The present disclosure, use pyridoxamine or a manufacturing method of pyridoxamine or a salt thereof with a producible recombinant microorganisms and recombinant microorganisms salts thereof, as well as pyridoxal or possible recombinant microorganism and recombinant microorganisms producing a salt thereof the method for producing a stomach pyridoxal or its salt.
BACKGROUND
[0002]
　Pyridoxamine and salts thereof Vitamin B 6 is a kind of, are known to have a saccharification reaction inhibitory effect. Pyridoxamine and salts thereof, for example, advanced glycation end products involved in various aging reaction; inhibiting accumulation in the body of the (Advanced Glycation End Products AGE). AGE is the generic term for substances produced by glycation of proteins, accumulation of AGE are diabetes, atherosclerosis, chronic renal failure, there is a worsening of the diseases such as Alzheimer's disease. Therefore, pyridoxamine and salts thereof are promising substances which are expected to allow the prevention and treatment of these diseases by preventing the accumulation of AGE.
[0003]
　Further, pyridoxamine and salts thereof are known to have activity as schizophrenia drugs, research towards its practical application have made various. Other, it has been advanced development of pyridoxamine and various health foods and cosmetics utilizing the physiological activities of the salts thereof.
　Pyridoxamine and salts thereof can be chemically synthesized. For example WO 2006/066806 discloses a method of chemically synthesizing the pyridoxamine dihydrochloride alanine and formic acid as the starting material. Furthermore, WO 2005/077902 discloses a method for chemically synthesizing pyridoxamine from pyridoxine.
[0004]
　On the other hand, it has also been studied to synthesize the pyridoxamine biologically. WO 2007/142222 discloses a method for obtaining a pyridoxamine from pyridoxal using a specific microorganism Achromobacter sp like. Furthermore, WO 2007/142222 by culturing the Acremonium Fushijioidesu in the presence pyridoxine, also discloses experiments a certain amount is converted into pyridoxal.
　JP-9-107985 discloses vitamin B belonging to the genus Rhizobium 6 a microorganism having an ability to produce, under aerobic conditions, cultured in medium, vitamins B produced 6 Vitamin B obtained from culture 6 production method of discloses.
[0005]
　Vitamin B 6 as the research on the synthesis, WO 2004/035010 is yaaD and vitamin B at least one of activity by culturing organisms with enhanced than the parent organism of yaaE Bacillus subtilis (Bacillus subtilis) 6 mixture It discloses a process for producing. JP 2000-23690 discloses vitamin B with cell-free extracts of Rhizobium meliloti (Rhizobium meliloti) IFO 14782 6 describes the production of the mixture. .. Journal of Molecular Catalysis B: Enzymatic, 2010, vol 67, p 104-110 is pyridoxamine modified so as to enable use of L- glutamic acid by sequence alterations - discloses pyruvate aminotransferase (PPAT) . The E. coli expressing the PPAT with this modified sequence, by incubating pyridoxal presence of saturating amounts, it has been described that could produce pyridoxamine.
[0006]
　Also, pyridoxal also, vitamin B 6 is a kind of a nutrient necessary for the survival of the organism. Vitamin B 6 is not only used in foods and food additives, has drugs, a wide range of applications such as cosmetics raw materials. Pyridoxal are useful applications as a single component. For example, pyridoxal phosphate active form of vitamin B 6 is used as a material, pyridoxal is a substance that may be intermediates for the production of pyridoxal phosphate. Pyridoxal, pharmaceuticals, useful as cosmetics, food, or intermediates for their manufacture. Sho 43-5712 Patent Publication No. pyridoxal is characterized by adding a carbonyl reagent in the reaction solution in the conversion enzyme reactions from pyridoxine by pyridoxine oxidase to pyridoxal - discloses a process for producing carbonyl derivative. Moreover, The Journal of Biological Chemistry, 1999 , Vol. 274, No. 33, Issue of August 13, pp. 23185-23190 also describes such convertase.
Summary of the Invention
Problems that the Invention is to Solve
[0007]
　However, not obtain a high reaction yield by pyridoxamine synthesis by chemical synthesis. For example, in the method described in WO 2006/066806, since the pyridoxamine dihydrochloride is synthesized through many chemical reactions, the reaction yield becomes low. The method described in WO 2005/077902 produces dimers and trimers of pyridoxamine, not much higher reaction yield.
　Meanwhile, in the method disclosed in WO 2007/142222 is a method using a microorganism of the particular species, the conversion efficiency from pyridoxal to pyridoxamine are large variations by microbial species, also intended much higher overall There was no. Further, in the culture experiments Acremonium Fushijioidesu in pyridoxine presence of described in WO 2007/142222, most of the product was pyridoxal instead of pyridoxamine. Further, in the description of JP-A-9-107985, JP-B vitamins resulting 6 is mostly a pyridoxol (pyridoxine), generation of pyridoxamine was only.
[0008]
　In WO 2004/035010, by culturing in a medium a microorganism obtained by highly expressed genes yaaD and yaaE, vitamin B 6 but have described that is obtained, the product pyridoxine, pyridoxal, pyridoxamine, a mixture of such pyridoxamine phosphate, it was not possible to obtain selectively the pyridoxamine.
　As described above, pyridoxamine or chemical synthesis (e.g., WO 2006/066806 and WO 2005/077902) a salt thereof, there is going through a number of reaction and purification steps, higher yield The resulting non. Although in the microorganisms are those having the pyridoxamine synthesizing ability (e.g. WO 2007/142222 Patent and Hei 9-107985 Patent Publication), takes time be newly discovered such a microorganism, also, vitamin B 6 not possible to selectively synthesize pyridoxamine or a salt thereof in the group, not even obtained high pyridoxamine production efficiency. Also, from the approach thus screening specific microbial species from microbial species that naturally occurring vitamin B 6 molecular biological information as to which enzymes or genes for highly producing a are important not be obtained.
[0009]
　Furthermore vitamin B 6 Although some examples using recombinant microorganisms prepared by using gene recombination technology with respect to production of (WO 2004/035010 Patent and Journal of Molecular Catalysis B:. Enzymatic , 2010, vol 67 , p. 104-110), vitamin B using a conventional medium during material production 6 or gave non-selectively (WO 2004/035010), or a saturated pyridoxal is an expensive raw material and pyridoxamine are obtained using an amount (Journal of Molecular Catalysis B:. . Enzymatic, 2010, vol 67, p 104-110), to produce better inexpensively and selectivity of pyridoxamine or a salt thereof have achieved Absent.
[0010]
　In view of the above situation, in a first aspect of the present disclosure, the recombinant microorganism can be inexpensively produced pyridoxine or a salt thereof pyridoxamine or a salt thereof with high production efficiency, and by using the recombinant microorganism pyridoxine or to provide a method for inexpensively produced pyridoxamine or high production efficiency a salt thereof from a salt thereof.
[0011]
　Further, the pyridoxal production, industrial efficient enzymatic production methods such as may be used in the production has not yet been put to practical use. In the method described in the methods and JP 2000-23690 JP described in WO 2004/035010, pyridoxine by enzymatic methods, mixtures of pyridoxal and pyridoxamine can be obtained, these methods select pyridoxal manner, i.e. as high purity pyridoxal rather than as a mixture with pyridoxine or pyridoxamine, not the method by which it is possible to obtain.
　In the method described in JP-B-43-5712, it is essential addition of carbonyl reagent to the reaction system. Carbonyl reagent, the generated pyridoxal is to convert the carbonyl derivative. However, in order to provide the next reaction pyridoxal as intermediates are often unsuitable method for generating a carbonyl derivative.
　The present inventors, in view of industrial production of pyridoxal was tried to prepare industrially available recombinant microorganism gene encoding pyridoxine dehydrogenase has been introduced. Host cells for such recombinant microorganism, it is preferable from the viewpoint of cost and ease of fabrication is a prokaryote, for example, when gene introduction is a eukaryotic genes is expressed in eukaryotic thereby introducing the gene encoding the protein into the prokaryotic expression involves many difficulties and often can not be expressed proteins remain retain activity. Therefore, in the second aspect of the present disclosure, expressing have introduced pyridoxine dehydrogenase provides recombinant microorganisms.
Means for Solving the Problems
[0012]
　A first aspect of the present disclosure include the following.
<1>
　encoding genes encoding pyridoxine dehydrogenase gene encodes pyridoxamine synthase having an enzyme activity to synthesize pyridoxamine from pyridoxal, and an amino acid regenerating enzyme having an enzyme activity to play an amino acid wherein the pyridoxamine synthase consumes has a gene,
　the pyridoxine gene encoding Shin dehydrogenase, the pyridoxamine or at least two of the synthase encoding gene and the amino acid regenerating enzyme encoding gene is a gene introduced from the outside of cells or bacteria inherent in is a gene whose expression is enhanced, the recombinant microorganism.
<2>
　the pyridoxamine synthase, pyridoxamine - pyruvic transaminase, pyridoxamine - oxaloacetic transaminase, aspartate transaminase, or pyridoxamine phosphate transaminase, recombinant microorganisms described <1>.
<3>
　the pyridoxine dehydrogenase includes at least one of the partial amino acid sequence of the following (a) and the partial amino acid sequence (b), and has a pyridoxine dehydrogenase activity, recombinant microorganisms described <1> or <2> . (A)
NX 1 X 2 EX 3 YG (SEQ ID NO: 97) 　　(wherein, X 1
Represents V, C, I, A, M, S, G or
　　L, X 2 represents G or
　　A, X 3 represents F or
L) (b) X 4 X 5 X 6 KGX 7 (SEQ ID NO: 　　98) (X 4 represents I, V, F or 　　L, X 5 represents S, T, N, C or 　　M, X 6 is C, V, a, I, W or an 　　F, X 7 is, G, a, represents a S or C) <4> 　the pyridoxine dehydrogenase is represented by enzyme number EC1.1.1.65, any of <1> to <3> the recombinant microorganism according to one or. <5>

　The pyridoxine gene encoding Shin dehydrogenase is derived from Saccharomyces cerevisiae, <1> ~ recombinant microorganism according to any one of <4>.
<6>
　gene encoding the pyridoxine dehydrogenase,
　or having any of the nucleotide sequences of SEQ ID NO: 7 and SEQ ID NO: 53 to SEQ ID NO: 59,
　in any of the nucleotide sequences of SEQ ID NO: 13 and SEQ ID NO: 75 to 81 18 nt ~ 3 'having a region to the end,
　DNA having a nucleotide sequence complementary to any of the nucleotide sequences of SEQ ID NO: 7 and SEQ ID NO: 53 to SEQ ID NO: 59, or SEQ ID NO: 13 and SEQ ID NO: 75 to a DNA which hybridizes with the DNA under stringent conditions with a 18 nt ~ 3 'region complementary to the nucleotide sequence to end in any of the nucleotide sequences of 81, encoding a protein and having a pyridoxine dehydrogenase activity the nucleotide sequence To, recombinant microorganism according to any one of <1> to <5>.
<7>
　gene encoding the pyridoxine dehydrogenase, or having the nucleotide sequence of SEQ ID NO: 7, a DNA which hybridizes with the DNA under stringent conditions with a nucleotide sequence complementary to a nucleotide sequence of SEQ ID NO: 7, and pyridoxine having a nucleotide sequence encoding a protein having dehydrogenase activity, recombinant microorganism according to any one of <1> to <6>.
<8>
　The pyridoxine gene encoding Shin dehydrogenase is derived from Schizosaccharomyces pombe, <1> ~ recombinant microorganism according to any one of <4>.
<9>
gene encoding the pyridoxine dehydrogenase, or having the nucleotide sequence of SEQ ID NO: 8, a DNA which hybridizes with the DNA under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 8, and pyridoxine having a nucleotide sequence encoding a protein having dehydrogenase activity, recombinant microorganism according to any one of <1> to <4> and <8>.
<10>
　gene encoding the pyridoxine dehydrogenase, SEQ ID NO: 1, any of the amino acid sequence of SEQ ID NO: 2 and SEQ ID NO: 22 to SEQ ID NO: 28, or SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 22 to 28 having a nucleotide sequence encoding the amino acid sequence, which has at least one 80% or more sequence identity to have and pyridoxine dehydrogenase activity of the amino acid sequences, according to any one of <1> to <9> recombinant microorganism.
<11>
　gene encoding the pyridoxine dehydrogenase, the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or at least one 80% or more sequence identity with the amino acid sequence of the amino acid sequence and SEQ ID NO: 2 of SEQ ID NO: 1 a and an amino acid sequence having pyridoxine dehydrogenase activity, having a nucleotide sequence encoding a <1> to recombinant microorganism according to any one of <10>.
<12>
　The pyridoxamine synthase partial amino acid sequence of the following (c), the partial amino acid sequence of (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) out includes at least one, and having an enzymatic activity to synthesize pyridoxamine from pyridoxal, <1> to recombinant microorganism according to any one of <11>. (C)
X 8 X 9 X 10 X 11 X 12 X 13 (SEQ ID NO: 　　99) (X 8 represents L, M, I or 　　V, X 9 represents H or 　　Q, X 10 is, G represents C or 　　a, X 11 represents E or 　　D, X 12 represents P or 　　a, X 13 represents V, I, L or a)

(D) X 14 X 15 TPSGTX 16 X 17 (SEQ ID NO:
　　100) (X 14 represents H or
　　S, X 15 represents D or
　　E, X 16 represents I, V or
　　L, X 17 represents N or
T) (e) X 18 DX 19 VSX 20 X 21 (SEQ ID NO: 　　101) (X 18 is, V, represents I or 　　a, X 19 is, a, represents T or S, 　　X 20 represents S, a or 　　G, X

21 is F, W, or an
V) (f) X 22 X 23 X 24 KCX 25 GX 26 X 27 P (SEQ ID NO: 　　102) (X 22 represents G or 　　S, X 23 is, P, S or 　　a, X 24 represents N, G, S, a or 　　Q, X 25 represents L or 　　M, X 26 represents a, S, C or 　　G, X 27 is P, T, represents an S or a) (g) X 28 X 29 X 30 X 31

