridoxamine-pyruvate transaminase of bradyrhizobium giganteum, the partial amino acid sequence (d) corresponds to amino acid residues that correspond in alignment to the amino acid residues at positions 144 to 152 from the N-terminus of pyridoxamine-pyruvate transaminase of bradyrhizobium giganteum, the partial amino acid sequence (e) corresponds to amino acid residues that correspond in alignment to the amino acid residues at positions 170 to 176 from the N-terminus of pyridoxamine-pyruvate transaminase of bradyrhizobium giganteum, the partial amino acid sequence (f) corresponds to amino acid residues that correspond in alignment to the amino acid residues at positions 194 to 203 from the N-terminus of pyridoxamine-pyruvate transaminase of bradyrhizobium giganteum, the partial amino acid sequence (g) corresponds to amino acid residues corresponding in alignment to amino acid residues 329 rd to 337 th from the N-terminus of pyridoxamine-pyruvate transaminase of bradyrhizobium japonicum in Lotus corniculatum, and the partial amino acid sequence (h) corresponds to amino acid residues corresponding in alignment to amino acid residues 343 rd to 353 rd from the N-terminus of pyridoxamine-pyruvate transaminase of bradyrhizobium japonicum in Lotus corniculatum. The partial amino acid sequence (c) is preferably present in the region of amino acid residues 55 to 80 from the N-terminus of the protein, more preferably in the region of amino acid residues 56 to 75 from the N-terminus. The partial amino acid sequence (d) is preferably present in the region of amino acid residues 134 to 162 from the N-terminus of the protein, more preferably in the region of amino acid residues 139 to 157 from the N-terminus. The partial amino acid sequence (e) is preferably present in the region of amino acid residues 160 to 186 from the N-terminus of the protein, more preferably in the region of amino acid residues 165 to 181 from the N-terminus. The partial amino acid sequence (f) is preferably present in the region from the 184 th to 213 th amino acid residues from the N-terminus of the protein, more preferably in the region from the 189 th to 208 th amino acid residues from the N-terminus. The partial amino acid sequence (g) is preferably present in the region of amino acid residues 319 to 347 from the N-terminus of the protein, more preferably in the region of amino acid residues 324 to 342 from the N-terminus. The partial amino acid sequence (h) is preferably present in the region of amino acid residues from 333 rd to 363 th from the N-terminus of the protein, more preferably in the region of amino acid residues from 338 th to 358 th from the N-terminus. In the present disclosure, alignment of sequences with each other can be performed based on default parameters using, for example, the BLAST (registered trademark) program.

In the present disclosure, "an amino acid residue corresponding to the X-th amino acid residue from the N-terminus of the enzyme a" in the amino acid sequence of the enzyme B refers to an amino acid residue in the amino acid sequence of the enzyme B corresponding to the X-th amino acid residue from the N-terminus of the amino acid sequence of the enzyme a when the amino acid sequence of the enzyme a and the amino acid sequence of the enzyme B are aligned.

As is clear from FIGS. 4-1 and 4-2, the partial amino acid sequence (c), the partial amino acid sequence (d), the partial amino acid sequence (e), the partial amino acid sequence (f), the partial amino acid sequence (g) and the partial amino acid sequence (h) are regions highly conserved among a group of pyridoxamine-pyruvate transaminase. Therefore, it is considered that a mutation of pyridoxamine-pyruvate transaminase, which includes at least one of the partial amino acid sequence (c) to the partial amino acid sequence (h), is highly likely to function as a pyridoxamine synthase in the present disclosure. Further, it is considered that an amino acid residue corresponding to the 197 th lysine residue from the N-terminus of pyridoxal in Leptospermum would be important for binding to pyridoxal, an amino acid residue corresponding to the 68 th glutamic acid residue from the N-terminus would be important for catalytic activity, an amino acid residue corresponding to the 171 th aspartic acid residue from the N-terminus and an amino acid residue corresponding to the 146 th threonine residue from the N-terminus assist binding to pyridoxal, an amino acid residue corresponding to the 336 th arginine residue from the N-terminus and an amino acid residue corresponding to the 345 th arginine residue from the N-terminus would be important for amino acid recognition (Journal of Biological Chemistry, 2008, vol.283, No. 2pp112-1120), is a functionally important residue in pyridoxine synthetases. It is considered that, if these residues are retained, the pyridoxol synthase in the present disclosure is likely to function. However, the amino acid residue corresponding to the 68 th glutamic acid residue from the N-terminus of pyridoxamine-pyruvate transaminase of bradyrhizobium in Lotus corniculatus may be aspartic acid in addition to glutamic acid. The amino acid residue corresponding to the 336 th arginine residue from the N-terminus of pyridoxamine-pyruvate transaminase of bradyrhizobium in crowtoe may be methionine or glutamine in addition to arginine.

Another expression of a region containing an amino acid residue corresponding to the alignment of the 68 th glutamic acid residue from the N-terminus of pyridoxamine-pyruvate transaminase of bradyrhizobium giganteum includes the following partial amino acid sequence (c-1). The pyridoxamine synthase may also comprise a partial amino acid sequence (c-1) in place of the partial amino acid sequence (c).

(c-1)X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉(SEQ ID NO. 45)

X₁The representation V, L, I or M is a representation of,

X₂the representation of I, L or V indicates that,

X₃the representation of L, M, I or V indicates that,

X₄represents a group of a compound represented by H or Q,

X₅the representation G, C or A is shown as,

X₆represents a group of a compound represented by E or D,

X₇the expression P or A is shown in the specification,

X₈the designation V, I, A or L is shown as,

X₉the representation of L, M, P or V indicates that,

X₁₀the compound is represented by the formula G or A,

X₁₁the structural formula of the compound is shown as L or I,

X₁₂represents a group of a compound represented by E or Q,

X₁₃is represented by the formula A or G,

X₁₄represents a group A or a group V,

X₁₅represents a group A or a group L,

X₁₆the designation A, L, H or Y is,

X₁₇the representation S, G or A is shown as,

X₁₈the representation L, F, V or A is shown as,

X₁₉indicating I, F, V or L.

The partial amino acid sequence (c-1) corresponds to amino acid residues corresponding to the amino acid residues from the 63 rd to the 81 th positions from the N-terminus of pyridoxamine-pyruvate transaminase of bradyrhizobium sieboldii in Lotus corniculatus in alignment. The partial amino acid sequence (c-1) is preferably present in the region of amino acid residues from position 53 to position 91 of the N-terminus of the protein, more preferably in the region of amino acid residues from position 58 to position 86 of the N-terminus.

Another expression of a region containing an amino acid residue corresponding to the alignment of the 146 th threonine residue from the N-terminus of pyridoxamine-pyruvate transaminase of bradyrhizobium giganteum includes the following partial amino acid sequence (d-1). The pyridoxamine synthase may also comprise a partial amino acid sequence (d-1) in place of the partial amino acid sequence (d).

(d-1)X₁X₂X₃X₄X₅X₆X₇X₈TPSGTX₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆(SEQ ID NO. 46)

X₁The representation V, I, L or M is a representation of,

X₂the expression V or I is shown in the specification,

X₃the designation S, A, V, C or F is,

X₄the representation V, I, A, L or T is shown as,

X₅represents a group of a compound represented by C or V,

X₆the representation H, N or A is shown as,

X₇represents a group of a compound represented by H or S,

X₈the expression D or E is shown in the specification,

X₉the designation I, V or L is shown as,

X₁₀the expression is N or T, and the expression is,

X₁₁the expression P or D is shown in the specification,

X₁₂the representation I, V, L or A is shown as,

X₁₃the representation of D, N, E, A, G, Q, V, R or P is shown,

X₁₄the representation A, E, Q or D is shown as,

X₁₅the compound is represented by I or L,

X₁₆represents G or A.