SX 32 GX 33 X 34 (SEQ ID NO:
103) (X 28 represents G or
　　D, X 29 represents V or
　　I, X 30 is V, T, A, S, M, I or L
　　represents, X 31 represents F, M, L, I or
　　V, X 32 represents S, G, a, T, I, L or
　　H, X 33 represents R, M or Q,
　　X 34 is, G, R, a, D , H or
K) (h) X 35 X 36 RX 37 X 38 HMGX 39 X 40A (SEQ ID NO:
　　104) (X 35 represents L or
　　V, X 36 represents T, I, V or
　　L, X 37 represents I, V or
　　L, X 38 is, G or S the
　　stands, X 39 is, P, represents a or
　　R, X 40 is, T, represents the V or
S) <13>
　the pyridoxamine synthase represented by enzyme number EC2.6.1.30, < 1> to recombinant microorganism according to any one of <12>.
<14>
　the gene encoding pyridoxamine synthase is derived from Mesorhizobium loti, <1> ~ recombinant microorganism according to any one of <13>.
<15>
　gene encoding the pyridoxamine synthase,
　or having any of the nucleotide sequences of SEQ ID NO: 9 and SEQ ID NO: 60 to SEQ ID NO: 66,
　Or having an area of up to end '18 nt ~ 3 in any of the nucleotide sequences of the region or SEQ ID NO: 82 to SEQ ID NO: 88 to the end' 18 nt ~ 3 in the nucleotide sequence of SEQ ID NO: 14,
　SEQ ID NO: 9 and SEQ ID NO: 60 through SEQ any DNA having a nucleotide sequence complementary to the nucleotide sequence, or regions of up to 18 nt ~ 3 'terminus of the nucleotide sequence of SEQ ID NO: 14 or SEQ ID NO: 82 among the numbers 66 a DNA which hybridizes with the DNA under stringent conditions with a 18 nt ~ 3 'region complementary to the nucleotide sequence to end in any of the nucleotide sequences of to SEQ ID NO: 88, and synthesizes the pyridoxamine from pyridoxal Tampa having the enzymatic activity Having a nucleotide sequence encoding a click proteins, recombinant microorganism according to any one of <1> to <14>.
<16>
　gene encoding the pyridoxamine synthase, or having the nucleotide sequence of SEQ ID NO: 9, a DNA which hybridizes with the DNA under stringent conditions with a nucleotide sequence complementary to a nucleotide sequence of SEQ ID NO: 9, and having a nucleotide sequence encoding a protein having an enzymatic activity to synthesize pyridoxamine from pyridoxal recombinant microorganism according to any one of <1> to <15>.
<17>
　The pyridoxamine gene encoding synthase, SEQ ID NO: 3 and SEQ ID NO: 29 to any of the amino acid sequence, or SEQ ID NO: 3 and at least one 80 of the amino acid sequence of SEQ ID NO: 29 to SEQ ID NO: 35 of SEQ ID NO: 35 % and having a sequence identity or more having a nucleotide sequence encoding the amino acid sequence, a having an enzyme activity to synthesize pyridoxamine from pyridoxal recombinant microorganism according to any one of <1> to <16> .
<18>
　amino acids with the gene encoding the pyridoxamine synthase comprises the amino acid sequence, or having the amino acid sequence 80% or more sequence identity with SEQ ID NO: 3 and the enzyme activity to synthesize pyridoxamine from pyridoxal SEQ ID NO: 3 having a nucleotide sequence encoding a sequence, recombinant microorganism according to any one of <1> to <17>.
<19>
　the an amino acid regenerating enzyme is alanine dehydrogenase, <12> - The recombinant microorganism according to any one of <18>.
<20>
　The alanine dehydrogenase, pyruvate and NH 3 are available for NADPH as a coenzyme in the reaction for generating the L- alanine, recombinant microorganisms described <19>.
<21>
　the alanine dehydrogenase partial amino acid sequence of the following (i), the partial amino acid sequence (j), wherein at least one of the partial amino acid sequence (k) and the partial amino acid sequence (l), and pyruvate and NH 3The recombinant microorganism according to <19> or <20> having an activity to produce L- alanine. (I)
EX 41 KX 42 X 43 EX 44 RX 45 X 46 (SEQ ID NO: 　　105) (X 41 represents I, T, S, F, N or 　　V, X 42 is N, M, A, V , L represents T or 　　D, X 43 represents H, N, L or 　　Q, X 44 is, Y, represents N or 　　F, X 45 represents V or 　　I, X 46 is, G or an a) (j) X 47 X 48 X 49

KVKEPX 50 (SEQ ID NO:
　　106) (X 47 represents M or
　　L, X 48 is I, represents L or
　　V, X 49 represents V, L, I or
　　M, X 50 is, Q, L, V represents N or
I) (k) LX 51 TYLHLA (SEQ ID NO:
　　107) (X 51 represents F or
Y) (l) X 52 DX 53 AX 54 DQGG (SEQ ID NO: 　　108) (X 52 represents V or 　　a, X 53 represents V or 　　I, X 54 represents I or V)

<22>
　the gene encoding the amino acid regeneration enzyme is Shewanella sp. From AC10, recombinant microorganism according to any one of <19> - <21>.
<23>
　the gene encoding the amino acid regeneration enzyme,
　or has any nucleotide sequence of SEQ ID NO: 11 and SEQ ID NO: 67 to SEQ ID NO: 74,
　any one of SEQ ID NO: 15 and SEQ ID NO: 89 to SEQ ID NO: 96 or having an area of up to 18 nt ~ 3 'terminus of the nucleotide sequence,
　DNA having a nucleotide sequence complementary to any of the nucleotide sequences of SEQ ID NO: 11 and SEQ ID NO: 67 to SEQ ID NO: 74, or SEQ ID NO: 15 and a DNA which hybridizes with the DNA under stringent conditions with a 18 nt ~ 3 'region complementary to the nucleotide sequence to end in any of the nucleotide sequence of SEQ ID NO: 89 to SEQ ID NO: 96, and alanine nucleotides encoding a protein with a reproduced enzymatic activity Having a row, recombinant microorganism according to any one of <19> - <22>.
<24>
　the gene encoding the amino acid regeneration enzyme, or has the nucleotide sequence of SEQ ID NO: 11, a DNA which hybridizes with the DNA under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 11, and having a nucleotide sequence encoding a protein having alanine regenerating enzyme activity, recombinant microorganism according to any one of <19> - <23>.
<25>
　Gene encoding the amino acid regeneration enzyme, SEQ ID NO: 5 and SEQ ID NO: 45 to any of the amino acid sequence, or SEQ ID NO: 5 and SEQ ID NO: 45 to at least one 80 of the amino acid sequence of SEQ ID NO: 52 of SEQ ID NO: 52 % with a nucleotide sequence encoding the amino acid sequence, having the above sequence identity have and alanine regenerating enzyme activity, recombinant microorganism according to any one of <19> - <24>.
<26>
　encoding genes encoding the amino acid regenerating enzyme comprises the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence and amino acid sequence having and alanine regenerating enzyme activity has 80% or more sequence identity with SEQ ID NO: 5, the having a nucleotide sequence that recombinant microorganism according to any one of <19> - <25>.
<27>
　the pyridoxamine synthase represented by enzyme number EC2.6.1.31 or EC2.6.1.1, recombinant microorganism according to any one of <1> to <11>.
<28>
　the pyridoxamine gene encoding synthase is derived from Escherichia coli, recombinant microorganism according to any one of <1> to <11> and <27>.
<29>
　Gene encoding the pyridoxamine synthase, or having the nucleotide sequence of SEQ ID NO: 10, a DNA which hybridizes with the DNA under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 10, and pyridoxamine from pyridoxal having a nucleotide sequence encoding a protein having an enzymatic activity to synthesize, <1> to <11> the recombinant microorganism according to any one of <27> and <28>.
<30>
　gene encoding the pyridoxamine synthase, amino acids having an amino acid sequence, or having the amino acid sequence 80% or more sequence identity with SEQ ID NO: 4 and enzyme activity to synthesize pyridoxamine from pyridoxal SEQ ID NO: 4 having a nucleotide sequence encoding a sequence, <1> to <11> and <27> - the recombinant microorganism according to any one of <29>.
<31>
the an amino acid regenerating enzyme is glutamate dehydrogenase, <27> - The recombinant microorganism according to any one of <30>.
<32>
　the gene encoding the amino acid regenerating enzyme is derived from Escherichia coli, recombinant microorganism according to <31>.
<33>
　the gene encoding the amino acid regeneration enzyme, or has the nucleotide sequence of SEQ ID NO: 12, a DNA which hybridizes with the DNA under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 12, and having a nucleotide sequence encoding a protein having a glutamate-regenerating enzyme activity, recombinant microorganisms described <31> or <32>.
<34>
　encoding genes encoding the amino acid regenerating enzyme comprises the amino acid sequence of SEQ ID NO: 6, or amino acid sequence and amino acid sequence having and glutamate regenerating enzyme activity has 80% or more sequence identity with SEQ ID NO: 6, the having a nucleotide sequence that recombinant microorganism according to any one of <31> - <33>.
<35>
　is a recombinant E. coli, the recombinant microorganism according to any one of <1> to <34>.
<36> <1>
　and treated to <35> culture or the recombinant microorganism or the culture of a recombinant microorganism or the recombinant microorganism according to any one of, a pyridoxine or a salt thereof contact is, to produce pyridoxamine or a salt thereof, pyridoxamine or a salt thereof.
<37>
　treatment of the recombinant microorganisms or culture of the recombinant microorganism or the recombinant microorganism or the culture comprises the pyridoxine dehydrogenase, the pyridoxamine synthase, and the amino acid regenerating enzyme, the <36> the method according.
<38>
　treatment of the recombinant microorganism of the workpiece or culture of the recombinant microorganism, heat treatment, cooling treatment, mechanical disruption of the cells, sonication, freeze-thaw treatment, drying treatment, pressurization or depressurization treatment, osmotic pressure treatment, cell autolysis, surfactant treatment, enzyme treatment, a cell separation process, treated by the process comprising one or more selected from the group consisting of purification treatment and extraction, <36> or the process according to <37>.
[0013]
　A second aspect of the present disclosure include the following.
<39>
　The following (1) to (7) Recombinant at least one of the polynucleotide described is introduced into a microorganism:
(1) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 1,
(2) in the amino acid sequence of SEQ ID NO: 1, from 1 to 50 amino acids are deleted, a polynucleotide encoding a protein consisting of the additional or substituted amino acid sequence, the protein pyridoxine or a salt thereof or pyridoxal or its comprises a synthetic activity of the salt, polynucleotide,
(3) a polynucleotide encoding a protein comprising the amino acid sequence having an amino acid sequence at least 60% sequence identity with SEQ ID NO: 1, wherein the protein is pyridoxine or a salt thereof, or pyridoxal or its Comprises a synthetic activity of the salt, polynucleotide,
(4) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 13,
(5) in the nucleotide sequence of SEQ ID NO: 13, 1 to 150 nucleotides are deleted, added or consists substituted by that nucleotide sequence a polynucleotide, the polynucleotide a polynucleotide encoding a protein having an activity of synthesizing the pyridoxine or a salt thereof or pyridoxal or its salt,
(6) A polynucleotide consisting of a nucleotide sequence having a nucleotide sequence at least 60% sequence identity with SEQ ID NO: 13, the polynucleotide encodes a protein having an activity of synthesizing the pyridoxine or a salt thereof or pyridoxal or its salt polynucleotides, which
(7) a polynucleotide hybridizing with a polynucleotide under stringent conditions comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 13, the polynucleotide pyridoxine or a salt thereof or pyridoxal or its polynucleotide encoding a protein having the synthetic activity of the salt.
<40>
　the recombinant microorganism is a recombinant bacterium, a recombinant microorganism according to <39>.
<41>
　the recombinant microorganism is a recombinant E. coli, the recombinant microorganism according to <40>.
<42>
　the protein is an enzyme classified in EC number 1.1.1.65, recombinant microorganism according to any one of <39> - <41>.
<43>
　the protein is pyridoxine dehydrogenase from Saccharomyces cerevisiae, the recombinant microorganism according to any one of <39> - <42>.
<44>
　<39> and recombinant microorganism as claimed in any one of ~ <43>, comprising contacting a pyridoxine or a salt thereof, pyridoxal or a salt thereof.
<45>
　Comprising contacting <39> - and treatment of any one recombinant microorganism culture obtained by culturing or culture according of <43>, and pyridoxine or a salt thereof, pyridoxal or its method for producing a salt.
The invention's effect
[0014]
　According to a first aspect of the present disclosure, the recombinant microorganism capable and inexpensively produce pyridoxine or pyridoxamine, or a salt thereof from a salt with a high production efficiency, and pyridoxamine or from pyridoxine or a salt thereof with a recombinant microorganism how to and inexpensively produced with high production efficiency of the salt is provided.
　According to a second aspect of the present disclosure, expressing have introduced pyridoxine dehydrogenase, a recombinant microorganism is provided.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015]
Shows the alignment of sequences [Figure 1-1] SEQ ID NO: 1 and SEQ ID NO: 22 to SEQ ID NO: 28.
Shows the alignment of sequences [Figure 1-2] SEQ ID NO: 1 and SEQ ID NO: 22 to SEQ ID NO: 28.
Shows the alignment of sequences [Figure 2-1] SEQ ID NO: 3 and SEQ ID NO: 29 to SEQ ID NO: 35.
Shows the alignment of sequences [Figure 2-2] SEQ ID NO: 3 and SEQ ID NO: 29 to SEQ ID NO: 35.
Shows the alignment of sequences [Figure 3-1] SEQ ID NO: 36 to SEQ ID NO: 44.
Shows the alignment of sequences [Figure 3-2] SEQ ID NO: 36 to SEQ ID NO: 44.
DESCRIPTION OF THE INVENTION
[0016]
　A first aspect of the present disclosure (hereinafter, simply referred to as the first embodiment), the gene encodes a gene encoding pyridoxine dehydrogenase, pyridoxamine synthase having an enzyme activity to synthesize pyridoxamine from pyridoxal, and the pyridoxamine synthase There has a gene encoding the amino acid regenerating enzyme having an enzyme activity for reproducing the amino acid consumption,
　Gene encoding the pyridoxine dehydrogenase, wherein at pyridoxamine at least two of the genes encoding the synthase and the gene encoding the amino acid regenerating enzyme is inherent in or originally cells a gene introduced from the outside of the cells its expression is a gene that has been enhanced, a recombinant microorganism (hereinafter, referred to as recombinant microorganism according to the first aspect) provides. A second aspect of the present disclosure (hereinafter, simply referred to as the second aspect) is (1) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 1, in the amino acid sequence of (2) SEQ ID NO: 1, 1 pieces to 50 amino acids are deleted, consists of addition or polynucleotide encoding a protein consisting of the amino acid sequence is substituted, (3) the amino acid sequence of SEQ ID NO: 1 and amino acid sequence having at least 60% sequence identity a polynucleotide encoding a protein, (4) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 13, (5) in the nucleotide sequence of SEQ ID NO: 13, nucleotides 1 to 150 nucleotides are deleted, are added or substituted polynucleotide consisting of SEQ, the least the nucleotide sequence of (6) SEQ ID NO: 13 Polynucleotide consisting of a nucleotide sequence having 60% sequence identity, or, is introducing a polynucleotide which hybridizes with a polynucleotide under stringent conditions comprising a nucleotide sequence complementary to a nucleotide sequence of (7) SEQ ID NO: 13 It provides a recombinant microorganism. Description of "present disclosure" is used to mean including first and second aspects. Note that the introduction in this disclosure, refers to the introduction so as to be expressed in bacterial cells. In addition, the term "recombinant" is used synonymously with "genetically modified".
[0017]

　far by biological methods by a chemical method, a method of and inexpensively produce pyridoxine or pyridoxamine, or a salt thereof from a salt thereof with high productivity has not been known. The structure of pyridoxamine are shown below.
[0018]
[Formula 1]

[0019]
　However, surprisingly, by using a treated product of the culture or the recombinant microorganism or the culture of a recombinant microorganism or the recombinant microorganism having the above configuration, pyridoxamine or a pyridoxine or a salt thereof the ability to inexpensively produced with high production efficiency of the salt present inventor has found. The reason is not necessarily clear, by the three types of enzymes are present together, is balanced and by-products such as and consumption produced in the process of the reaction in the process of generating a pyridoxine or pyridoxamine or a salt salt thereof that the amount of such raw materials, the cooperative action of the three enzymes, is speculated that the order is advantageous for the generation of pyridoxine or pyridoxamine, or a salt thereof from a salt thereof. Also, at least two of the genes encoding the three enzymes, respectively, but inherent in or bacteria is introduced from the outside of the cells by the expression is enhanced, it has been introduced or enhanced gene it is possible to express a high expression level of the enzyme from is estimated that further improve the production efficiency of pyridoxamine, or a salt thereof.
[0020]
　Such above three cooperative action of the enzyme, which has not been found so far, by using a pyridoxine or a salt thereof by which performs each time a reaction of the multi-step as chemical synthesis methods without the need, it has become possible to produce the pyridoxamine, or a salt thereof with high efficiency. Moreover, the use of the recombinant microorganism according to the first aspect, the vitamin B 6 can be avoided by-produced in large quantities of other substances contained in the group. Also, pyridoxine is industrially cheaply available starting materials as compared to pyridoxal, the ability pyridoxine starting material, it becomes possible to inexpensively manufacture the pyridoxamine, or a salt thereof.
[0021]
　
　pyridoxine dehydrogenase is an enzyme which is also known as pyridoxal reductase or pyridoxine 4-dehydrogenase. Pyridoxine dehydrogenase may be an enzyme represented by enzyme number EC1.1.1.65, it may be an enzyme expressed by another enzyme number. Pyridoxine dehydrogenase is an enzyme having an enzyme activity that catalyzes the reaction or which the reverse reaction to convert the addition of hydrogen to the pyridoxal to pyridoxine. Incidentally, pyridoxal and pyridoxine but may also be present as a salt by the environment around, for simplicity of notation, references in this disclosure to salt for a description of the activity of the enzyme is denoted omitted. Pyridoxine dehydrogenase, when adding hydrogen to pyridoxal consumes NADH (reduced nicotinamide adenine dinucleotide) or NADPH (reduced nicotinamide adenine dinucleotide phosphate) as coenzyme when removing hydrogen from pyridoxine consumes NAD + as a coenzyme (oxidized nicotinamide adenine dinucleotide) or NADP + (oxidized nicotinamide adenine dinucleotide phosphate) to. In general conditions, equilibrium reactions the enzyme catalyzes because they heavily biased to pyridoxine producer, a high yield be used for the reaction to consume pyridoxine pyridoxine dehydrogenase been considered impossible to achieve It was. However, in the first aspect, combined with certain other enzymes as described above, the unexpected effect that the productivity of pyridoxine or pyridoxamine, or a salt thereof as the raw material a salt thereof can be achieved at a high production efficiency obtained.
[0022]
　Pyridoxine dehydrogenase is, for example, preferably derived from a microorganism belonging to Ascomycetes (Ascomycota) Gate, or may be derived from a microorganism belonging to Saccharomyces (Saccharomycotina) subphylum or Chawantake (Pezizomycotina) subphylum. More specifically, pyridoxine dehydrogenase, for example, (having the amino acid sequence of SEQ ID NO: 1) pyridoxine dehydrogenase from Saccharomyces cerevisiae (Saccharomyces cerevisiae), Saccharomyces Yubayanusu pyridoxine dehydrogenase from (Saccharomyces Eubayanus) (SEQ ID NO: 22 having the amino acid sequence), Torulaspora Deruburutsuki (having the amino acid sequence of pyridoxine dehydrogenase (SEQ ID NO: 23 derived from Torulaspora delbrueckii)), having the amino acid sequence of pyridoxine dehydrogenase (SEQ ID NO: 24 derived from the Zygosaccharomyces Bairii (Zygosaccharomyces Bailii)) Kluyveromyces marxianus (having the amino acid sequence of SEQ ID NO: 26) pyridoxine dehydrogenase from (Kluyveromyces marxianus), (having the amino acid sequence of SEQ ID NO: 25) pyridoxine dehydrogenase from Aspergillus oryzae (Aspergillus oryzae), Candida albicans (Candida albicans) pyridoxine dehydrogenase (having the amino acid sequence of SEQ ID NO: 27) derived from (have an amino acid sequence of SEQ ID NO: 28) pyridoxine dehydrogenase from Yarrowia Riporiteika (Yarrowia lipolytica), or the like. Among these microorganisms, Aspergillus oryzae belongs to pezizomycotina, otherwise belong to the Saccharomyces Amon.
[0023]
　The pyridoxine dehydrogenase EC1.1.1.65 may be, for example, pyridoxine dehydrogenase such as from Saccharomyces cerevisiae (Saccharomyces cerevisiae) and Schizosaccharomyces pombe (Schizosaccharomyces pombe). Incidentally, pyridoxine dehydrogenase Saccharomyces cerevisiae has the amino acid sequence of SEQ ID NO: 1, pyridoxine dehydrogenase Schizosaccharomyces pombe has the amino acid sequence of SEQ ID NO: 2.
[0024]
　
　The pyridoxamine synthase used in the first embodiment, refers to any enzyme having an enzyme activity to synthesize pyridoxamine from pyridoxal. Incidentally, pyridoxal and pyridoxamine are may also be present as a salt by the environment around, for simplicity of notation, references in this disclosure to salt for a description of the activity of the enzyme is denoted omitted. Examples of pyridoxamine synthase, for example, pyridoxamine - (enzymes expressed, for example enzyme number EC2.6.1.30) pyruvate transaminase, enzymes expressed by aspartate transaminase (e.g. EC2.6.1.1 ), pyridoxamine - oxaloacetic enzyme represented by acetic acid transaminase (e.g. EC2.6.1.31) and pyridoxamine phosphate transaminase (eg, an enzyme expressed by EC2.6.1.54) can be mentioned.
　Note aspartate transaminase represented by EC2.6.1.1 is a holoenzyme that the pyridoxal phosphate as a coenzyme, to transfer the amino group of the aspartic acid to 2-oxoglutarate, generate glutamate and oxaloacetate It has the enzymatic activity to be. In the aspartate transaminase apoenzyme which is not bound to pyridoxal phosphate state, an amino group of glutamic acid or aspartic acid can be synthesized metastasized pyridoxamine to pyridoxal known (JOURNAL OF BIOLOGICAL CHEMISTRY, 1962 January month, Vol.237, No.1, p.127-132). That apoenzyme of aspartate transaminase represented by EC2.6.1.1 is pyridoxamine of EC2.6.1.31 - a oxaloacetic transaminase. Therefore, aspartate transaminase, when pyridoxine or amount Searle phosphoric acid is not present is small coenzymes are present as apoenzyme synthesize pyridoxamine.
　Pyridoxamine synthase in the synthesis of pyridoxal or pyridoxamine, or a salt thereof from a salt thereof, oxidizing the amino group moiety of a particular amino acid (= O), to produce a pyridoxamine or a salt thereof by transferring the amino group . For example, pyridoxamine - pyruvic transaminase are available either in L- alanine and D- alanine, pyridoxamine - oxaloacetic aspartate transaminase states acid transaminase and apoenzyme D- aspartate, L- aspartic acid, D- glutamic acid, and any of L- glutamic acid are available, pyridoxamine phosphate transaminase is available D- glutamic acid.
[0025]
　The pyridoxamine - pyruvate transaminase, e.g., Proteobacteria49, Actinobacteria gate may be one derived from a microorganism belonging to the spirochete Gate or Firmicutes. Pyridoxamine - pyruvate transaminase, e.g., Mezorizobiumu Roti (Mesorhizobium loti), tailed Black Mycobacterium anthropi (Ochrobactrum anthropi) and Pseudomonas (Pseudomonas) genus (e.g., Pseudomonas sp.MA-1) pyridoxamine such as from - pyruvate transaminase it may be. Incidentally, for example, pyridoxamine of Mesorhizobium loti - pyruvic transaminase has an amino acid sequence of SEQ ID NO: 3.
[0026]
　Pyridoxamine - pyruvate transaminase, other, for example, Mesorhizobium sp. YR577 (Mesorhizobium sp YR577.) Pyridoxamine from - (having the amino acid sequence of SEQ ID NO: 29) pyruvic transaminase, pyridoxamine from pseudotype amino Arthrobacter Sari sila butoxy Dance (Pseudaminobacter salicylatoxidans) - pyruvic transaminase (SEQ ID NO: 30 having the amino acid sequence), derived from Bauldia litoralis pyridoxamine - having the amino acid sequence of pyruvate transaminase (SEQ ID NO: 31), pyridoxamine from Skermanella stibiiresistens - having the amino acid sequence of pyruvate transaminase (SEQ ID NO: 32), Rhizobium sp . AC44 / 96 (. Rhizobium sp AC44 / 96) from To pyridoxamine - (having the amino acid sequence of SEQ ID NO: 33) pyruvic transaminase, Erwinia Toretana (Erwinia Toletana) from To pyridoxamine - pyruvic transaminase (SEQ ID NO: 34 amino acids having the sequence), pyridoxamine from Herbiconiux ginsengi - may be a pyruvate transaminase (having the amino acid sequence of SEQ ID NO: 35).
[0027]
　The pyridoxamine - oxaloacetic transaminase, for example, E. coli (Escherichia coli), Rabbit (Oryctolagus cuniculus) and brown rat (Rattus norvegicus) pyridoxamine from such - may be oxaloacetic transaminase. Incidentally, for example, pyridoxamine of Escherichia coli - oxaloacetic transaminase has an amino acid sequence of SEQ ID NO: 4. The aspartate transaminase, for example, E. coli (Escherichia coli), or may be aspartate transaminase such as from Trichoderma viride (Trichoderma viride). Further, the pyridoxamine phosphate transaminase, e.g. Clostridium butyricum (Clostridium butyricum) may be a pyridoxamine phosphate transaminase derived.
[0028]
　
　Amino acid regenerating enzyme used in the first embodiment, it refers to any enzyme which has the enzymatic activity to play an amino acid wherein the pyridoxamine synthase consumed. Pyridoxamine has an amino group, when pyridoxamine synthesis are considered amino acids are consumed as provided source of amino groups. Incidentally, amino acids are can be present as a salt by the environment around, for simplicity of notation, references in this disclosure to salt for a description of the activity of the enzyme is denoted omitted.
　For example, if the pyridoxamine synthase consume L- alanine can be used an enzyme that can play L- alanine as an amino acid regenerating enzyme. Further, when the pyridoxamine synthase to consume L- glutamic acid or L- aspartic acid, an enzyme capable of playing L- glutamic acid or L- aspartic acid can be used as an amino acid regenerating enzyme.
　Examples of such amino-regenerating enzyme, alanine dehydrogenase (eg, an enzyme expressed by the enzyme number 1.4.1.1), glutamate dehydrogenase (eg, enzyme number 1.4.1.2, or 1.4 .1.3, or enzymes expressed by 1.4.1.4), such as a modified alanine dehydrogenase and NADP + / NADPH made available as a coenzyme by modification of amino acid sequences. In the first aspect, pyruvate and NH using NADH or NADPH as a coenzyme 3 using an enzyme activity to produce L- alanine referred to as alanine regenerating enzyme activity, the NADH or NADPH as a coenzyme Te oxoglutarate and NH 3May be referred to as glutamate play enzyme activity that the enzyme activity of producing L- glutamic acid.
　Preferred combination with pyridoxamine synthase, pyridoxamine as pyridoxamine synthase - and oxaloacetate transaminase or aspartate transaminase, - and pyruvate transaminase, pyridoxamine in combination with alanine dehydrogenase as the amino acid regenerating enzyme, and as pyridoxamine synthase combination with glutamate dehydrogenase as an amino acid regenerating enzyme, and the like. Amino regenerating enzyme is inherent in gene or cell introduced from extracellular preferably has an enzymatic activity that play an amino acid pyridoxamine synthetase encoded by a gene whose expression is enhanced consumed.
[0029]
　The amino acid regeneration enzyme is, for example, Shewanella (Shewanella) sp. It may be alanine dehydrogenase (having the amino acid sequence of SEQ ID NO: 36) derived from ac10, (having the amino acid sequence of SEQ ID NO: 37) alanine dehydrogenase from Aeromonas hydrophila (Aeromonas hydrophila), Rhizobium sp. Alanine dehydrogenase from LPU83 (having the amino acid sequence of SEQ ID NO: 38), (having the amino acid sequence of SEQ ID NO: 39) alanine dehydrogenase from Pseudomonas mendocina (Pseudomonas mendocina) in Bradyrhizobium japonicum (Bradyrhizobium japonicum) derived from alanine dehydrogenase (having an amino acid sequence or SEQ ID NO: 41 SEQ ID NO: 40) (having the amino acid sequence of SEQ ID NO: 42) Streptomyces aureofaciens alanine dehydrogenase from (Streptomyces aureofaciens), Anabaena Shirindorika (Anabaena alanine dehydrogenase (sequence derived from cylindrica) Having the amino acid sequence of issue 43), it may be such as Bacillus subtilis (having the amino acid sequence alanine dehydrogenase (SEQ ID NO: 44 derived from Bacillus subtilis)). Alternatively, the amino acid regenerating enzyme may be a glutamate dehydrogenase from in, for example, E. coli (Escherichia coli). Wherein the amino acid regenerating enzyme (e.g. alanine dehydrogenase or glutamate dehydrogenase), it is preferable during the reaction to regenerate the amino acid is available NADPH (reduced nicotinamide adenine dinucleotide phosphate) as coenzyme.
　For example, in the case of alanine dehydrogenase, pyruvate and NH 3 is preferably in from generating the L- alanine reaction is alanine dehydrogenase is available NADPH as a coenzyme. When using amino acid regenerating enzyme available as a coenzyme NADPH is, NADPH / NADP + ratio is changed by consumption of NADPH, pyridoxine dehydrogenase affect the equilibrium and rate of the reaction catalyzed, pyridoxamine or a salt thereof efficiency of production can be further improved. Also, most of the alanine dehydrogenase is NADH (reduced nicotinamide adenine dinucleotide) is available as a coenzyme, it is impossible to utilize NADPH. When using alanine dehydrogenase is available NADPH as coenzyme, NADPH / NADP + ratio is changed by the consumption of the above as NADPH, pyridoxine dehydrogenase affect the equilibrium and rate of the reaction catalyzed, pyridoxamine or it is possible to improve the production efficiency of a salt thereof. Further, the amino acid regenerating enzyme (e.g. alanine dehydrogenase or glutamate dehydrogenase), both in the reaction to regenerate the amino acid of NADH (reduced nicotinamide adenine dinucleotide) and NADPH (reduced nicotinamide adenine dinucleotide phosphate) it is also preferable is available as a coenzyme. Examples of such alanine dehydrogenase, but include proteins having one of the amino acid sequence of SEQ ID NO: 5 and SEQ ID NO: 45 to SEQ ID NO: 52 below, but is not limited thereto.
[0030]
　The pyridoxine dehydrogenase, pyridoxamine synthase, and amino acid regenerating enzyme are both known amino acid sequence having the enzymatic activity (e.g., amino acid sequence, for example the above-exemplified microorganisms or the like is encoded by the naturally occurring gene in an organism of from natural microbial amino acid sequence) that is encoded by a gene having may be a protein having remained unaltered, within a range which does not impair the enzymatic activity for such amino acid sequence (above enzyme activity) it may be a protein having an amino acid sequence plus the sequence modifications. Such modifications, insertion of an amino acid residue, deletion, and addition of additional amino acid residues to substitutions and amino acid sequences N-terminus or C-terminus or both. Insertion of amino acid residues, if there are one or more of the deletions and substitutions, insertions, each of deletions and substitutions, if present, for example, 1 to 30 amino acid residues or 1-20 amino acid residues, or 1-10 amino acid residues, or may be 1-5 amino acid residues, insertion of amino acid residues, deletion and the total number of substituents is, for example 1-50 amino acid residues, or 1-30 amino acid residues, or 1-10 amino acid residues or may be a 1-5 amino acid residues. As the number of amino acid residues added to the ends, first end per example 1-50 amino acid residues, if present, or 1-30 amino acid residues, or 1 to 10 amino acid residues, or 1-5, it may be an amino acid residue. Amino acid residues such additional, may form a signal sequence for secretion or the like into the extracellular. Examples of the signal sequence, and the like OmpA signal sequence of E. coli.
　Alternatively, each enzyme, known amino acid sequence having the enzymatic activity (e.g., an amino acid sequence encoded by a gene naturally present in the organism) known amino acid sequence having protein or the enzymatic activity having itself (e.g. has an amino acid sequence having amino acid sequence encoded by a naturally occurring gene) 80% or more with respect to, or more than 85%, or 90%, or 95% sequence identity to an organism, the desired enzyme activity (described above enzyme activity) may be a protein having. Here, sequence identity can be assessed in default parameters using e.g. BLAST (registered trademark, National Library of Medicine) program.
[0031]
　For example, pyridoxine dehydrogenase, for example, may be a protein having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, substitution of amino acid residues to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, deletions, insertion and may be a protein having the amino acid sequence of performing one or more of the additional N-terminal or C-terminus or additional amino acid residues to both of the amino acid sequence. Amino acid residue substitutions, deletions, for example of the order of addition of additional amino acid residues to the N-terminus or C-terminus or both insertion and amino acid sequences are as described above.
　Alternatively, pyridoxine dehydrogenase, SEQ ID NO: 1 or SEQ ID NO: of the amino acid sequence and amino acid sequence of SEQ ID NO: 2 proteins or SEQ ID NO: 1 having the amino acid sequence of the 2 least one for example 80%, or 85% or more , or 90% or more, or may be a protein having an amino acid sequence having 95% or more sequence identity.
　As described above, when using a protein having an amino acid sequence that is similar to one or more of the amino acid sequence of the amino acid sequence and SEQ ID NO: 2 of SEQ ID NO: 1, the protein having activity as pyridoxine dehydrogenase it should have. Pyridoxine dehydrogenase activity, e.g., pyridoxine and required NAD as substrate + or NADP + the aqueous solution to the test object of proteins containing added or the resulting pyridoxal quantified by high performance liquid chromatography, or produced pyridoxal and tris hydroxymethyl in such absorbance measurements at 415nm such to form a Schiff base between the amine such as aminomethane can be measured by quantifying the Schiff base.
[0032]
　Alternatively, pyridoxine dehydrogenase, SEQ ID NO: 1, SEQ ID NO: 2, and SEQ amino acid sequence of the protein or SEQ ID NO: 1 having any of the amino acid sequence of ID NO: 22 to SEQ ID NO: 28, the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 22 amino acid sequence, the amino acid sequence of SEQ ID NO: 23, the amino acid sequence of SEQ ID NO: 24, SEQ amino acid sequence of ID NO: 25, the amino acid sequence of SEQ ID NO: 26, SEQ amino acid sequence of ID NO: 27, and at least one of the amino acid sequence of SEQ ID NO: 28 One against, for example, 80% or more, or 85% or more, or 90% or more, or may be a protein having an amino acid sequence having 95% or more sequence identity. The protein should also have activity as pyridoxine dehydrogenase.
[0033]
　Alternatively, pyridoxine dehydrogenase includes at least one of the partial amino acid sequence of the following (a) and the partial amino acid sequence (b), may be a pyridoxine dehydrogenase and having pyridoxine dehydrogenase activity. (A)
NX 1 X 2 EX 3 YG (SEQ ID NO: 97) 　　(wherein, X 1 represents V, C, I, A, M, S, G or 　　L, X 2 represents G or A , 　　X 3 represents F or L) (b) X 4 X 5 X 6 KGX 7 (SEQ ID NO: 　　98) (X 4 represents I, V, F or 　　L, X 5 is S, T, N, represents C or 　　M, X 6