The partial amino acid sequence (d-1) corresponds to amino acid residues corresponding in alignment to the 138 th to 158 th amino acid residues from the N-terminus of pyridoxamine-pyruvate transaminase of bradyrhizobium giganteum. The partial amino acid sequence (d-1) is preferably present in the region of amino acid residues from position 128 to 168 of the N-terminus of the protein, more preferably in the region of amino acid residues from position 133 to 163 of the N-terminus.

Another expression of a region containing an amino acid residue corresponding to the alignment of the 171 th aspartic acid residue from the N-terminus of pyridoxamine-pyruvate transaminase of bradyrhizobium giganteum includes the following partial amino acid sequence (e-1). The pyridoxamine synthase may also comprise a partial amino acid sequence (e-1) in place of the partial amino acid sequence (e).

(e-1)X₁X₂X₃X₄X₅X₆DX₇VSX₈X₉X₁₀X₁₁X₁₂(SEQ ID NO: 47)

X₁The representation G, D or A is shown as,

X₂the designation A, G, K, T, Q, R or E is the same as,

X₃the designation Y, N, L or F is,

X₄the representation of L, F, M or V indicates that,

X₅the designation I, L or Y is,

X₆the representation of V, A or I is,

X₇the representation A, S or T is shown as,

X₈the representation of S, A or G is,

X₉the representation of F, W or V indicates that,

X₁₀the designation G, A or L is shown as,

X₁₁the expression G or S is used for expressing,

X₁₂indicating M, V or L.

The partial amino acid sequence (e-1) corresponds to amino acid residues corresponding in alignment to amino acid residues from position 165 to position 179 from the N-terminus of pyridoxamine-pyruvate transaminase of bradyrhizobium sieboldii. The partial amino acid sequence (e-1) is preferably present in the region of amino acid residues 155 to 189 from the N-terminus of the protein, more preferably in the region of amino acid residues 160 to 184 from the N-terminus.

Another expression including an amino acid residue region corresponding to the alignment of the 197 th lysine residue from the N-terminus of pyridoxamine-pyruvate transaminase of bradyrhizobium giganteum includes the following partial amino acid sequence (f-1). The pyridoxamine synthase may also comprise a partial amino acid sequence (f-1) in place of the partial amino acid sequence (f).

(f-1)X₁X₂X₃X₄X₅X₆X₇X₈X₉KCX₁₀GX₁₁X₁₂PX₁₃X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉S (SEQ ID NO: 48)

X₁The representation of A, S, V or I is,

X₂the representation D, G or A is shown as,

X₃the representation I, L, F, V or M is a representation of,

X₄the designation Y, F, L or C is the number,

X₅the expression V or I is shown in the specification,

X₆represents a group of a or a, wherein,

X₇the expression G or S is used for expressing,

X₈the representation P, S or A is shown as,

X₉the representation N, G, S, Q or A is shown as,

X₁₀represents L or M, and is represented by,

X₁₁the representation of A, S, C or G is,

X₁₂the representation P, T, S or A is shown as,

X₁₃the representation G, A or the representation S,

X₁₄represents a group of a compound represented by L or V,

X₁₅the representation T, S or A is shown as,

X₁₆the designation M, I, L, V or F is,

X₁₇the representation of M, L, V, A or I is,

X₁₈the representation G, A, H or the representation S,

X₁₉indicating V, I or a.

The partial amino acid sequence (f-1) corresponds to amino acid residues corresponding in alignment to amino acid residues 188 to 211 from the N-terminus of pyridoxamine-pyruvate transaminase of bradyrhizobium sieboldii. The partial amino acid sequence (f-1) is preferably present in the region of amino acid residues 178 to 221 from the N-terminus of the protein, more preferably in the region of amino acid residues 183 to 216 from the N-terminus.

Another expression of a region containing an amino acid residue corresponding to the alignment of the 336 th arginine residue from the N-terminus of pyridoxamine-pyruvate transaminase of bradyrhizobium giganteum includes the following partial amino acid sequence (g-1). The pyridoxamine synthase may also comprise a partial amino acid sequence (g-1) in place of the partial amino acid sequence (g).

(g-1)X₁X₂X₃X₄X₅SX₆GX₇X₈(SEQ ID NO. 49)

X₁The representation Y, F, H or the representation S,

X₂the expression G or D is shown in the specification,

X₃the expression V or I is shown in the specification,

X₄the designation V, T, A, S, M, I or L is shown as,

X₅the representation of F, M, L, I or V indicates that,

X₆the designation S, G, A, T, I, L or H is shown in,

X₇the representation of R, M or Q,

X₈represents G, R, A, D, H or K.

The partial amino acid sequence (g-1) corresponds to amino acid residues corresponding to the 328 th to 337 th amino acid residues of pyridoxamine-pyruvate transaminase of bradyrhizobium japonicum from the N-terminus in an alignment. The partial amino acid sequence (g-1) is preferably present in the region of amino acid residues 318 to 347 from the N-terminus of the protein, more preferably in the region of amino acid residues 323 to 342 from the N-terminus.

Another expression of a region containing an amino acid residue corresponding to the alignment of the 345 th arginine residue from the N-terminus of pyridoxamine-pyruvate transaminase of bradyrhizobium giganteum includes the following partial amino acid sequence (h-1). The pyridoxamine synthase may also comprise a partial amino acid sequence (h-1) in place of the partial amino acid sequence (h).

(h-1)X₁X₂X₃X₄X₅RX₆X₇HMGX₈X₉AX₁₀X₁₁(SEQ ID NO: 50)

X₁The representation of L, Q, K, A, F, Y or W is,

X₂the representation G, N, H or D is shown as,

X₃the representation of K, R or N is,

X₄represents a group of a compound represented by L or V,

X₅the designation T, I, V or L is shown as,

X₆the designation I, V or L is shown as,

X₇the expression G or S is used for expressing,

X₈the expression P, A or R is used for,

X₉the representation T, V or the representation S,

X₁₀the representation of Q, R, E, K, H, Y or G is,

X₁₁represents P or G.

The partial amino acid sequence (h-1) corresponds to amino acid residues corresponding in alignment to amino acid residues from 340 th to 355 th positions from the N-terminus of pyridoxamine-pyruvate transaminase of bradyrhizobium in Lotus corniculatus. The partial amino acid sequence (h-1) is preferably present in the region of amino acid residues 330 to 365 from the N-terminus of the protein, more preferably in the region of amino acid residues 335 to 360 from the N-terminus.

The pyridoxamine synthase may be, for example, a protein having the amino acid sequence of SEQ ID NO.2, or a protein having an amino acid sequence obtained by substituting, deleting or inserting an amino acid residue in the amino acid sequence of SEQ ID NO.2, or adding an additional amino acid residue to the N-terminus or the C-terminus or both of the amino acid sequence. Examples of the degree of substitution, deletion, insertion, and addition of an additional amino acid residue to the N-terminus, the C-terminus, or both of the amino acid sequences are as described above.

Alternatively, the pyridoxamine synthase may be a protein having the amino acid sequence of SEQ ID NO. 2; or a protein having an amino acid sequence having, for example, 80% or more, or 85% or more, or 90% or more, or 95% or more sequence identity with the amino acid sequence of SEQ ID NO. 2.

When a protein having an amino acid sequence similar to that of SEQ ID NO.2 is used, the protein should have an activity as a pyridoxamine synthase (an enzyme activity for synthesizing pyridoxamine from pyridoxal, which is also referred to as pyridoxamine synthase activity in the present disclosure). The enzyme activity for synthesizing pyridoxamine from pyridoxal (pyridoxamine synthase activity) can be measured, for example, by the following method: a protein to be tested is added to an aqueous solution containing pyridoxal as a substrate and a desired amino acid (for example, L-alanine in the case of a protein having an amino acid sequence similar to that of SEQ ID NO. 2), and the amount of pyridoxamine produced is quantified by high performance liquid chromatography or the like.