Represents C, V, A, I, W or
　　F, X 7 is G, A, represents a S or C) may be a protein having the amino acid sequence.
　Figure 1-1 and Figure 1-2 shows the alignment of SEQ ID NO: 1 and SEQ ID NO: 22 to SEQ ID NO: 28. In Figure 1-1 and Figure 1-2, the pyridoxine dehydrogenase ScPlr is Saccharomyces cerevisiae, SePlr is a pyridoxine dehydrogenase Saccharomyces eubayanus, TdPlr is a pyridoxine dehydrogenase Torulaspora delbrueckii, ZbPlr is a pyridoxine dehydrogenase Zygosaccharomyces bailii, KmPlr the Kluyveromyces the pyridoxine dehydrogenase marxianus, the pyridoxine dehydrogenase AoPlr are Aspergillus oryzae, CaPlr is a pyridoxine dehydrogenase Candida albicans, YlPlr the Yarrowi Representing the pyridoxine dehydrogenase lipolytica. Partial amino acid sequence (a) corresponds to pyridoxine amino acid residue corresponding on alignment to the 55 th to 61 th amino acid residue from the N-terminus of the dehydrogenase of Saccharomyces cerevisiae, partial amino acid sequence (b) is, pyridoxine of Saccharomyces cerevisiae corresponding to amino acid residues corresponding on alignment from the N-terminus of the dehydrogenase 86 th to 91 th amino acid residue. Sequence alignment between the present disclosure, for example, BLAST (registered trademark, National Library of It can be performed in default parameters using Medicine) program. Partial amino acid sequence (a) is preferably present in the 45 th to the region of 71 amino acid residues from the N-terminus of the protein is present in the region of 46-th to 66 th amino acid residue from the N-terminal it is more preferable. Partial amino acid sequence (b) is preferably present in the region of 76-th to 101 th amino acid residue from the N-terminus of the protein is present in the region of 79-th to 96 th amino acid residue from the N-terminal it is more preferable.
[0034]
　In this disclosure, the term "corresponding amino acid residues in the X amino acid residues from the N-terminus of the enzyme A" in the amino acid sequence of the enzyme B, and the amino acid sequence of the enzyme A were aligned to the amino acid sequence of the enzyme B Occasionally, it refers to the amino acid residues on the amino acid sequence of the enzyme B corresponding the N-terminal amino acid sequence of the enzyme a and X amino acid residue.
[0035]
　As can be seen from FIGS. 1-1 and FIG. 1-2, the partial amino acid sequence (a) and the partial amino acid sequence (b) is a highly conserved region among the group of pyridoxine dehydrogenase. Accordingly, variation of pyridoxine dehydrogenase comprising at least one of the partial amino acid sequence (a) and the partial amino acid sequence (b) is likely to function as pyridoxine dehydrogenase in the present disclosure is considered to be high. Also, Saccharomyces cerevisiae pyridoxine 60th alignment on the corresponding amino acid residue to tyrosine residue from the N-terminus of the dehydrogenase, and Saccharomyces cerevisiae pyridoxine amino acid residue corresponding on alignment to 89 lysine residues from the N-terminus of dehydrogenase of are all believed to NADPH binding residues (Journal of Biological Chemistry 1990, 274 (33), 23185-23190), the function of pyridoxine dehydrogenase is an important residue. If it is these residues stored, probability that functions as pyridoxine dehydrogenase in the present disclosure is considered to be high.
[0036]
　Another representation of the region containing the Saccharomyces cerevisiae pyridoxine amino acid residue corresponding on alignment from the N-terminus to 60th tyrosine residue dehydrogenase include the following partial amino acid sequence (a-1). Pyridoxine dehydrogenase may comprise partial amino acid sequence of (a-1) in place of the partial amino acid sequence (a). (A-1)
X 1 X 2 NX 3 X 4 EX 5 YGX 6 X 7 (SEQ ID NO: 　　109) X 1 represents F, L, I, M, Y or 　　V, X 2 is F, I, Y, L, represents a W or 　　V, X 3 is, V, C, I, a , M, S, represents G or 　　L, X 4 represents a G or 　　a, X 5 is F or L It 　　represents, X

6 represents P, K, R, G, E, T, A, N or
　　S, X 7 represents D, N, H, K, E, P, L or I.
　Partial amino acid sequence (a-1) is equivalent to pyridoxine amino acid residue corresponding on alignment from the N-terminus to the 53 th to 63 th amino acid residue of the dehydrogenase Saccharomyces cerevisiae. Partial amino acid sequence (a-1) is preferably present in the region of amino acid residues from the N-terminus of the 43 th to 73 th protein, the 45 th to the region of 68 amino acid residues from the N-terminus it is more preferably present.
[0037]
　Another representation of the region containing the Saccharomyces cerevisiae pyridoxine amino acid residue corresponding on alignment from the N-terminus to 89th lysine residue of dehydrogenase, include the following partial amino acid sequence (b-1). Pyridoxine dehydrogenase may comprise partial amino acid sequence (b-1) in place of the partial amino acid sequence (b). (B-1)
X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 KGX 9 (SEQ ID NO: 　　110) X 1 represents R, K, A, S or 　　N, X 2 is, K, S, E, Q, represents a D or 　　a, X 3 is, D, H, N, Y , E, K, C, represents Q or 　　R, X 4 is, V, T, I, M or L It 　　represents, X

5 represents V, I, L, F, M, or
　　T, X 6 represents I, V, F or
　　L, X 7 represents S, T, N, C or
　　M, X 8 is represents C, V, a, I, W or
　　F, X 9 represents G, a, S or C.
　Partial amino acid sequence (b-1) is equivalent to pyridoxine amino acid residue corresponding on alignment from the N-terminus to the 81 th to 91 th amino acid residue of the dehydrogenase Saccharomyces cerevisiae. Partial amino acid sequence (a-1) is preferably present in the region of amino acid residues from the N-terminus of the 71 th to 101 th of the protein, the 74 th to the region of 96 amino acid residues from the N-terminus it is more preferably present.
[0038]
　Further, pyridoxamine synthase, for example, may be a protein having the amino acid sequence of SEQ ID NO: 3, N-terminal substitutions, deletions, insertions and amino acid sequence of amino acid residues to the amino acid sequence of SEQ ID NO: 3 or it may be a protein having one or more amino acid sequences were performed of adding the C-terminus or additional amino acid residues to both. Amino acid residue substitutions, deletions, for example of the order of addition of additional amino acid residues to the N-terminus or C-terminus or both insertion and amino acid sequences are as described above.
　Alternatively, pyridoxamine synthase, SEQ ID NO: 3 protein or having the amino acid sequence of SEQ ID NO: 3 amino acid sequence with respect to, for example, 80% or more, or 85% or more, or 90%, or 95% or more sequence identity to it may be a protein having an amino acid sequence having.
　As described above, when using a protein having an amino acid sequence similar to the amino acid sequence of SEQ ID NO: 3, the protein is an enzyme activity to synthesize pyridoxamine from activity (pyridoxal as pyridoxamine synthase, in a first aspect It should have also referred to) and pyridoxamine synthase activity. Enzyme activity to synthesize pyridoxamine from pyridoxal, for example, as a substrate pyridoxal and necessary amino acids (in the case of a protein having an amino acid sequence similar to the amino acid sequence of SEQ ID NO: 3, for example L- alanine) of the subject test in an aqueous solution containing protein was added, the amount of generated pyridoxamine can be measured by quantifying by high performance liquid chromatography.
[0039]
　Alternatively, pyridoxamine synthase any amino acid sequence of the protein or SEQ ID NO: 3 having the amino acid sequence, the amino acid sequence of SEQ ID NO: 29, the amino acid sequence of SEQ ID NO: 30 of SEQ ID NO: 3 and SEQ ID NO: 29 to SEQ ID NO: 35, amino acid sequence of SEQ ID NO: 31, SEQ amino acid sequence of ID NO: 32, the amino acid sequence of SEQ ID NO: 33, the amino acid sequence of SEQ ID NO: 34, and the amino acid sequence of SEQ ID NO: 35 at least one relative, for example, 80% or more, or 85 % or more, or 90% or more, or may be a protein having an amino acid sequence having 95% or more sequence identity. The protein should also have activity as pyridoxamine synthase. In this case, the enzyme activity to synthesize pyridoxamine from pyridoxal, for example, by pyridoxal and L- alanine aqueous tested proteins, including as a substrate was added, the amount of generated pyridoxamine quantified by high performance liquid chromatography It can be measured.
[0040]
　Alternatively, pyridoxamine synthase partial amino acid sequence of the following (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) at least one comprises, and may be a pyridoxamine synthase having an enzyme activity to synthesize pyridoxamine from pyridoxal among. In this case, the enzyme activity to synthesize pyridoxamine from pyridoxal, for example, by pyridoxal and L- alanine aqueous tested proteins, including as a substrate was added, the amount of generated pyridoxamine quantified by high performance liquid chromatography It can be measured. (C)
X 8 X 9 X 10 X 11 X 12 X 13 (SEQ ID NO: 　　99) (X 8 represents L, M, I or 　　V, X 9 represents H or 　　Q, X 10 is, G It represents C or 　　a, X 11 represents E or 　　D, X 12

Represents P or
　　A, X 13 is V, I, represents L or
A) (d) X 14 X 15 TPSGTX 16 X 17 (SEQ ID NO: 　　100) (X 14 represents H or 　　S, X 15 represents a D or 　　E, X 16 is, I, represents V or 　　L, X 17 represents N or T) (e) X 18 DX 19 VSX 20 X 21 (SEQ ID NO: 　　101) (X 18 is, V, represents I or 　　a, X 19

Is, A, represents T or
　　S, X 20 is, S, represents A or
　　G, X 21 is, F, W, or an
V) (f) X 22 X 23 X 24 KCX 25 GX 26 X 27 P (SEQ ID NO: 　　102) (X 22 represents G or 　　S, X 23 represents P, S or 　　a, X 24 represents N, G, S, a or 　　Q, X 25 is L or an 　　M, X 26 represents a, S, C or 　　G, X 27 represents P, T, S or a)

(G) X 28 X 29 X 30 X 31 SX 32 GX 33 X 34 (SEQ ID NO:
103) (X 28 represents G or
　　D, X 29 represents V or
　　I, X 30 is V, T represents a, S, M, I or
　　L, X 31 represents F, M, L, I or
　　V, X 32 represents S, G, a, T, I, L or
　　H, X 33 is R, represents M or
　　Q, X 34 represents G, R, a, D, H or
K) (h) X 35 X 36RX 37 X 38 HMGX 39 X 40 A (SEQ ID NO:
　　104) (X 35 represents L or
　　V, X 36 represents T, I, V or
　　L, X 37 represents I, V or L ,
　　X 38 represents G or
　　S, X 39 is, P, represents a or
　　R, X 40 represents T, V or S)
[0041]
　Figure 2-1 and Figure 2-2 shows the alignment of the sequence of SEQ ID NO: 3 and SEQ ID NO: 29 to SEQ ID NO: 35. In Figure 2-1 and Figure 2-2, pyridoxamine of MlPPAT is Mesorhizobium loti - pyruvate transaminase, MsPPAT the Mesorhizobium sp. Pyruvate transaminase, PsPPAT is pyridoxamine of Pseudaminobacter salicylatoxidans - - pyridoxamine of YR577 pyruvate transaminase, BlPPAT is pyridoxamine of Bauldia litoralis - pyruvate transaminase, SsPPAT is pyridoxamine of Skermanella stibiiresistens - pyruvate transaminase, RsPPAT is Rhizobium sp. Pyruvate transaminase, EtPPAT the pyridoxamine of Erwinia toletana - - AC44 / 96 pyridoxamine in pyruvate transaminase, HgPPAT the pyridoxamine of Herbiconiux ginsengi - represents a pyruvate transaminase. Partial amino acid sequence (c) is, pyridoxamine of Mesorhizobium loti - corresponds to amino acid residues corresponding on alignment to the N-terminus to the 65th ~ 70th amino acid residue of pyruvic transaminase, partial amino acid sequence (d) are, Mesorhizobium loti of pyridoxamine - corresponds to amino acid residues corresponding to the alignment to 144 th to 152 th amino acid residue from the N-terminus of pyruvic transaminase, partial amino acid sequence (e) is pyridoxamine of Mesorhizobium loti - pyruvate transaminase N corresponds to amino acid residues corresponding on alignment from end to 170 th to 176 th amino acid residue, partial amino acid sequence (f) is the Mesorhizobium loti Pyridoxamine - 19 from the N-terminus of pyruvate transaminase Corresponds to the corresponding amino acid residues on alignments to the fourth to 203 th amino acid residue, partial amino acid sequence (g) is pyridoxamine of Mesorhizobium loti - 329 th from N-terminal of pyruvic transaminase to 337 th amino acid residue the equivalent to the corresponding amino acid residue on the alignments, partial amino acid sequence (h) is the Mesorhizobium loti pyridoxamine - from the N-terminus of pyruvate transaminase in 343 th to 353 th amino acid residue with an amino acid residue corresponding on alignment Equivalent to. Partial amino acid sequence (c) is preferably present in the 55 th to 80 th in the region of amino acid residues from the N-terminus of the protein is present in the region of 56 th to 75 th amino acid residue from the N-terminal it is more preferable. Partial amino acid sequence (d) is preferably present in the region of 134 th to 162 th amino acid residue from the N-terminus of the protein is present in the region of 139 th to 157 th amino acid residue from the N-terminal it is more preferable. Partial amino acid sequence (e) is preferably present in the 160th ~ region of 186 amino acid residue from the N-terminus of the protein is present in the region of 165 th to 181 th amino acid residue from the N-terminal it is more preferable. Partial amino acid sequence (f) is preferably present in the region of 184 th to 213 th amino acid residue from the N-terminus of the protein is present in the region of 189 th to 208 th amino acid residue from the N-terminal it is more preferable. Partial amino acid sequence (g) is preferably present in the region of 319 th to 347 th amino acid residue from the N-terminus of the protein is present in the region of 324 th to 342 th amino acid residue from the N-terminal it is more preferable. Partial amino acid sequence (h) is preferably present in the region of 333 th to 363 th amino acid residue from the N-terminus of the protein is present in the region of 338 th to 358 th amino acid residue from the N-terminal it is more preferable.
[0042]
　As can be seen from FIGS. 2-1 and 2-2, 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 partial amino acid sequence (h), the group of pyridoxamine - a highly conserved region between the pyruvate transaminase. Therefore, pyridoxamine contains at least one of the partial amino acid sequence (c) ~ partial amino acid sequence (h) - Variation of pyruvate transaminase probability that functions as pyridoxamine synthase in the present disclosure is considered to be high. Further, pyridoxamine of Mesorhizobium loti - amino acid residue corresponding on alignment from the N-terminus to 197 lysine residues pyruvate transaminase is important for binding to pyridoxal, on alignments 68th glutamic acid residue from the N-terminus corresponding amino acid residues are important for catalytic activity, the amino acid residue corresponding on the alignment of amino acid residues and N-terminal corresponding on alignment to 171 th aspartic acid residue from the N-terminus to 146th threonine residue to assist the binding of pyridoxal, amino acid residue corresponding on alignment from the corresponding amino acid residues and N-terminus on alignments 336 arginine residue from the N-terminus to the 345th arginine residues are important to the amino acid recognition and considered Gills are and (Journal of Biological Chemistry, 2008, vol. 283, No. 2 pp1120-1127), on the function of pyridoxine synthase is an important residue. If it is these residues stored, probability that functions as pyridoxine synthase in the present disclosure is considered to be high. However, pyridoxamine of Mesorhizobium loti - amino acid residue corresponding on alignment from the N-terminus of pyruvate transaminase 68th glutamic acid residue may be aspartic acid other than glutamic acid. Further, pyridoxamine of Mesorhizobium loti - amino acid residue corresponding on alignment from the N-terminus of pyruvate transaminase 336 arginine residues may be methionine or glutamine in addition to arginine.
[0043]
　Mesorhizobium loti of pyridoxamine - from the N-terminus of pyruvate transaminase 68th glutamic acid residue as a separate representation of the region containing the corresponding amino acid residues on alignments include the following partial amino acid sequence (c-1) it is. Pyridoxamine synthase may contain the partial amino acid sequence (c-1) in place of the partial amino acid sequence (c). (C-1)
X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19(SEQ ID NO:
　　111) X 1 is represents V, L, I or
　　M, X 2 is I, represents L or
　　V, X 3 represents L, M, I or
　　V, X 4 is H or represents
　　Q, X 5 is, G, represents C or
　　a, X 6 represents E or
　　D, X 7 represents P or
　　a, X 8 represents V, I, a or L ,
　　X 9 represents L, M, P or
　　V, X 10 represents G or
　　a, X 11 represents L or
　　I, X 12 represents E or Q,
　　X 13 represents A or
　　G, X 14 represents A or
　　V, X 15 represents A or
　　L, X 16 represents A, L, H or
　　Y, X 17 is, S, It represents G or
　　a, X 18 represents L, F, V or
　　a, X 19 represents I, F, V or L.
　Partial amino acid sequence (c-1) is pyridoxamine of Mesorhizobium loti - corresponding to amino acid residues corresponding on alignment from the N-terminus of pyruvate transaminase in 63 th to 81 th amino acid residue. Partial amino acid sequence (c-1) is preferably present in the region of 53 th to 91 th amino acid residue from the N-terminus of the protein, in the region of 58-th to 86 th amino acid residue from the N-terminal it is more preferably present.
[0044]
　Mesorhizobium loti of pyridoxamine - from the N-terminus of pyruvate transaminase 146th threonine residue as another representation of the region containing the corresponding amino acid residues on alignments include the following partial amino acid sequence (d-1). Pyridoxamine synthase may contain the partial amino acid sequence (d-1) in place of the partial amino acid sequence (d). (D-1)
X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 TPSGTX 9 X 10 X 11 X 12 X　13 X 14 X 15 X 16 (SEQ ID NO: 　　112) X 1 is, V, I, It represents an L or M,

　　X 2 represents V or
　　I, X 3 is S, A, V, represents C or
　　F, X 4 is V, I, A represents L or
　　T, X 5 is a C or V
　　represents, X 6 is H, represents N or
　　a, X 7 represents H or
　　S, X 8 represents D or
　　E, X 9 represents I, V or
　　L, X 10 is It represents N or
　　T, X 11 represents P or
　　D, X 12 represents I, V, L or
　　a, X 13 is, D, N, E, a , G, Q, V, R or It represents a
　　P, X 14 represents A, E, and Q or
　　D, X 15 represents I or
　　L, X 16 represents G or A.
　Partial amino acid sequence (d-1), pyridoxamine of Mesorhizobium loti - corresponding to amino acid residues corresponding on alignment from the N-terminus of pyruvate transaminase in 138 th to 158 th amino acid residue. Partial amino acid sequence (d-1) is preferably present in the region of 128 th to 168 th amino acid residue from the N-terminus of the protein, in the region of 133 th to 163 th amino acid residue from the N-terminal it is more preferably present.
[0045]
　Mesorhizobium loti of pyridoxamine - from the N-terminus of pyruvate transaminase in 171 th aspartic acid residue as a separate representation of the region containing the corresponding amino acid residues on alignments include the following partial amino acid sequence (e-1). Pyridoxamine synthase may contain the partial amino acid sequence (e-1) in place of the partial amino acid sequence (e). (E-1)
X 1 X 2 X 3 X 4 X 5 X 6 DX 7 VSX 8 X 9 X 10 X 11 X 12 (SEQ ID NO: 　　113) X 1 is, G, D, or 　　A X 2 is, A, G represents K, T, Q, R or 　　E, X 3 represents Y, N, L or F,

　　X 4 is, L, F, represent M or
　　V, X 5 is, I, represents L or
　　Y, X 6 is, V, represents A or
　　I, X 7 is, A, represents S or T,
　　X 8 is, S, represents a or
　　G, X 9 is, F, represents W or
　　V, X 10 represents G, a or
　　L, X 11 represents G or
　　S, X 12 is M, representing a V or L.
　Partial amino acid sequence (e-1) is, pyridoxamine of Mesorhizobium loti - corresponding to amino acid residues corresponding on alignment from the N-terminus of pyruvate transaminase 165 th to 179 th amino acid residue. Partial amino acid sequence (e-1) is preferably present in the region of amino acid residues from the N-terminus of the 155-th to 189-th protein, in the region of 160 th to 184 th amino acid residue from the N-terminal it is more preferably present.
[0046]
　Mesorhizobium loti of pyridoxamine - from the N-terminus of pyruvate transaminase 197th lysine residue as another representation of the region containing the corresponding amino acid residues on alignments include the following partial amino acid sequence (f-1) it is. Pyridoxamine synthase may include a partial amino acid sequence (f-1) in place of the partial amino acid sequence (f). (F-1)
X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 KCX 10 GX 11 X 12 PX 13 X 14 X 15 X 16 X 17 X 18 X 19S (SEQ ID NO:
114) X 1 represents A, S, V or
I, X 2 is, D, represents G or
A, X 3 represents I, L, F, V or
　　M, X 4 represents Y, F, L or
　　C, X 5 represents V or
　　I, X 6 represents T or
　　a, X 7 represents G or
　　S, X 8 is, P, S or represents
　　a, X 9 represents N, G, S, and Q or
　　a, X 10 represents L or
　　M, X 11 represents a, S, C or
　　G, X 12 is, P, T, S or a,
　　X 13 is, G, represents A or
　　S, X 14 represents L or
　　V, X 15 is, T, S or
　　A, X 16 represents M, I, L, V or F,
　　X 17 represents M, L, V, a or
　　I, X 18 represents G, a, H or
　　S, X 19 represents V, I or a.
　Partial amino acid sequence (f-1) is, pyridoxamine of Mesorhizobium loti - corresponding to amino acid residues corresponding on alignment from the N-terminus of pyruvate transaminase in 188 th to 211 th amino acid residue. Partial amino acid sequence (f-1) is preferably present in the region of 178 th to 221 th amino acid residue from the N-terminus of the protein, in the region of 183 th to 216 th amino acid residue from the N-terminal it is more preferably present.
[0047]
　Mesorhizobium loti of pyridoxamine - from the N-terminus of pyruvate transaminase 336 arginine residue as another representation of the region containing the corresponding amino acid residues on alignments include the following partial amino acid sequence (g-1) is. Pyridoxamine synthase may contain the partial amino acid sequence (g-1) in place of the partial amino acid sequence (g). (G-1)
X 1 X 2 X 3 X 4 X 5 SX 6 GX 7 X 8 (SEQ ID NO: 　　115) X 1 represents Y, F, H or 　　S, X 2 represents G or D, 　　X 3 represents V or 　　I, X 4 represents V, T, a, S, M, I or 　　L, X 5 represents F, M, L, I or V,