Alternatively, the pyridoxamine synthase may be, for example, a protein having the amino acid sequence of SEQ ID NO. 4, or a protein having an amino acid sequence obtained by substituting, deleting or inserting an amino acid residue in the amino acid sequence of SEQ ID NO. 4, or adding an additional amino acid residue to the N-terminus or the C-terminus or both of the amino acid sequence. Examples of the degree of substitution, deletion, insertion, and addition of an additional amino acid residue to the N-terminus, the C-terminus, or both of the amino acid sequences are as described above.

Alternatively, the pyridoxamine synthase may be a protein having the amino acid sequence of SEQ ID NO. 4; or a protein having an amino acid sequence having, for example, 80% or more, or 85% or more, or 90% or more, or 95% or more sequence identity with the amino acid sequence of SEQ ID NO. 4.

When a protein having an amino acid sequence similar to that of SEQ ID NO. 4 is used, the protein should have an activity as a pyridoxamine synthase (an enzyme activity for synthesizing pyridoxamine from pyridoxal, which is also referred to as pyridoxamine synthase activity in the present disclosure). The enzyme that synthesizes pyridoxamine from pyridoxal (pyridoxamine synthase activity) can be measured, for example, by the following method: a protein to be tested is added to an aqueous solution containing pyridoxal as a substrate and a desired amino acid (for example, L-glutamic acid, L-aspartic acid or a salt thereof in the case of a protein having an amino acid sequence similar to that of SEQ ID NO. 4), and the amount of pyridoxamine produced is quantified by high performance liquid chromatography or the like.

The hydrogen peroxide catabolic enzyme may be, for example, a protein having the amino acid sequence of seq id No. 3, or a protein having an amino acid sequence obtained by substituting, deleting, inserting, or adding at least one of an amino acid residue to the amino acid sequence of seq id No. 3, or an additional amino acid residue to the N-terminus or the C-terminus or both of the amino acid sequence. Examples of the degree of substitution, deletion, insertion, and addition of an additional amino acid residue to the N-terminus, the C-terminus, or both of the amino acid sequences are as described above.

Alternatively, the hydrogen peroxide catabolic enzyme may be a protein having the amino acid sequence of SEQ ID No. 3; or a protein having an amino acid sequence having, for example, 80% or more, or 85% or more, or 90% or more, or 95% or more sequence identity with the amino acid sequence of SEQ ID NO. 3.

When a protein having an amino acid sequence similar to that of SEQ ID NO. 3 is used, the protein should have an activity as a hydrogen peroxide catabolic enzyme. The hydrogen peroxide catabolic enzyme activity can be measured, for example, by the following method: a protein to be tested is added to an aqueous solution of hydrogen peroxide as a substrate, and the decrease in the amount of hydrogen peroxide is quantified using, as an index, the decrease in absorbance at 240nm, or the like.

< Gene encoding pyridoxol oxidase, Gene encoding pyridoxamine synthase, and Gene encoding Hydrogen peroxide catabolic enzyme >

The gene encoding pyridoxol oxidase may be any of the genes encoding pyridoxol oxidase described above. The gene encoding pyridoxamine synthase may be any of the genes encoding pyridoxamine synthase described above. The gene encoding a hydrogen peroxide catabolic enzyme may be any gene encoding a hydrogen peroxide catabolic enzyme as described above. The enzyme encoded by these genes is not limited to a known amino acid sequence having the enzyme activity (for example, an amino acid sequence encoded by a gene naturally occurring in an organism), and may be an enzyme having a modified amino acid sequence different from the known amino acid sequence.

Such a gene may be a known gene such as a gene naturally possessed by the microorganism exemplified above (microorganism to be the source) as the microorganism having each enzyme, or a gene obtained by modifying a nucleotide sequence obtained by modifying a known amino acid sequence of the enzyme as described above within a range in which a desired enzyme activity is obtained, so that the modified amino acid sequence encodes a modified amino acid sequence. Examples of such modified amino acid sequences include amino acid sequences similar to any of the amino acid sequences of SEQ ID Nos. 1 to 4 as described above. In addition, the nucleotide sequence of a gene encoding a specific amino acid sequence may be varied within the scope of the degeneracy of the codon. In this case, it is preferable to use codons that are frequently used in microorganisms that serve as hosts of recombinant microorganisms, from the viewpoint of the expression efficiency of genes.

The nucleotide sequence of the gene may be designed based on a codon table based on the amino acid sequence to be encoded. The designed nucleotide sequence can be obtained by modifying a known nucleotide sequence using a gene recombination technique, or by chemically synthesizing the nucleotide sequence.

Examples of Methods for modifying a nucleotide sequence include a site-specific mutation method (Kramer, W.and frata, H.J., Methods in Enzymology, vol.154, P.350(1987)), a recombinant PCR method (PCRTechnology, Stockton Press (1989), a method for chemically synthesizing a DNA of a specific portion, a method for treating a gene with hydroxylamine, a method for treating a strain holding a gene with ultraviolet irradiation or with a chemical such as nitrosoguanidine or nitrous acid, and a method using a commercially available mutation introduction kit.

For example, the gene encoding pyridoxine oxidase, the gene encoding pyridoxamine synthase, and the gene encoding hydrogen peroxide catabolic enzyme may be unmodified DNAs having known nucleotide sequences encoding polynucleotides having the enzymatic activity (e.g., nucleotide sequences of genes naturally occurring in organisms such as the microorganisms exemplified above), or DNAs having nucleotide sequences obtained by sequence modification of such nucleotide sequences within a range that the encoded enzyme does not lose its enzymatic activity (the enzymatic activity described above). Such modifications include insertion, deletion, substitution of nucleotides, and addition of additional nucleotides to the 5 '-end or the 3' -end or both of the nucleotide sequence. When one or more of nucleotide insertion, deletion and substitution is present, the insertion, deletion and substitution may each be, for example, 1 to 90 nucleotides, or 1 to 60 nucleotides, or 1 to 30 amino acid residues, or 1 to 20 amino acid residues, or 1 to 15 nucleotides, or 1 to 10 nucleotides, or 1 to 5 nucleotides, respectively, when present, and the total number of nucleotide insertion, deletion and substitution may be, for example, 1 to 100 nucleotides, or 1 to 50 nucleotides, or 1 to 30 nucleotides, or 1 to 10 nucleotides, or 1 to 5 nucleotides. The nucleotide insertion or deletion may be present locally, but it is preferable that no large-scale frame shift occurs with respect to the entire nucleotide sequence. In addition, as the number of nucleotides added to the end, in the presence, each end is, for example, 1 to 150 nucleotides, or 1 to 100 nucleotides, or 1 to 50 nucleotides, or 1 to 30 nucleotides, or 1 to 10 nucleotides, or 1 to 5 nucleotides. Such additional nucleotides may encode a signal sequence for secretion outside the cell, etc.

Alternatively, the gene encoding each enzyme may be a DNA having a known nucleotide sequence itself encoding a polynucleotide having the enzyme activity (for example, a nucleotide sequence of a gene naturally present in an organism), or a DNA having a nucleotide sequence having 80% or more, or 85% or more, or 90% or more, or 95% or more sequence identity to the known nucleotide sequence and encoding an enzyme having a desired enzyme activity (the above-mentioned enzyme activity). Here, sequence identity can be evaluated based on default parameters using, for example, BLAST (registered trademark) program.

Alternatively, the gene encoding each enzyme may be a DNA having a known nucleotide sequence (for example, a nucleotide sequence of a gene naturally occurring in an organism) itself encoding a polynucleotide having the enzyme activity, or a DNA having a nucleotide sequence hybridizing with a DNA having a nucleotide sequence complementary to the known nucleotide sequence under stringent conditions and encoding an enzyme having a desired enzyme activity (the above-mentioned enzyme activity). Hybridization under stringent conditions can be performed as follows.

In the hybridization, a DNA containing a nucleotide sequence complementary to a reference nucleotide sequence or a partial sequence thereof is used as a probe, and the probe is hybridized with a target DNA, and after washing under stringent conditions, it is confirmed whether or not the probe is significantly hybridized with the target nucleic acid. The length of the probe may be, for example, 20 or more, preferably 50 or more, more preferably 100 or more, and still more preferably 200 or more nucleotides in a row. It is also preferable to use, as a probe, a DNA complementary over the entire length and having the same nucleotide length as the nucleotide sequence as the reference. The conditions for hybridization include those commonly used by those skilled in the art for detecting a specific hybridization signal. Preferably, stringent hybridization conditions and stringent washing conditions are indicated. For example, the probe is incubated with a solution containing 6 XSSC (sodium citrate sodium chloride) (1 XSSC composition: 0.15M NaCl, 0.015M sodium citrate, pH7.0), 0.5% SDS, 5 XDenhardt (Denhardt) and 100mg/ml herring sperm DNA at 55 ℃ overnight. The filter is exemplified by subsequent washing in 0.2 XSSC at 42 ℃ and the like. The stringent conditions are 0.1 XSSC and 50 ℃ in the step of washing the filter, and the more stringent conditions are 0.1 XSSC and 65 ℃ in the same step.

For example, the gene encoding pyridoxol oxidase may be, for example, a DNA having the nucleotide sequence of SEQ ID NO. 5 (the nucleotide sequence of the pyridoxol oxidase gene of Microbacterium flavum), or a DNA having a nucleotide sequence that hybridizes with a DNA having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO. 5 under stringent conditions and encodes a protein having pyridoxol oxidase activity.

Alternatively, the gene encoding pyridoxol oxidase may be, for example, a DNA having the nucleotide sequence of seq id No. 5, or a DNA having a nucleotide sequence obtained by substituting, deleting, inserting, or adding one or more additional nucleotides to the 5 '-end or the 3' -end or both of the nucleotide sequence of seq id No. 5 and encoding a protein having pyridoxol oxidase activity. Examples of nucleotide substitutions, deletions, insertions, and the degree of addition of additional nucleotides to the N-terminus or the C-terminus or both of the nucleotide sequence are as described above.

Alternatively, the pyridoxol oxidase may be a DNA having the nucleotide sequence of SEQ ID NO. 5 or a DNA having a nucleotide sequence which has, for example, 80% or more, or 85% or more, or 90% or more, or 95% or more sequence identity to the nucleotide sequence of SEQ ID NO. 5 and encodes a protein having pyridoxol oxidase activity.

The gene encoding pyridoxamine synthase may be, for example, a DNA having the nucleotide sequence of SEQ ID NO. 6 (the nucleotide sequence of the pyridoxamine-pyruvate transaminase gene of bradyrhizobium in Lotus corniculatus) or the nucleotide sequence of SEQ ID NO. 8 (the nucleotide sequence of the pyridoxamine-oxaloacetate transaminase gene of Escherichia coli), or a DNA having a nucleotide sequence which hybridizes under stringent conditions to a DNA having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO. 6 or 8 and encodes a protein having pyridoxamine synthase activity.

Alternatively, the gene encoding pyridoxamine synthase may be, for example, a DNA having the nucleotide sequence of seq id No. 6 or 8, or a DNA having a nucleotide sequence obtained by substituting, deleting, inserting, or adding one or more additional nucleotides to the 5 '-end or the 3' -end or both of the nucleotide sequence of seq id No. 6 or 8 and encoding a protein having pyridoxamine synthase activity. Examples of nucleotide substitutions, deletions, insertions, and the degree of addition of additional nucleotides to the N-terminus or the C-terminus or both of the nucleotide sequence are as described above.

Alternatively, the pyridoxamine synthase may be a DNA having the nucleotide sequence of SEQ ID NO. 6 or 8 or a DNA having a nucleotide sequence which has, for example, 80% or more, or 85% or more, or 90% or more, or 95% or more sequence identity with the nucleotide sequence of SEQ ID NO. 6 or 8 and which encodes a protein having pyridoxamine synthase activity.

Alternatively, the gene encoding pyridoxamine synthase may be, for example, a DNA having the nucleotide sequence of any one of seq id No. 6 and seq id No. 25 to 31, or a DNA having a nucleotide sequence that hybridizes under stringent conditions to a DNA having a nucleotide sequence complementary to the nucleotide sequence of any one of seq id No. 6 and seq id No. 25 to 31 and encodes a protein having pyridoxamine synthase activity.

Alternatively, the gene encoding pyridoxamine synthase may be, for example, a DNA having the nucleotide sequence of any one of seq id No. 6 and seq id No. 25 to 31, or a DNA having a nucleotide sequence obtained by substituting, deleting, inserting, or adding at least one of additional nucleotides to the 5 'end or the 3' end or both of the nucleotide sequence of any one of seq id No. 6 and seq id No. 25 to 31, and encoding a protein having pyridoxamine synthase activity. Examples of nucleotide substitutions, deletions, insertions, and the degree of addition of additional nucleotides to the N-terminus or the C-terminus or both of the nucleotide sequence are as described above.

Alternatively, the pyridoxamine synthase may be a DNA having the nucleotide sequence of any one of SEQ ID NO. 6 and SEQ ID NO. 25 to SEQ ID NO. 31, or a DNA having the nucleotide sequence of SEQ ID NO. 6, the nucleotide sequence of SEQ ID NO. 25 (the nucleotide sequence of the gene encoding pyridoxamine-pyruvate transaminase of Mesorhizobium strain YR577), the nucleotide sequence of SEQ ID NO. 26 (the nucleotide sequence of the gene encoding pyridoxamine-pyruvate transaminase of Pseudoamino acid bacterium salicylate), the nucleotide sequence of SEQ ID NO. 27 (the nucleotide sequence of the gene encoding pyridoxamine-pyruvate transaminase of Bacillus shore), the nucleotide sequence of SEQ ID NO. 28 (the nucleotide sequence of the gene encoding pyridoxamine-pyruvate transaminase of anti-Staphyloccocus antimonicola, or the nucleotide sequence of SEQ ID NO. 28 (the nucleotide sequence of the gene encoding anti-pyridoxamine-pyruvate transaminase), At least one of the nucleotide sequence of SEQ ID NO. 29 (nucleotide sequence of the gene encoding pyridoxamine-pyruvate transaminase of Rhizobium strain AC44/96), the nucleotide sequence of SEQ ID NO. 30 (nucleotide sequence of the gene encoding pyridoxamine-pyruvate transaminase of Erwinia torpedo.), and the nucleotide sequence of SEQ ID NO. 31 (nucleotide sequence of the gene encoding pyridoxamine-pyruvate transaminase of Raffinobacter), has, for example, 80% or more, or 85% or more, or 90% or more, or 95% or more sequence identity, and encodes a protein having pyridoxamine synthase activity.

When the expression is carried out in a recombinant microorganism using a prokaryotic organism as a host such as Escherichia coli, codons may be optimized for easy expression. For example, the nucleotide sequence may be modified so that a codon having the highest frequency of use among codons encoding the respective amino acids in a prokaryote as a host is used at a high frequency as the codon for the amino acid. From such a viewpoint, as the DNA containing the gene encoding pyridoxamine synthase, for example, a DNA having a region from the 18 th nucleotide to the 3 'end in the nucleotide sequence of seq id No. 10 or a region from the 18 th nucleotide to the 3' end of any of the nucleotide sequences of seq id No. 32 to seq id No. 38 may be used, or a DNA having a nucleotide sequence that hybridizes under stringent conditions with a DNA having a nucleotide sequence complementary to the region from the 18 th nucleotide to the 3 'end in the nucleotide sequence of seq id No. 10 or the region from the 18 th nucleotide to the 3' end of any of the nucleotide sequences of seq id No. 32 to seq id No. 38 and encodes a protein having pyridoxamine synthase activity. The nucleotide sequence of SEQ ID NO. 10 has an upstream region of 17 nucleotides from the 5' end, and the initiation codon is located from the 18 th nucleotide to the 20 th nucleotide. Therefore, the region from the 18 th nucleotide to the 3' end of the nucleotide sequence may be used as the gene region encoding pyridoxamine synthase. Similarly, the nucleotide sequences of SEQ ID NO. 32 to SEQ ID NO. 38 have 17 nucleotides from the 5' end thereof as an upstream region, and the initiation codon is located at 18 th to 20 th nucleotides. Therefore, the region from the 18 th nucleotide to the 3' end of these nucleotide sequences can also be used as a gene region encoding pyridoxamine synthase.