　　X 6 represents S, G, A, T, I, L or
　　H, X 7 is R, represents M or
　　Q, X 8 represents G, R, A, D, H or K.
　Partial amino acid sequence (g-1) is, pyridoxamine of Mesorhizobium loti - corresponding to amino acid residues corresponding on alignment from the N-terminus of pyruvate transaminase in 328 th to 337 th amino acid residue. Partial amino acid sequence (g-1) is preferably present in the region of 318 th to 347 th amino acid residue from the N-terminus of the protein, in the region of 323 th to 342 th amino acid residue from the N-terminal it is more preferably present.
[0048]
　Mesorhizobium loti of pyridoxamine - from the N-terminus of pyruvate transaminase 345th arginine residue as another representation of the region containing the corresponding amino acid residues on alignments include the following partial amino acid sequence (h-1) it is. Pyridoxamine synthase may contain the partial amino acid sequence (h-1) instead of the partial amino acid sequence (h). (H-1)
X 1 X 2 X 3 X 4 X 5 RX 6 X 7 HMGX 8 X 9 AX 10 X 11 (SEQ ID NO: 　　116) X 1 is, L, Q, K, A , F, Y or W 　　represents, X 2 is, G, N, H or 　　D, X 3 is, K, represents the R or 　　N, X

4 represents L or
　　V, X 5 represents T, I, V or
　　L, X 6 is, I, represents V or
　　L, X 7 represents G or
　　S, X 8 is P represents a or
　　R, X 9 is, T, represents the V or
　　S, X 10 represents Q, R, E, K, H, Y or
　　G, X 11 represents P or G.
　Partial amino acid sequence (h-1) is, pyridoxamine of Mesorhizobium loti - corresponding to amino acid residues corresponding on alignment from the N-terminus of pyruvate transaminase 340 th to 355 th amino acid residue. Partial amino acid sequence (h-1) is preferably present in the region of amino acid residues from the N-terminal 330-th to 365 th of the protein, the 335 th to the region of 360 amino acid residue from the N-terminal it is more preferably present.
[0049]
　Alternatively, pyridoxamine synthase, for example, may be a protein having the amino acid sequence of SEQ ID NO: 4, N-terminal substitutions, deletions, insertions and amino acid sequence of amino acid residues to the amino acid sequence of SEQ ID NO: 4 or it may be a protein having one or more amino acid sequences were performed of adding the C-terminus or additional amino acid residues to both. Amino acid residue substitutions, deletions, for example of the order of addition of additional amino acid residues to the N-terminus or C-terminus or both insertion and amino acid sequences are as described above.
　Alternatively, pyridoxamine synthase, SEQ ID NO: protein having the amino acid sequence of 4 or SEQ ID NO: 4 amino acid sequence to, for example, 80% or more, or 85% or more, or 90%, or 95% or more sequence identity to it may be a protein having an amino acid sequence having.
　As described above, when using a protein having an amino acid sequence similar to the amino acid sequence of SEQ ID NO: 4, the protein has an activity (enzyme activity to synthesize pyridoxamine from pyridoxal) as pyridoxamine synthase it should. Enzyme activity to synthesize pyridoxamine from pyridoxal, for example, pyridoxal and necessary amino acids (in the case of a protein having an amino acid sequence similar to the amino acid sequence of SEQ ID NO: 4, for example L- glutamic acid or L- aspartic acid or a salt thereof as a substrate ) the aqueous solution tested proteins, including adding, the amount of generated pyridoxamine can be measured by quantifying by high performance liquid chromatography.
[0050]
　In addition, amino acid regeneration enzyme is, for example, Shewanella sp. It may be used a protein having AC10 derived amino acid regenerating enzyme amino acid sequence (SEQ ID NO: 36) amino acid sequence was the amino acid substitutions D198A against (SEQ ID NO: 5). This protein is alanine dehydrogenase represented by enzyme number 1.4.1.1. Not limited thereto, relative to alanine dehydrogenase represented by enzyme number 1.4.1.1, NADP + can be used alanine dehydrogenase with a corresponding amino acid substitutions. Below Journal of Molecular Catalysis B: Enzymatic 30 (2004) 173-176 as described in, alanine dehydrogenase most things NAD as a coenzyme + can be used to (oxidized + ( oxidized nicotinamide adenine dinucleotide phosphate) can not be used. However, a protein having the amino acid sequence of SEQ ID NO: 5 was performed amino acid substitutions of the D198A as coenzyme NAD + and NADP + Both can be used (see supra). Note that a protein having the amino acid sequence of SEQ ID NO: 5, the reverse reaction (pyruvate and NH 3 at the time of the L- alanine reproduced from), of course, both of NADH and NADPH can be used.
[0051]
　Shewanella sp. AC10 it is possible to perform the same amino acid substitutions also alanine dehydrogenase other than from alanine dehydrogenase. Here, in the amino acid sequence of alanine dehydrogenase, the corresponding residue on the alignment from the N-terminus of the amino acid sequence of SEQ ID NO: 36 to 198 th aspartic acid residue, the replacement of the alanine residue, NADP + corresponding amino acid It is referred to as a replacement. Alanine dehydrogenase as the amino acid regenerating enzyme is Aeromonas hydrophila (Aeromonas hydrophila) NADP alanine dehydrogenase from + corresponding amino acid substitutions protein having (in this case, D198A) the amino acid sequence was carried out (SEQ ID NO: 45), Rhizobium sp. LPU83 NADP alanine dehydrogenase from (Rhizobium sp. LPU83) + corresponding amino acid substitutions protein having (in this case, D198A) the amino acid sequence was carried out (SEQ ID NO: 46), the alanine dehydrogenase from Pseudomonas mendocina (Pseudomonas mendocina) NADP + corresponding amino acid substitutions protein having (in this case, D198A) the amino acid sequence was carried out (SEQ ID NO: 47), the alanine dehydrogenase from Bradyrhizobium japonicum (Bradyrhizobium japonicum) NADP +Corresponding amino acid substitution protein having (in this case, D198A) the amino acid sequence was carried out (SEQ ID NO: 48 or SEQ ID NO: 49), NADP alanine dehydrogenase from Streptomyces aureofaciens (Streptomyces aureofaciens) + corresponding amino acid substitution ( proteins with this case D198A) the amino acid sequence was carried out (SEQ ID NO: 50), Anabaena Shirindorika (Anabaena cylindrica NADP alanine dehydrogenase from) + corresponding amino acid substitution (in this case, D198A) the amino acid sequence was carried out (SEQ ID NO: proteins with 51), NADP alanine dehydrogenase from Bacillus subtilis (Bacillus subtilis) + corresponding amino acid Conversion (in this case, D196A) may be a protein having the amino acid sequence was performed (SEQ ID NO: 52).
[0052]
　Incidentally, NAD + in the case of available reaction conditions, the amino acid regenerating enzyme NADP + can be used without the corresponding amino acid substitutions, other than this, expressed in enzyme number 1.4.1.1 that alanine dehydrogenase can be generally used.
[0053]
　The amino acid regenerating enzyme, for example having the amino acid sequence of SEQ ID NO: 6, or may be a glutamate dehydrogenase from Escherichia coli. Glutamate dehydrogenase, 2-oxoglutarate and NH using NADH or NADPH as a coenzyme 3 having the enzymatic activity of producing glutamic acid L- from.
[0054]
　The amino acid regenerating enzyme, for example, may be a protein having the amino acid sequence of SEQ ID NO: 5, N-terminal substitutions, deletions, insertions and amino acid sequence of amino acid residues to the amino acid sequence of SEQ ID NO: 5 or it may be a protein having one or more amino acid sequences were performed of adding the C-terminus or additional amino acid residues to both. Amino acid residue substitutions, deletions, for example of the order of addition of additional amino acid residues to the N-terminus or C-terminus or both insertion and amino acid sequences are as described above.
　Alternatively, amino acid regeneration enzymes, SEQ ID NO: protein having the amino acid sequence of 5 or SEQ ID NO: 5 of the amino acid sequence to, for example, 80% or more, or 85% or more, or 90%, or 95% or more sequence identity to it may be a protein having an amino acid sequence having.
　As described above, when using a protein having an amino acid sequence similar to the amino acid sequence of SEQ ID NO: 5, the protein should have alanine regenerating enzyme activity. Further, it is preferable to retain the Ala residue corresponding on sequence alignment from the N-terminus of the amino acid sequence of SEQ ID NO: 5 198 th Ala residues or thereto.
[0055]
　Alternatively, amino acid regenerating enzyme is a protein having any of the amino acid sequence of SEQ ID NO: 5 and SEQ ID NO: 45 to SEQ ID NO: 52, or SEQ ID NO: 5, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 and the amino acid sequence of SEQ ID NO: 52, for example, 80% or more, or 85% or more, or 90% or more, or having an amino acid sequence having 95% or more sequence identity a protein may be.
　As described above, SEQ ID NO: 5, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, having an amino acid sequence similar to SEQ ID NO: 49, SEQ ID NO: 50, the amino acid sequence of SEQ ID NO: 51 and SEQ ID NO: 52 when using a protein, said protein should have alanine regenerating enzyme activity. Further, it is preferable to retain the Ala residue corresponding on sequence alignment from the N-terminus of the amino acid sequence of SEQ ID NO: 5 198 th Ala residues or thereto.
[0056]
　Alternatively, amino acid regenerating enzyme, for example, may be a protein having the amino acid sequence of SEQ ID NO: 6, N-terminal substitutions, deletions, insertions and amino acid sequence of amino acid residues to the amino acid sequence of SEQ ID NO: 6 or it may be a protein having one or more amino acid sequences were performed of adding the C-terminus or additional amino acid residues to both. Amino acid residue substitutions, deletions, for example of the order of addition of additional amino acid residues to the N-terminus or C-terminus or both insertion and amino acid sequences are as described above.
　Alternatively, amino acid regeneration enzymes, SEQ ID NO: protein having the amino acid sequence of 6 or SEQ ID NO: 6 amino acid sequence to example 80% or more, or 85%, or 90%, or 95% or more sequence identity to it may be a protein having an amino acid sequence having.
　As described above, when using a protein having an amino acid sequence similar to the amino acid sequence of SEQ ID NO: 6, the protein should have a glutamate regenerating enzyme activity.
[0057]
　Amino regenerating enzyme activity, for example, the aqueous solution to be tested for protein added containing NADP or NADPH as occasion substrate and Required to quantify the amount of desired amino acid as a reaction product in high performance liquid chromatography that It can be measured by. For example, alanine regenerating enzyme activity, for example pyruvic acid, NH 3 and an aqueous solution to be tested proteins, including NADH or NADPH addition, by the amount of L- alanine as the reaction product is quantified by high performance liquid chromatography It can be measured. Furthermore, glutamate regenerating enzyme activity, for example, 2-oxoglutarate, NH 3 aqueous solution to the protein to be tested containing and NADH or NADPH addition, to quantify the amount of L- glutamic acid as the reaction product by high performance liquid chromatography It can be measured by.
[0058]
　Alternatively, alanine dehydrogenase partial amino acid sequence (i), the partial amino acid sequence (j), wherein at least one of the partial amino acid sequence (k) and the partial amino acid sequence (l), and pyruvate and NH 3 from L- it may be an enzyme having the activity to form alanine. For even such alanine dehydrogenase, Shewanella sp. By replacing the alanine residue amino acid residues corresponding on alignment from the N-terminus of AC10 198 th aspartic acid residue, as a coenzyme NAD + and NADP + Both can be made available. (I)
EX 41 KX 42 X 43 EX 44 RX 45 X 46 (SEQ ID NO: 　　105) (X 41 represents I, T, S, F, N or 　　V, X 42 is N, M, A, V , L represents T or 　　D, X 43 represents H, N, L or Q,

　　X 44 is, Y, represents N or
　　F, X 45 is, V, or an
　　I, X 46 represents G or
A) (j) X 47 X 48 X 49 KVKEPX 50 (SEQ ID NO: 　　106) ( X 47 represents M or 　　L, X 48 is I, represents L or 　　V, X 49 is V, L, represents I or 　　M, X 50 is Q, L, V, N or I expressed) (k) LX 51 TYLHLA (SEQ ID NO: 　　107) (X 51 represents F or Y) (l) X 52

DX 53 AX 54 DQGG (SEQ ID NO:
　　108) (X 52 represents V or
　　A, X 53 represents V or
　　I, X 54 represents I or V)
[0059]
　Figure 3-1 and Figure 3-2 shows the alignment of SEQ ID NO: 36 to SEQ ID NO: 44. In Figure 3-1 and Figure 3-2, SsAdh the Shewanella sp. Alanine dehydrogenase of AC10, AhAdh is an alanine dehydrogenase of Aeromonas hydrophila, RsAdh is Rhizobium sp. Alanine dehydrogenase LPU83, PmAdh is alanine dehydrogenase Pseudomonas mendocina, one of alanine dehydrogenase BjAdh1 is Bradyrhizobium japonicum, BjAdh2 is another one of the alanine dehydrogenase of Bradyrhizobium japonicum, SaAdh alanine dehydrogenase Streptomyces aureofaciens the, AcAdh is alanine dehydrogenase Anabaena cylindrica, BsAdh represents alanine dehydrogenase Bacillus subtilis. Partial amino acid sequence (i) is, Shewanella sp. Corresponds to the corresponding amino acid residues on the alignment from the N-terminus of the alanine dehydrogenase AC10 8 th to 17 th amino acid residue, partial amino acid sequence (j) is, Shewanella sp. Corresponds to the corresponding amino acid residues on the alignment from the N-terminus of the alanine dehydrogenase AC10 to 70 th to 78 th amino acid residue, partial amino acid sequence (k) is, Shewanella sp. Corresponds to the corresponding amino acid residues on the alignment from the N-terminus of the alanine dehydrogenase AC10 to 91 th to 98 th amino acid residue, partial amino acid sequence (l) is, Shewanella sp. From N-terminal alanine dehydrogenase AC10 to 265 th to 273 th amino acid residue corresponding to the corresponding amino acid residue on the alignments. Partial amino acid sequence (i) is present in the region of the second to 27-th amino acid residue from the N-terminus of the protein It is preferable, it is more preferably present in the region of the third to 22 amino acid residues from the N-terminus. Partial amino acid sequence (j) is preferably present in the 60 th to the region of 88 amino acid residues from the N-terminus of the protein is present in the region of 65-th to 83 th amino acid residue from the N-terminal it is more preferable. Partial amino acid sequence (k) is preferably present in the region of amino acid residues from the N-terminus of the 81 th to 108 th of the protein is present in the region of 86-th to 103 th amino acid residue from the N-terminal it is more preferable. Partial amino acid sequence (l) is preferably present in the 255th ~ region of 283 amino acid residue from the N-terminus of the protein, there N-terminus to 260 th to the region of 278 amino acid residues it is more preferable.
[0060]
　As can be seen from FIGS. 3-1 and FIG. 3-2, the partial amino acid sequence (i), the partial amino acid sequence (j), the partial amino acid sequence (k) and the partial amino acid sequence (l) is between the group of alanine dehydrogenase it is a highly conserved region. Thus, variations of alanine dehydrogenase comprising at least one of the partial amino acid sequence (i) ~ partial amino acid sequence (l) is the probability that functions as an amino acid-regenerating enzyme according to the present disclosure is considered to be high. In addition, Shewanella sp. Amino acid residue corresponding on the alignment of amino acid residues and N-terminal corresponding on alignment to 15 arginine residues from the N-terminal alanine dehydrogenase AC10 to 75 lysine residues are important for binding and NADH, amino acid residue corresponding on alignment from the 96th histidine residues and the N-terminal from the N-terminus to 270 th aspartic acid residue is considered to be important structural form of the protein (J. Mol. Biol., 2008, 377, 1161-1173, Enzyme and Microbial Technology, 2018, 110, 61-68), the function of alanine dehydrogenase is an important residue. If it is these residues stored, probability functioning as amino-regenerating enzyme according to the present disclosure is considered to be high.
[0061]
　Shewanella sp. From N-terminal alanine dehydrogenase AC10 to 15 arginine residues another representation of the region containing the amino acid residues corresponding on alignments include the following partial amino acid sequence (i-1). Alanine dehydrogenase may comprise partial amino acid sequence a (i-1) in place of the partial amino acid sequence (i). (I-1)
GX 1 PX 2 EX 3 KX 4 X 5 EX 6 RX 7 X 8 X 9 X 10 PX 11 X 12 X 13 X 14 X 15 X 16 (SEQ ID NO: 　　117) X 1 is, V, I, represents L or C,