Alternatively, the gene encoding pyridoxamine synthase may be, for example, a DNA having a region from the 18 th nucleotide to the 3 'end in the nucleotide sequence of seq id No. 10 or a region from the 18 th nucleotide to the 3' end of any of the nucleotide sequences of seq id No. 32 to seq id No. 38, or a DNA having a nucleotide sequence obtained by substituting, deleting, inserting, or adding at least one of additional nucleotides to the 5 'end or the 3' end or both of the nucleotide sequence by the region from the 18 th nucleotide to the 3 'end of the nucleotide sequence of seq id No. 10 or the region from the 18 th nucleotide to the 3' end of any of the nucleotide sequences of seq id No. 32 to seq id No. 38 and encoding a protein having pyridoxamine synthase activity. Examples of nucleotide substitutions, deletions, insertions, and the degree of addition of additional nucleotides to the N-terminus or the C-terminus or both of the nucleotide sequence are as described above.

Alternatively, the pyridoxamine synthase may be DNA having a region from the 18 th nucleotide to the 3 ' end in the nucleotide sequence of SEQ ID NO. 10 or a region from the 18 th nucleotide to the 3 ' end of any of the nucleotide sequences of SEQ ID NO. 32 to SEQ ID NO. 38, or DNA having a nucleotide sequence from the 18 th nucleotide to the 3 ' end of the nucleotide sequence of SEQ ID NO. 10 (codon-optimized nucleotide sequence of the gene encoding pyridoxamine-pyruvate transaminase of bradyrhizobium japonicum in Lotus corniculatus), a region from the 18 th nucleotide to the 3 ' end of the nucleotide sequence of SEQ ID NO. 32 (codon-optimized nucleotide sequence of the gene encoding pyridoxamine-pyruvate transaminase of Mesorhizobium yR577), or a region from the 18 th nucleotide to the 3 ' end of the nucleotide sequence of SEQ ID NO. 33 (pyridoxamine-pyruvate transaminase encoding Pseudoaminobacter salicylate) A codon-optimized nucleotide sequence of the pyruvate transaminase gene), a region from the 18 th nucleotide to the 3 'terminus of the nucleotide sequence of SEQ ID NO. 34 (a codon-optimized nucleotide sequence of the pyridoxamine-pyruvate transaminase gene encoding Bacillus shore), a region from the 18 th nucleotide to the 3' terminus of the nucleotide sequence of SEQ ID NO. 35 (a codon-optimized nucleotide sequence of the pyridoxamine-pyruvate transaminase gene encoding S.antimonicola), a region from the 18 th nucleotide to the 3 'terminus of the nucleotide sequence of SEQ ID NO. 36 (a codon-optimized nucleotide sequence of the pyridoxamine-pyruvate transaminase gene encoding Rhizobium strain AC44/96), and a region from the 18 th nucleotide to the 3' terminus of the nucleotide sequence of SEQ ID NO. 37 (a codon-optimized nucleotide sequence of the pyridoxamine-pyruvate transaminase gene encoding Tolydoheia) And a region from nucleotide 18 to the 3' -end of the nucleotide sequence of SEQ ID NO. 38 (codon-optimized nucleotide sequence of a gene encoding pyridoxamine-pyruvate transaminase of Raffinophila ginseng), wherein at least one of the regions has a sequence identity of 80% or more, or 85% or more, or 90% or more, or 95% or more, and encodes a protein having pyridoxamine synthase activity.

The gene encoding a hydrogen peroxide catabolic enzyme may be, for example, a DNA having the nucleotide sequence of SEQ ID NO. 7 (the nucleotide sequence of the catalase gene of Listeria cilleri), or a DNA having a nucleotide sequence that hybridizes with a DNA having a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO. 7 under stringent conditions and encodes a protein having hydrogen peroxide catabolic enzyme activity.

Alternatively, the gene encoding a hydrogen peroxide catabolic enzyme may be, for example, a DNA having the nucleotide sequence of seq id No. 7, or a DNA having a nucleotide sequence obtained by subjecting the nucleotide sequence of seq id No. 7 to substitution, deletion, insertion of nucleotides, or addition of one or more additional nucleotides to the 5 '-end or the 3' -end or both of the nucleotide sequence, and encoding a protein having hydrogen peroxide catabolic enzyme activity. Examples of nucleotide substitutions, deletions, insertions, and the degree of addition of additional nucleotides to the N-terminus or the C-terminus or both of the nucleotide sequence are as described above.

Alternatively, the hydrogen peroxide catabolic enzyme may be a DNA having the nucleotide sequence of SEQ ID NO. 7 or a DNA having a nucleotide sequence that has, for example, 80% or more, or 85% or more, or 90% or more, or 95% or more sequence identity with the nucleotide sequence of SEQ ID NO. 7 and encodes a protein having hydrogen peroxide catabolic enzyme activity.

< recombinant microorganism having Gene encoding pyridoxol oxidase and Gene encoding pyridoxamine synthase >

In the recombinant microorganism according to the present disclosure, each of the gene encoding pyridoxol oxidase and the gene encoding pyridoxamine synthase may be a gene endogenous to the bacterial cell and having enhanced expression, or may be a gene introduced into the bacterial cell from the outside of the bacterial cell. In addition, both a gene derived from a bacterial cell and having enhanced expression and a gene introduced into the bacterial cell from the outside of the bacterial cell may be present in the recombinant microorganism. In addition, in the recombinant microorganism according to the present disclosure, a gene encoding pyridoxol oxidase endogenous to the bacterial cell and having enhanced expression thereof, and/or a gene encoding pyridoxol oxidase introduced into the bacterial cell from the outside of the bacterial cell, may be present in addition to the gene encoding pyridoxol oxidase endogenous to the bacterial cell and having no enhanced expression (for example, a gene encoding pyridoxol oxidase having an unmodified promoter). Similarly, in addition to the gene encoding pyridoxamine synthase endogenous to the bacterial cell but having enhanced expression, and/or the gene encoding pyridoxamine synthase introduced into the bacterial cell from outside the bacterial cell, there may be a gene encoding pyridoxamine synthase endogenous to the bacterial cell and not having enhanced expression (for example, a gene having an unmodified promoter). In the present disclosure, since the gene encoding pyridoxol oxidase and the gene encoding pyridoxamine synthase are each subjected to one or more of enhancement of expression of a gene endogenous to the bacterial cell and gene transfer from outside the bacterial cell into the bacterial cell, even if a gene whose expression is not enhanced originally exists in the bacterial cell, a significantly higher expression level can be obtained than an expression level derived from the original gene whose expression is not enhanced, and thus high production efficiency of pyridoxamine can be achieved.

When the recombinant microorganism according to the present disclosure further has a gene encoding a hydrogen peroxide catabolic enzyme, the gene encoding the hydrogen peroxide catabolic enzyme may be a gene endogenous to the bacterial cell, a gene endogenous to the bacterial cell but having enhanced expression, or a gene introduced into the bacterial cell from the outside of the bacterial cell. However, since the amount of hydrogen peroxide produced increases as the reaction rate increases, it is preferable to increase the expression level of the hydrogen peroxide catabolic enzyme by at least one of the following methods: enhancing the expression of a gene encoding a hydrogen peroxide catabolic enzyme endogenous to the bacterial cell; and introducing a gene encoding a hydrogen peroxide catabolic enzyme into the cell from outside the cell.

In other words, the gene encoding pyridoxol oxidase and the gene encoding pyridoxamine synthase may be genes endogenous to the genome of the host microorganism and enhanced in expression by, for example, substitution of a promoter, or may be genes introduced into the bacterial cell from the outside of the bacterial cell using a vector such as a plasmid. In addition to this, there may be a gene endogenous to the genome of the host microorganism before recombination and not subjected to expression enhancement. In the present disclosure, a gene encoding pyridoxol oxidase and a gene encoding pyridoxamine synthase are each introduced extracellularly from a host microorganism, or the expression of the genes in cells endogenous to the host microorganism is enhanced by, for example, replacing a promoter, thereby increasing the expression of the genes and imparting pyridoxamine-producing ability by the combination of the 2 enzymes. Here, when the gene encoding pyridoxol oxidase and the gene encoding pyridoxamine synthase are introduced or expression-enhanced, either or both of the introduction from outside the cell of the host microorganism and the enhancement of the expression of the genes by, for example, replacement of a promoter may be performed.

The recombinant microorganism to which the present disclosure relates may or may not have a gene encoding a hydrogen peroxide catabolic enzyme. When the recombinant microorganism according to the present disclosure further has a gene encoding a hydrogen peroxide catabolic enzyme, the gene encoding the hydrogen peroxide catabolic enzyme may be a gene endogenous to the genome of the host microorganism before recombination, a gene endogenous to the genome of the host microorganism and having its expression enhanced by an operation such as promoter replacement, or a gene introduced into the cell from the outside of the cell using a vector such as a plasmid. When a gene encoding a hydrogen peroxide catabolic enzyme is introduced or expression is enhanced, either or both of the introduction from outside the cell of the host microorganism and the enhancement of the expression of the gene by replacement of a promoter or the like may be performed.

If the gene expression is not increased as described above for the gene encoding pyridoxol oxidase and the gene encoding pyridoxamine synthase, a sufficient production capacity for producing pyridoxamine or a salt thereof at a high level cannot be obtained. Therefore, in the recombinant microorganism according to the present disclosure, both the gene encoding pyridoxol oxidase and the gene encoding pyridoxamine synthase are subjected to at least one of the introduction from outside the cell body and the enhancement of the expression of the gene endogenous to the cell body. The introduction of the enzyme gene from the outside of the cell is not necessarily limited to the supplementation of an enzyme gene not present in the host microorganism, and may be carried out for the purpose of enhancing the expression of an enzyme gene endogenous to the host microorganism. The enzyme gene not present in the host microorganism can be easily confirmed using an enzyme database such as KEGG and BRENDA.

When the expression of a gene endogenous to a host microorganism is enhanced by replacing the promoter of the gene with another promoter, the other promoter (newly introduced promoter) is not particularly limited as long as the expression of the gene can be enhanced in the host microorganism (can be enhanced as compared with before the replacement of the promoter), and may be a constitutive promoter or an inducible promoter. Replacement of the promoter can be carried out using conventional gene recombination techniques. The sequence of the promoter endogenous to the host microorganism may be partially or completely left as long as it does not adversely affect the expression by the newly introduced promoter, which is a practical problem.

When the host microorganism is, for example, a prokaryote, examples of promoters which can be used as newly introduced promoters include trp promoter, lac promoter, GAPDH promoter, PL promoter and PR promoter derived from phage lambda, glucose synthase promoter (gnt) derived from bacillus subtilis, alkaline protease promoter (apr), neutral protease promoter (npr), and α -amylase promoter (amy) derived from escherichia coli. Further, a promoter sequence which is independently modified or designed such as tac promoter may be used.

When the host microorganism is, for example, a filamentous bacterium, examples of promoters which can be used as newly introduced promoters include cellobiohydrolase (cbh) promoter, endoglucanase (egl) promoter, xylanase III (xyn3) promoter, U6 promoter, alpha-amylase (amy) promoter, and the like.

Examples of promoters which can be used as newly introduced promoters when the host microorganism is, for example, yeast include an alcohol dehydrogenase (ADH1) promoter, a phosphoglycerate kinase (PGK1) promoter, a peptide chain elongation factor (TEF) promoter, a glycerol-3-phosphate dehydrogenase (GPD) promoter, a galactokinase (GAL1) promoter, a metallothionein (CUP1) promoter, an inhibitory acid phosphatase (PHO5) promoter, and a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter. The source of the promoter sequence is not limited to yeast which is a host microorganism. Exogenous promoters such as the Cytomegalovirus (CMV) promoter may also be used.

When a gene (foreign (heterologous) gene) is introduced from outside the cell of a host microorganism, the method is not particularly limited as long as the gene can be introduced into the cell and the enzyme encoded by the gene can be expressed, and examples thereof include transformation with a plasmid holding an enzyme gene, introduction of an enzyme gene into a genome, and a combination thereof. When a gene is introduced, an expression vector incorporating the gene may be introduced into the cell. The expression vector is not particularly limited as long as the nucleotide sequence of the gene is incorporated, but from the viewpoint of improving transformation efficiency, translation efficiency, and the like, a plasmid vector or a phage vector having the following configuration is more preferable.

The expression vector is not particularly limited as long as it contains the nucleotide sequence of the gene and can transform the host microorganism. If necessary, a nucleotide sequence constituting another region (hereinafter also simply referred to as "another region") may be included in addition to the nucleotide sequence. Examples of the other region include a control region required for the production of a desired enzyme by a recombinant microorganism obtained by transformation, a region required for autonomous replication, and the like.

In addition, from the viewpoint of ease of screening the recombinant microorganism, a nucleotide sequence encoding a screening gene which can be used as a screening marker may be further contained.

Examples of the control region required for producing a desired enzyme include a promoter sequence (including an operator sequence for controlling transcription), a ribosome binding sequence (SD sequence), and a transcription termination sequence.

In the case of using yeast as a host microorganism, the expression vector preferably further contains a promoter sequence in addition to the nucleotide sequence of the gene in view of the expression efficiency of the gene. Any promoter sequence may be used as long as it can express the gene in a transformant of a host microorganism such as yeast. Examples of the promoter include those described above as examples of promoters which can be used as newly introduced promoters when the host microorganism is yeast.

In addition, the aforementioned expression vector may contain a secretion signal. Thus, when a desired enzyme is produced by a recombinant microorganism, the enzyme can be secreted extracellularly.

The secretion signal is not particularly limited as long as it can secrete a desired enzyme from the yeast serving as the host microorganism. From the viewpoint of secretion efficiency, an alpha factor signal sequence, a sucrase signal sequence, an acid phosphatase signal sequence, a glucoamylase signal sequence, and the like are preferably used.

Specific examples of expression vectors having the above-described promoter sequence and secretion signal include pRS423, pRS424, YEplac195 and the like.

The expression vector that can be used when a filamentous bacterium is used as a host microorganism preferably further contains a promoter sequence in addition to the nucleotide sequence of the gene in view of the expression efficiency of the gene. Any promoter sequence may be used as long as it can express the gene in a transformant of a host microorganism such as a filamentous fungus. Examples of the promoter include those described above as examples of promoters which can be used as newly introduced promoters when the host microorganism is a filamentous bacterium.

Suitable expression vectors for filamentous bacteria are described In van den Hondel, c.a.m.j.j. et al (1991) In: bennett, J.W.and Lasure, L.L, (eds.) More genes managers in fungi.academic Press, PP.396-428.

Other expression vectors commonly used in the art, such as pUC18, pBR322, pUC100, pSL1180(Pharmacia Inc.), pFB6, Aspergillus (Aspergillus) pRAX, and Trichoderma (Trichoderma) pTEX, can also be used.