　　X 2 is, T, represents K or
　　R, X 3 represents I, V, T, S, F or
　　N, X 4 represents N, M, A, D, V, and L or T,
　　X 5 represents H, N, and Q or
　　L, X 6 is, Y, represents N or
　　F, X 7 represents V or
　　I, X 8 represents G or
　　a, X 9 is M, represents L or
　　I, X 10 represents V, T, I or
　　S, X 11 represents S, a, T, Q, H, N, L or
　　G, X 12 is, S, It represents a, N, G or
　　V, X 13 represents V or a,
　　X 14 represents R, K, N, Q, S, A, L or
　　H, X 15 represents E, Q, D, V or
　　A, X 16 is L, A, V, F or representing the Y.
　Partial amino acid sequence (i-1) is, Shewanella sp. From N-terminal alanine dehydrogenase AC10 4 th to 26 th amino acid residue corresponding to amino acid residues corresponding the alignment. Partial amino acid sequence (i-1) is preferably present in the second to 36-th region of amino acid residues from the N-terminus of the protein, in the region of the third to 31 amino acid residues from the N-terminus it is more preferably present.
[0062]
　Shewanella sp. From N-terminal alanine dehydrogenase AC10 to 75 lysine residues as another representation of the region containing the amino acid residues corresponding on alignments include the following partial amino acid sequence (j-1). Alanine dehydrogenase may comprise partial amino acid sequence (j-1) in place of the partial amino acid sequence (j). (J-1)
X 1 X 2 X 3 X 4 KVKEPX 5 X 6 X 7 EX 8 X 9 X 10 (SEQ ID NO: 　　118) X 1 represents D, E, and Q or 　　K, X 2 is, M or represents 　　L, X 3 is I, represents L or 　　V, X 4 represents V, L, I or M,

　　X 5 represents Q, L, I, V or
　　N, X 6 represents A, T, S, P, M, K, R, Q, V, I or
　　E, X 7 is V, I, E, N, Q, T, a, D, H, M, N, V, a, S, I, D, G, represents W or
　　K, X 8 is, R, C, Y or W
　　represents, X 9 is, a, E, represents R, Q, K, T, S, M, N, V, P, G, C or
　　H, X 10 is, M, L, K, R , Q, E, W, F or Y.
　Partial amino acid sequence (j-1) is, Shewanella sp. From N-terminal alanine dehydrogenase AC10 to 69 th to 84 th amino acid residue corresponding to the corresponding amino acid residue on the alignments. Partial amino acid sequence (j-1) is preferably present in the region of amino acid residues from the N-terminus of the 59 th to 94 th protein, in the region of 64-th to 89 th amino acid residue from the N-terminal it is more preferably present.
[0063]
　Shewanella sp. From N-terminal alanine dehydrogenase AC10 to 96th histidine residue as another representation of the region containing the amino acid residues corresponding on alignments include the following partial amino acid sequence (k-1). Alanine dehydrogenase may comprise partial amino acid sequence a (k-1) in place of the partial amino acid sequence (k). (K-1)
X 1 X 2 X 3 X 4 X 5 X 6 LX 7 TYLHLAX 8 X 9 X 10 X 11 X 12 X 13 X 14 X 15 LX 16 X 17 X 18 X 19(SEQ ID NO:
　　119) X 1 represents L or
　　F, X 2 represents R, K, C, S, H, G or
　　Q, X 3 is, H, E, P, S , D, R , K, represents Q or
　　a, X 4 represents D, G, Q, H, E, S, N or
　　M, X 5 represents Q or
　　H, X 6 is I, L, V , T, represents C or
　　a, X 7 represents F or
　　Y, X 8 represents P or
　　a, X 9 represents D, S, N, H or
　　E, X 10 is L represents M, P, R, V, Q, E or
　　K, X 11 represents P, a, V, Q, K, E, D, T, N, R, S or M,
　　X 12 is, Q, represents C or
　　L, X 13 is, T, represents A or
　　V, X 14 represents E, I, Q, A, R, K, T, D or
　　N, X 15 represents E, D, L, a, G, H, Y or
　　S, X 16 represents I, M, V, L, T or
　　K, X 17 is T, K, S, D, represents H, E, a, N, R, G or
　　Q, X 18 represents S, G, C, a or
　　K, X 19 represents G, K, R, and Q or D.
　Partial amino acid sequence (k-1) is, Shewanella sp. From N-terminal alanine dehydrogenase AC10 to 85 th to 111 th amino acid residue corresponding to the corresponding amino acid residue on the alignments. Partial amino acid sequence (k-1) is preferably present in the region of 75-th to 121 th amino acid residue from the N-terminus of the protein, the 80 th to the region of 116 amino acid residue from the N-terminal it is more preferably present.
[0064]
　Shewanella sp. From N-terminal alanine dehydrogenase AC10 270 th aspartic acid residue as a separate representation of the region containing the amino acid residues corresponding on alignments include the following partial amino acid sequence (l-1). Alanine dehydrogenase may comprise partial amino acid sequence (l-1) in place of the partial amino acid sequence (l). (L-1)
X 1 X 2 X 3 X 4 X 5 DX 6 AX 7 DQGGX 8 X 9 X 10 X 11 (SEQ ID NO: 　　120) X 1 is, G, represents the R or 　　S, X 2 is, S, represents a or 　　G, X 3 represents a or 　　V, X 4