In the case of using a prokaryotic organism such as Escherichia coli, Bacillus subtilis, or actinomycetes as a host microorganism, it is preferable that the expression vector further contains a promoter sequence in addition to the nucleotide sequence of the gene in view of the expression efficiency of the gene. In addition to the promoter sequence, a ribosome binding sequence, a transcription termination sequence, and the like may be contained.

Examples of the promoter sequence include the promoters described above as examples of promoters that can be used as newly introduced promoters when the host microorganism is a prokaryote.

Examples of the ribosome binding sequence include a sequence derived from Escherichia coli or Bacillus subtilis, but the ribosome binding sequence is not particularly limited as long as it functions in a desired host microorganism such as Escherichia coli or Bacillus subtilis.

Examples of the ribosome binding sequence include a sequence obtained by preparing a consensus sequence having 4 or more consecutive nucleotides among sequences complementary to the 3' -terminal region of 16S ribosomal RNA by DNA synthesis.

A transcription termination sequence is not necessarily required, and a rho-factor-independent transcription termination sequence such as a lipoprotein terminator, a trp operon terminator and the like can be used.

The sequence order of these control regions on the expression vector is not particularly limited, and in consideration of transcription efficiency, it is desirable that the promoter sequence, ribosome binding sequence, gene encoding the target enzyme, and transcription termination sequence are arranged in this order from the 5' -end upstream side.

Specific examples of expression vectors that can be used when the host microorganism is a prokaryote include pBR322, pUC18, Bluescript II SK (+), pKK223-3, pSC101, which have a region autonomously replicable in Escherichia coli, and pUB110, pTZ4, pC194, ρ 11, φ 1, φ 105, which have a region autonomously replicable in Bacillus subtilis.

In addition, as examples of expression vectors that can autonomously replicate in 2 or more host microorganisms, pHV14, TRp7, YEp7, pBS7, and the like can be used as expression vectors.

When a gene is introduced from the outside of the host microorganism, the gene may have a nucleotide sequence that is not present in the genome of the host microorganism, or may have a nucleotide sequence that is present in the genome of the host microorganism. Even if the same gene originally exists in the genome of the host microorganism, stronger gene expression can be obtained by introducing the gene from outside the microorganism, and the production efficiency of pyridoxamine or a salt thereof can be further improved by utilizing the enhanced enzyme activity.

Methods for preparing genomic DNA, cleaving and ligating DNA, transforming DNA, PCR (Polymerase Chain Reaction), designing and synthesizing oligonucleotides used as primers, and the like, which are required for introducing genes into a cell from the outside of the cell (from the outside of the cell) can be carried out by a conventional method known to those skilled in the art. These methods are described in Sambrook, J.et al, "Molecular Cloning A Laboratory Manual, second edition," Cold Spring Harbor Laboratory Press, (1989), etc. Examples thereof include a method using competent cells and a method using electroporation.

The host microorganism is not particularly limited as long as it can express a gene encoding pyridoxol oxidase and a gene encoding pyridoxamine synthase in the presence of these genes. Examples of the host microorganism include yeast, filamentous fungi, and prokaryotes. Examples of the yeast include a yeast belonging to the genus Saccharomyces such as Saccharomyces cerevisiae, a yeast belonging to the genus Schizosaccharomyces such as Schizosaccharomyces pombe (Schizosaccharomyces pombe), a yeast belonging to the genus Hansenula (Hansenula), and a yeast belonging to the genus Pichia. Examples of the filamentous fungi include filamentous fungi of the genus Trichoderma (Trichoderma), such as Trichoderma reesei and Trichoderma viride, filamentous fungi of the genus Aspergillus (Aspergillus), such as Aspergillus niger and Aspergillus oryzae, filamentous fungi of the genus Humicola, such as Humicola insolens, and filamentous fungi of the genus Acremonium, such as Acremonium cellulolyticum and Acremonium, such as Acremonium cocerucate. Examples of the prokaryote include bacteria belonging to the genus Escherichia such as Escherichia coli, bacteria belonging to the genus Shewanella such as Shewanella strain AC10, bacteria belonging to the genus Mesorhizobium such as Mesorhizobium, bacteria belonging to the genus Rhizobium such as Rhizopus such as alfalfa, Bacillus subtilis such as Bacillus subtilis, and actinomycetes such as Streptomyces (Streptomyces lividans) such as Streptomyces lividans.

By introducing (transforming) an expression vector containing a desired gene into these host microorganisms, for example, the desired gene can be introduced into the host microorganisms. The introduced gene can be expressed at a high level in the resulting recombinant microorganism by utilizing, for example, the activity of a promoter contained in an expression vector.

As a method for transferring a recombinant DNA such as an expression vector into cells of a host microorganism, for example, when the host microorganism is Escherichia coli, a competent cell method by calcium treatment, an electroporation method, or the like can be used. By culturing the recombinant microorganism thus obtained, an enzyme encoded by the introduced gene can be stably produced in a high expression level.

In addition, DNA encoding these enzymes can be removed from recombinant microorganisms, or can be transferred into other microorganisms. In addition, the following operations can also be easily performed: using this DNA as a template, a DNA fragment encoding the enzyme is amplified by PCR, treated with a restriction enzyme or the like, and then combined with another vector DNA fragment to be reintroduced into the host microorganism.

< Process for producing pyridoxamine or a salt thereof >

In one embodiment, the process for producing pyridoxamine or a salt thereof comprises a step of contacting the recombinant microorganism, the culture of the recombinant microorganism, or a treated product of the recombinant microorganism or the culture, according to the present disclosure, with pyridoxine or a salt thereof in the presence of oxygen to produce pyridoxamine or a salt thereof.

Examples of the salt of pyridoxine include a salt of pyridoxine with an acid. Examples of the acid include hydrochloric acid, sulfuric acid, nitric acid, and phosphoric acid. The salt of pyridoxine is, for example, pyridoxine hydrochloride.

Examples of the salt of pyridoxamine include a salt of pyridoxamine with an acid. Examples of the acid include hydrochloric acid, sulfuric acid, nitric acid, acetic acid, and phosphoric acid. From the viewpoint of use as a medicine, a pyridoxamine dihydrochloride, which is the most advanced for application to medical uses, is preferable.

The method for producing pyridoxamine or a salt thereof using the recombinant microorganism, the culture of the recombinant microorganism, or the treated product of the recombinant microorganism or the culture is also referred to simply as "a method for producing pyridoxamine or a salt thereof using a recombinant microorganism".

The culture of the recombinant microorganism refers to a product obtained by culturing the recombinant microorganism and containing the cell body, a surrounding medium, and the like. When the enzyme is secreted extracellularly, the use of such a culture makes it easier to contact the substrate with the enzyme, and the efficiency of producing pyridoxamine or a salt thereof can be improved. However, a culture may not be used, and for example, a previously prepared cell of a dried or frozen recombinant microorganism may be directly added to the reaction system.

When culturing a recombinant microorganism, any of a synthetic medium and a natural medium can be used as the medium as long as it contains a suitable amount of carbon sources, nitrogen sources, inorganic substances and other nutrients. The culture can be carried out in a liquid medium containing the above-mentioned culture components by a usual culture method such as shaking culture, aeration-agitation culture, continuous culture or fed-batch culture (fed-batch culture).

More specifically, the conditions for culturing the recombinant microorganism may be known as the conditions for culturing the original host microorganism.

As the components for the medium, known ones can be used. For example, organic nutrient sources such as meat extract, yeast extract, malt extract, peptone, NZ amine and potato, carbon sources such as glucose, maltose, sucrose, starch and organic acids, nitrogen sources such as ammonium sulfate, urea and ammonium chloride, inorganic nutrient sources such as phosphate, magnesium, potassium and iron, and vitamins can be used in combination as appropriate.

In the culture of the recombinant microorganism transformed with the expression vector containing the selection marker, for example, a medium containing a drug corresponding to the selection marker is used when the selection marker has drug resistance, and a medium containing no nutrient corresponding to the selection marker is used when the selection marker is auxotrophic.

The culture conditions are appropriately selected depending on the type of the recombinant microorganism, the culture medium, and the culture method, and are not particularly limited as long as the recombinant microorganism grows and can produce pyridoxol oxidase and pyridoxamine synthase.

The pH of the medium may be selected, for example, within a range of 4 to 8, or may be within a range of 5 to 8.

The culture temperature is, for example, 20 to 45 ℃ and preferably 24 to 37 ℃. The culture may be carried out aerobically or anaerobically, depending on the type of microorganism.

The culture time is, for example, 1 to 7 days. The incubation time can be set so that the amount of the target enzyme produced is maximized.

The treated product of the recombinant microorganism is a product obtained by optionally treating the recombinant microorganism within a range in which activities of pyridoxol oxidase and pyridoxamine synthase produced by the recombinant microorganism are not lost. Examples of such treatment include 1 or more treatments selected from the group consisting of heating treatment, cooling treatment, mechanical disruption, ultrasonic treatment, freeze-thaw treatment, drying treatment, pressure or reduced pressure treatment, osmotic pressure treatment, autodigestion, surfactant treatment, and enzyme treatment (e.g., cell lysis treatment). Even if the recombinant microorganism itself dies due to such treatment, it can be used for the reaction as long as the activity of the enzyme produced by the microorganism remains.

The treated product of the culture is a product obtained by optionally treating a culture of the recombinant microorganism within a range in which the activity of pyridoxol oxidase and pyridoxamine synthase produced by the recombinant microorganism is not lost. Examples of such treatment include 1 or more treatments selected from the group consisting of heating treatment, cooling treatment, mechanical disruption of cells, ultrasonic treatment, freeze-thaw treatment, drying treatment, pressure or reduced pressure treatment, osmotic pressure treatment, autodigestion of cells, surfactant treatment, enzyme treatment (for example, cell disruption treatment), cell separation treatment, purification treatment, and extraction treatment. For example, cells of the recombinant microorganism may be separated from a culture medium or the like, and the separated cells may be added to the reaction system. Such separation may be performed by filtration, centrifugation, or the like. Alternatively, a purification treatment for separating pyridoxine oxidase, pyridoxamine synthase, and (when present) hydrogen peroxide catabolic enzyme from inclusions may be performed, and a solution containing the enzymes obtained by the purification treatment may be added to the reaction system. Alternatively, an extract obtained by extracting the culture with an organic solvent such as methanol or acetonitrile or a mixed solvent of an organic solvent and water may be added to the reaction system. Such a purified product or extract may not comprise cells of a recombinant microorganism. Even if the cells of the microorganism are not present, the enzyme can be used for the reaction as long as the activity of the enzyme remains.

The disruption or lysis treatment of the cells as described above can be carried out by disrupting the cell membrane of the recombinant microorganism by a known method such as lysozyme treatment, freeze-thawing, and ultrasonication.

The recombinant microorganism, the culture of the recombinant microorganism, or the treated product of the recombinant microorganism or the culture is preferably contacted with pyridoxine or a salt thereof under the following conditions.

The contacting is preferably carried out in a solution containing pyridoxine or a salt thereof as a substrate. The pH of the solution is not particularly limited as long as the enzymatic activities of pyridoxol oxidase and pyridoxamine synthase can be maintained, but is preferably 6.0 to 9.0, and more preferably 7.0 to 8.5. The temperature of the solution is not particularly limited as long as the enzymatic activities of pyridoxine oxidase and pyridoxamine synthase can be maintained, and is preferably 20 to 70 ℃, and more preferably 25 to 50 ℃.

As the medium of the solution, water or an aqueous medium, an organic solvent, or a mixed solution of water or an aqueous medium and an organic solvent can be used. As the aqueous medium, for example, a phosphate buffer, HEPES (N-2-hydroxyethylpiperazine-N-ethanesulfonic acid) buffer, Tris [ Tris (hydroxymethyl) aminomethane ] hydrochloric acid buffer, or the like can be used. The organic solvent may be any organic solvent as long as it does not inhibit the reaction, and for example, acetone, ethyl acetate, dimethyl sulfoxide, xylene, methanol, ethanol, butanol, or the like can be used.

The contact of the recombinant microorganism, the culture of the recombinant microorganism, or the treated product of the recombinant microorganism or the culture with pyridoxine or a salt thereof is performed in the presence of oxygen. This is because oxygen is consumed when pyridoxine oxidase oxidizes pyridoxine or a salt thereof. As the oxygen concentration in the reaction system, for example, a solution containing pyridoxine or a salt thereof and the recombinant microorganism, a culture of the recombinant microorganism, or a treated product of the recombinant microorganism or the culture can be allowed to open to the atmosphere, that is, brought into contact with the atmosphere to effect a reaction; or, the reaction is carried out by contacting the reaction product with a gas containing 0.1 to 20%, 0.5 to 10%, or 1 to 5% by volume of oxygen. The amount of oxygen dissolved in such a solution may be 0.1mg to 13mg oxygen/L, 0.5mg to 10mg oxygen/L, or 1mg to 8mg oxygen/L.

When the recombinant microorganism has a gene encoding a hydrogen peroxide catabolic enzyme capable of producing oxygen, the reaction can proceed even if the oxygen concentration is reduced or under conditions in which the supply of oxygen is reduced or blocked. For example, the reaction can be carried out under a condition where the reaction system is sealed or under a condition where the reaction system is purged with nitrogen (nitrogen substitution).

The contact of the recombinant microorganism, the culture of the aforementioned recombinant microorganism, or the treated matter of the aforementioned recombinant microorganism or the aforementioned culture with pyridoxine or a salt thereof referred to in the present disclosure is preferably performed under shaking or stirring. For example, such contacting may be performed in solution. For example, pyridoxine or a salt thereof may be added in the form of a substrate solution or in the form of a solid to a solution containing the recombinant microorganism, a culture of the recombinant microorganism, or a treated product of the recombinant microorganism or the culture. Further, an amino acid consumed by pyridoxamine synthase may be added to a solution containing the recombinant microorganism, a culture of the recombinant microorganism, or a treated product of the recombinant microorganism or the culture. The amino acid may be added to a solution containing the recombinant microorganism, a culture of the recombinant microorganism, or a treated product of the recombinant microorganism or the culture together with pyridoxine or a salt thereof in a state of being contained in a substrate solution containing pyridoxine or a salt thereof. The amino acid may be added to a solution containing the recombinant microorganism, a culture of the recombinant microorganism, or a treated product of the recombinant microorganism or the culture in a state of being contained in a substrate solution different from a substrate solution containing pyridoxine or a salt thereof, or in a solid form.

An acid or a base may be added at the start of the reaction or during the reaction in order to maintain the pH of the reaction solution in an appropriate range. Examples of the alkali that can be added to the reaction solution include alkali metal hydroxides such as lithium hydroxide, sodium hydroxide, and potassium hydroxide, and substances such as ammonium hydroxide, calcium hydroxide, dipotassium hydrogen phosphate, disodium hydrogen phosphate, potassium pyrophosphate, and ammonia that are dissolved in water and have alkaline liquid properties. Examples of the acid that can be added to the reaction solution include hydrochloric acid, sulfuric acid, nitric acid, acetic acid, phosphoric acid, and the like.

The above contact may be carried out, for example, under an air atmosphere, or under a partially deoxygenated atmosphere. The deoxidation atmosphere can be achieved by substitution with an inert gas, pressure reduction, boiling, or a combination thereof. At least, substitution with an inert gas, that is, use of an inert gas atmosphere is preferable. Examples of the inert gas include nitrogen, helium, argon, carbon dioxide, and the like, and nitrogen is preferable. However, since oxygen is consumed when pyridoxine is oxidized by pyridoxine oxidase, an atmosphere in which oxygen is not removed is preferable.