Is I, L represents M or
　　V, X 5 represents V or
　　A, X 6 represents V or
　　I, X 7 represents I or
　　V, X 8 is C or I
represents, X 9 represents V, I, a, F, S or
C, X 10 represents E or
　　a, X 11 represents T or D.
　Partial amino acid sequence (l-1) is, Shewanella sp. From N-terminal alanine dehydrogenase AC10 to 261 th to 277 th amino acid residue corresponding to the corresponding amino acid residue on the alignments. Partial amino acid sequence (l-1) is preferably present in the region of 251 th to 287 th amino acid residue from the N-terminus of the protein, in the region of 256 th to 282 th amino acid residue from the N-terminal it is more preferably present.
[0065]
　
　gene encoding pyridoxine dehydrogenase may be any gene encoding pyridoxine dehydrogenase described above. Genes encoding pyridoxamine synthase may be any gene encoding pyridoxamine synthase described above. Gene encoding the amino acid regenerating enzyme may be any gene encoding the amino acid regenerating enzyme described above. Enzymes encoded by these genes are known amino acid sequence having the enzymatic activity (e.g., an amino acid sequence encoded by a gene naturally present in the organism) not limited to, a different modification to the known amino acid sequence it may be an enzyme having the amino acid sequence.
[0066]
　Such genes, such as genes exemplified microorganisms (microbes from which it is derived) has naturally on a microorganism having the enzyme, may be a known gene, a range of desired enzymatic activity is obtained the inner, it may be a gene obtained by modifying the nucleotide sequence to encode a modified modified amino acid sequence from the known amino acid sequence of the enzyme as described above. Examples of such modified amino acid sequence, as described above, amino acid sequence similarity and the like in any of the amino acid sequence of SEQ ID NO: 1-6. Further, the nucleotide sequence of the gene that codes for a specific amino acid sequence can be varied within the scope of codon degeneracy. In this case, better to use a frequently used codon in a microorganism as a host of the recombinant microorganism is preferred in terms of expression efficiency of the gene.
[0067]
　Incidentally, the nucleotide sequence of the gene can also be designed from the amino acid sequence to be encoded based on the codon table. Nucleotide sequences designed, to a known nucleotide sequence may be obtained by modified using genetic recombination techniques, it may be obtained by chemically synthesizing a nucleotide sequence.
　As a method for modifying nucleotide sequences, such as site-specific mutagenesis (Kramer, W. And frita, H.J., Methods in Enzymology, vol.154, P.350 (1987)), recombinant PCR method (PCR Technology, Stockton Press (1989), a method of chemically synthesizing the DNA of a particular part, the gene ultraviolet irradiation processing method, a strain carrying a gene hydroxylamine treatment, or a chemical agent in the process, such as nitrosoguanidine or nitrous acid how to, and a method of using a commercially available mutagenesis kit.
[0068]
　For example, the gene encoding the pyridoxine dehydrogenase genes encoding the pyridoxamine synthetase, and genes encoding the amino acid regenerating enzyme are both known nucleotide sequence encoding a polynucleotide having the enzymatic activity (e.g., may be a DNA having remained unaltered nucleotide sequence having a naturally occurring gene is) in an organism such as a microorganism exemplified above, for such nucleotide sequences, the encoded enzyme the enzyme activity it may be a DNA having the nucleotide sequence plus the sequence modifications in a range not losing the (enzyme activity described above). Such modifications, insertion of a nucleotide, deletion, and addition of substitutions and nucleotide sequence 5 'terminus or 3' terminus or additional nucleotides to both. Insertion of nucleotides, if there are one or more of the deletions and substitutions, insertions, deletions and each substitution, when present, for example, 1 to 90 nucleotides or 1 to 60 nucleotides, or 1 to 30 amino acids, residues or 1-20 amino acid residues, or 1-15 nucleotides or 1-10 nucleotides, or may be 1-5 nucleotides, insertion of nucleotides, deletion and the total number of substituents is, for example, 1 to 100 nucleotides,,, or 1-50 nucleotides or 1-30 nucleotides, or one to 10 nucleotides, or may be 1-5 nucleotides. Insertion or deletion of nucleotides may be present in the local, but is preferably not generated a large frame shift as a whole nucleotide sequence. Further, the number of nucleotides added to the ends, first end per example 1 to 150 nucleotides, if present, or 1-100 nucleotides, or 1-50 nucleotides, or 1 to 30 nucleotides, or 1 to 10 nucleotides, or it may be from 1 to 5 nucleotides. Such additional nucleotides may encode a signal sequence for secretion or the like into the extracellular.
[0069]
　Alternatively, the gene encoding each enzyme is known nucleotide sequence encoding a polynucleotide having the enzymatic activity (e.g., the nucleotide sequence of the gene naturally present in the organism) DNA having itself or the known nucleotide sequence having a nucleotide sequence having 80% or more, or 85%, or 90%, or 95% sequence identity with respect to, encoding an enzyme having the desired enzymatic activity (above enzyme activity) DNA it may be. Here, sequence identity can be assessed in default parameters using e.g. BLAST (registered trademark, National Library of Medicine) program.
[0070]
　Alternatively, the gene encoding each enzyme is known nucleotide sequence encoding a polynucleotide having the enzymatic activity (e.g., the nucleotide sequence of the gene naturally present in the organism) DNA having itself or the known nucleotide sequence having a nucleotide sequence that hybridizes with the DNA under stringent conditions with a nucleotide sequence complementary to, or may be a DNA encoding an enzyme having the desired enzymatic activity (above enzyme activity). Further, the hybridization under stringent conditions can be carried out as follows.
　In hybridization, a DNA composed from a reference nucleotide sequence complementary to the nucleotide sequence or subsequence thereof as a probe, hybridization is performed with respect to the DNA of interest, probe after washing under stringent conditions and the target to check that you are significantly hybridize to a nucleic acid. The length of the probe, for example a continuous 20 or more nucleotides, preferably 50 nucleotides or more, more preferably 100 nucleotides or more, more preferably it is possible to use more than 200 nucleotides. Have the same nucleotide length as the reference become nucleotide sequence, it is also preferable to use as a probe a DNA complementary over the entire length. And conditions for hybridization can be exemplified by conditions under which the skilled worker is generally used to detect specific hybridization signals. Preferably, it means stringent hybridization conditions and stringent washing conditions. For example, 6 × SSC (saline sodium citrate) (1 × SSC composition: 0.15 M NaCl, 0.015M sodium citrate, pH7.0), 0.5% SDS, 5 × Denhardt and 100 mg / ml herring sperm DNA conditions that incubation overnight at 55 ° C. with the probe and the like in a solution containing. Then the like can be exemplified by washing the filters in 0.2 × SSC in 42 ° C.. Stringent conditions are conditions of 0.1 × SSC, 50 ° C. in a filter cleaning step, as further stringent conditions, and the like conditions of 0.1 × SSC, 65 ° C. in the same process it can.
[0071]
　For example, the gene encoding pyridoxine dehydrogenase, for example, may be a DNA having the nucleotide sequence of SEQ ID NO: 7 (nucleotide sequence of the gene encoding pyridoxine dehydrogenase Saccharomyces cerevisiae), complementary to the nucleotide sequence of SEQ ID NO: 7 specific hybridizes with DNA under stringent conditions with a nucleotide sequence, the protein may be a DNA having a nucleotide sequence encoding a and having a pyridoxine dehydrogenase activity.
　Alternatively, the gene encoding pyridoxine dehydrogenase, for example, may be a DNA having the nucleotide sequence of SEQ ID NO: 7, nucleotides to the nucleotide sequence of SEQ ID NO: 7 of the substitution, deletion, insertion and nucleotide sequences 5 'terminus or 3' terminus or a nucleotide sequence of performing one or more of the addition of additional nucleotides to both, or may be a DNA having a nucleotide sequence encoding a protein having a pyridoxine dehydrogenase activity. Nucleotide substitutions, deletions, for example of the order of addition of additional nucleotides to the N-terminus or C-terminus or both insertion and nucleotide sequences are as described above.
　Alternatively, pyridoxine dehydrogenase, SEQ ID NO: 7 nucleotide sequence with DNA or the nucleotide sequence of SEQ ID NO: 7, for example, 80% or more, or 85%, or 90%, or 95% or more sequence identity a nucleotide sequence may be a DNA having a nucleotide sequence encoding a protein having a pyridoxine dehydrogenase activity.
[0072]
　Alternatively, the gene encoding pyridoxine dehydrogenase, for example, may be a DNA having the nucleotide sequence of SEQ ID NO: 8 (nucleotide sequence of the gene encoding pyridoxine dehydrogenase Schizosaccharomyces pombe), complementary to the nucleotide sequence of SEQ ID NO: 8 specific hybridizes with DNA under stringent conditions with a nucleotide sequence, the protein may be a DNA having a nucleotide sequence encoding a and having a pyridoxine dehydrogenase activity.
　Alternatively, the gene may be, for example, a DNA having the nucleotide sequence of SEQ ID NO: 8 nucleotide substitutions to the nucleotide sequence of SEQ ID NO: 8, 5 of deletion, insertion and nucleotide sequences encoding pyridoxine dehydrogenase 'terminus or 3' terminus or a nucleotide sequence of performing one or more of the addition of additional nucleotides to both, or may be a DNA having a nucleotide sequence encoding a protein having a pyridoxine dehydrogenase activity. Nucleotide substitutions, deletions, for example of the order of addition of additional nucleotides to the N-terminus or C-terminus or both insertion and nucleotide sequences are as described above.
　Alternatively, pyridoxine dehydrogenase nucleotide sequence to example 80% or more DNA or SEQ ID NO: 8 having the nucleotide sequence of SEQ ID NO: 8, or 85%, or 90%, or 95% or more sequence identity a nucleotide sequence may be a DNA having a nucleotide sequence encoding a protein having a pyridoxine dehydrogenase activity.
[0073]
　Alternatively, the gene encoding pyridoxine dehydrogenase, for example, may be a DNA having any nucleotide sequence of SEQ ID NO: 7 and SEQ ID NO: 53 to SEQ ID NO: 59, SEQ ID NO: 7 and SEQ ID NO: 53 to SEQ ID NO: 59 hybridizes with DNA under stringent conditions with a nucleotide sequence complementary to any of the nucleotide sequences of a protein may be a DNA having a nucleotide sequence encoding a and having a pyridoxine dehydrogenase activity.
　Alternatively, the gene encoding pyridoxine dehydrogenase, for example, may be a DNA having any nucleotide sequence of SEQ ID NO: 7 and SEQ ID NO: 53 to SEQ ID NO: 59, SEQ ID NO: 7 and SEQ ID NO: 53 to SEQ ID NO: nucleotide substitutions relative to any of the nucleotide sequences of 59, deletion, insertion and nucleotide sequence 5 'terminus or 3' terminus or one or more nucleotide sequences was carried out the addition of additional nucleotides to both a is, or may be a DNA having a nucleotide sequence encoding a protein having a pyridoxine dehydrogenase activity. Nucleotide substitutions, deletions, for example of the order of addition of additional nucleotides to the N-terminus or C-terminus or both insertion and nucleotide sequences are as described above.
　Alternatively, pyridoxine dehydrogenase encoding DNA, or nucleotide sequence of SEQ ID NO: 7, the pyridoxine dehydrogenase nucleotide sequence (Saccharomyces eubayanus of SEQ ID NO: 53 having any of the nucleotide sequences of SEQ ID NO: 7 and SEQ ID NO: 53 to SEQ ID NO: 59 nucleotide sequences of a gene), nucleotide sequence (nucleotide sequence of the gene encoding pyridoxine dehydrogenase Torulaspora delbrueckii), the nucleotide sequence of the gene encoding pyridoxine dehydrogenase nucleotide sequence of SEQ ID NO: 55 (Zygosaccharomyces bailii of SEQ ID NO: 54), SEQ the nucleotide sequence of the number 57 (Kluyveromyces The nucleotide sequence of the gene encoding pyridoxine dehydrogenase arxianus), the nucleotide sequence of SEQ ID NO: 56 (nucleotide sequence of the gene encoding pyridoxine dehydrogenase Aspergillus oryzae), encoding pyridoxine dehydrogenase nucleotide sequence (Candida albicans of SEQ ID NO: 58 gene the nucleotide sequence of), and the nucleotide sequence of SEQ ID NO: 59 (Yarrowia lipolytica of pyridoxine nucleotide sequence of the gene encoding the thin dehydrogenase) at least one for example 80% or more of, or more than 85%, or 90% or more, or a nucleotide sequence having 95% or more sequence identity, pyridoxine protraction hydro It may be a DNA having a nucleotide sequence encoding a protein having Genaze activity.
[0074]
　When expressing in a recombinant microorganism prokaryotic as a host such as E. coli, it may be codon optimized to facilitate expression. For example, what is the highest frequency of use among the codons encoding each amino acid in a prokaryotic as the host may be modified nucleotide sequence to be used frequently as a codon for the amino acids. From this point of view, as a DNA containing a gene encoding pyridoxine dehydrogenase, e.g., region up to 18 nt ~ 3 'end of either the nucleotide sequence of SEQ ID NO: 13 and SEQ ID NO: 75 to SEQ ID NO: 81 may be used DNA having a DNA having a 18th nucleotides to the 3 'region complementary to the nucleotide sequence to end of either of the nucleotide sequences of SEQ ID NO: 13 and SEQ ID NO: 75 to SEQ ID NO: 81 hybridizes under stringent conditions, the protein may be used a DNA having a nucleotide sequence encoding a and having a pyridoxine dehydrogenase activity. The nucleotide sequence of SEQ ID NO: 13 and SEQ ID NO: 75 to SEQ ID NO: 81 are both 17 nucleotides from its 5 'end has a upstream region, there is a start codon 18 nt to 20 nt. Thus, may be used range up to 18 nt ~ 3 'end of these nucleotide sequences as a gene region encoding pyridoxine dehydrogenase.
　Alternatively, the gene encoding pyridoxine dehydrogenase, for example, may be a DNA having a region up to 18 nt ~ 3 'end of either the nucleotide sequence of SEQ ID NO: 13 and SEQ ID NO: 75 to SEQ ID NO: 81 and, 'substituted nucleotides to a region of to the end, 5 of deletion, insertion and nucleotide sequence' 18 nt ~ 3 of any nucleotide sequence of SEQ ID NO: 13 and SEQ ID NO: 75 to SEQ ID NO: 81 ends or 3 'end or a nucleotide sequence of performing one or more of the addition of additional nucleotides to both, or may be a DNA having a nucleotide sequence encoding a protein having a pyridoxine dehydrogenase activity. Nucleotide substitutions, deletions, for example of the order of addition of additional nucleotides to the N-terminus or C-terminus or both insertion and nucleotide sequences are as described above.
　Alternatively, pyridoxine dehydrogenase, 18 th nucleotide sequence of DNA, or SEQ ID NO: 13 having an area of ​​up to 18 nt ~ 3 'end of either the nucleotide sequence of SEQ ID NO: 13 and SEQ ID NO: 75 to SEQ ID NO: 81 nucleotides to the 3 '(gene codon optimized nucleotide sequences encoding pyridoxine dehydrogenase Saccharomyces cerevisiae) region up end, the nucleotide sequence of SEQ ID NO: 75 18 nt ~ 3' region up end (Saccharomyces Eubayanus of pyridoxine encoding dehydrogenase codon optimized nucleotide sequence of the gene), a region of up to 18 nt ~ 3 'terminus of the nucleotide sequence of SEQ ID NO: 76 (Toru aspora pyridoxine dehydrogenase gene codon optimized nucleotide sequences encoding the delbrueckii), the nucleotide sequence of SEQ ID NO: 77 18 nt ~ 3 'end to a region (Zygosaccharomyces bailii of pyridoxine encoding Shin dehydrogenase gene codon optimized nucleotide sequence), the nucleotide sequence of SEQ ID NO: 79 18 nt ~ 3 'end to a region (Kluyveromyces marxianus of pyridoxine dehydrogenase gene codon optimized nucleotide sequence encoding a), 18 th nucleotide of the nucleotide sequence of SEQ ID NO: 78 co the pyridoxine dehydrogenase region (Aspergillus oryzae up to 3 'end Gene codon optimized nucleotide sequence over de), 18 nt ~ 3 'encoding pyridoxine dehydrogenase region up end (Candida albicans gene codon optimized nucleotide sequence of the nucleotide sequence of SEQ ID NO: 80), and the sequence region of up to 18 nt ~ 3 'terminus of the nucleotide sequence of ID NO: 81 (Yarrowia lipolytica Piridoki of
[0075]
　Genes encoding pyridoxamine synthase, for example, the nucleotide sequence of SEQ ID NO: 9 - may be a DNA having a (pyridoxamine of Mesorhizobium loti nucleotide sequence of the gene coding for pyruvate transaminase), the nucleotide sequence of SEQ ID NO: 9 and a DNA which hybridizes with the DNA under stringent conditions with a nucleotide sequence complementary, may be a DNA and having a nucleotide sequence encoding a protein having a pyridoxamine synthase activity.
　Alternatively, genes encoding the pyridoxamine synthase, for example, may be a DNA having the nucleotide sequence of SEQ ID NO: 9, nucleotides to the nucleotide sequence of SEQ ID NO: 9 substitution, deletion, insertion and nucleotide sequences 5 'terminus or 3' terminus or a nucleotide sequence of performing one or more of the addition of additional nucleotides to both, even DNA having a nucleotide sequence encoding a protein having pyridoxamine synthase activity good. Nucleotide substitutions, deletions, for example of the order of addition of additional nucleotides to the N-terminus or C-terminus or both insertion and nucleotide sequences are as described above.
　Alternatively, pyridoxamine synthase, SEQ ID NO: 9 nucleotide sequence having DNA or the nucleotide sequence of SEQ ID NO: 9 for example, 80% or more, or 85%, or 90%, or 95% or more sequence identity to a nucleotide sequence having, or may be a DNA having a nucleotide sequence encoding a protein having pyridoxamine synthase activity.
[0076]
　Alternatively, genes encoding the pyridoxamine synthase, for example, the nucleotide sequence of SEQ ID NO: 10 (in Escherichia coli pyridoxamine - oxaloacetic nucleotide sequence of the gene coding for acid transaminase) may be a DNA having the SEQ ID NO: 10 a DNA which hybridizes with the DNA under stringent conditions with a nucleotide sequence complementary to a nucleotide sequence, the protein may be a DNA having a nucleotide sequence encoding a and having a pyridoxamine synthase activity.
　Alternatively, genes encoding the pyridoxamine synthase, for example, may be a DNA having the nucleotide sequence of SEQ ID NO: 10, nucleotides to the nucleotide sequence of SEQ ID NO: 10 substitutions, deletions, insertion and nucleotide sequences 5 'terminus or 3' terminus or a nucleotide sequence of performing one or more of the addition of additional nucleotides to both, even DNA having a nucleotide sequence encoding a protein having pyridoxamine synthase activity good. Nucleotide substitutions, deletions, for example of the order of addition of additional nucleotides to the N-terminus or C-terminus or both insertion and nucleotide sequences are as described above.
　Alternatively, pyridoxamine synthase nucleotide sequence to example 80% or more DNA or SEQ ID NO: 10 having the nucleotide sequence of SEQ ID NO: 10, or 85%, or 90%, or 95% or more sequence identity to a nucleotide sequence having, or may be a DNA having a nucleotide sequence encoding a protein having pyridoxamine synthase activity.
[0077]
　Alternatively, genes encoding the pyridoxamine synthase, for example, may be a DNA having any nucleotide sequence of SEQ ID NO: 9 and SEQ ID NO: 60 to SEQ ID NO: 66, SEQ ID NO: 9 and SEQ ID NO: 60 through SEQ a DNA which hybridizes with the DNA under stringent conditions with a nucleotide sequence complementary to any of the nucleotide sequences of No. 66, a protein may be a DNA having a nucleotide sequence encoding a and having a pyridoxamine synthase activity .
　Alternatively, genes encoding the pyridoxamine synthase, for example, may be a DNA having any nucleotide sequence of SEQ ID NO: 9 and SEQ ID NO: 60 to SEQ ID NO: 66, SEQ ID NO: 9 and SEQ ID NO: 60 through SEQ nucleotide substitutions to any of the nucleotide sequences of No. 66, deletion, insertion and nucleotide sequences of the 5 'terminus or 3' terminus or one or more nucleotides was carried out the addition of additional nucleotides to both a sequence may be a DNA having a nucleotide sequence encoding a protein having pyridoxamine synthase activity. Nucleotide substitutions, deletions, for example of the order of addition of additional nucleotides to the N-terminus or C-terminus or both insertion and nucleotide sequences are as described above.
　Alternatively, pyridoxamine synthase, DNA having any nucleotide sequence of SEQ ID NO: 9 and SEQ ID NO: 60 to SEQ ID NO: 66, or the nucleotide sequence of SEQ ID NO: 9, SEQ ID NO: 60 (Mesorhizobium sp YR577 of pyridoxamine -. Pyruvate the nucleotide sequence of the nucleotide sequence of the gene) encoding the transaminase, SEQ ID NO: 61 (Pseudaminobacter salicylatoxidans of pyridoxamine - nucleotide sequence of the nucleotide sequence of the gene coding for pyruvate transaminase), pyridoxamine of SEQ ID NO: 62 (Bauldia litoralis - pyruvate transaminase the nucleotide sequence of the coding nucleotide sequence of the gene), SEQ ID NO: 63 ( Skermanella stibiiresistens of pyridoxamine - nucleotide sequence of pyruvate nucleotide sequence of the gene encoding acid transaminase), SEQ ID NO: 64 (Rhizobium sp of AC44 / 96 pyridoxamine -. The nucleotide sequence of the nucleotide sequence of the gene coding for pyruvate transaminase), SEQ ID NO: 65 - the nucleotide sequence of (Erwinia toletana of pyridoxamine nucleotide sequence of the gene encoding pyruvate transaminase), and SEQ ID NO: 66 - at least one of the nucleotide sequences of (Herbiconiux ginsengi of pyridoxamine nucleotide sequence of the gene encoding pyruvate transaminase) for example, 80% or more One Or 85% or more, or 90% or more, or a nucleotide sequence having 95% or more sequence identity, or may be a DNA having a nucleotide sequence encoding a protein having pyridoxamine synthase activity.
[0078]
　When expressing in a recombinant microorganism prokaryotic as a host, such as E. coli, the codons as described above in order to facilitate expression may be optimized. From this point of view, any as DNA containing a gene encoding the pyridoxamine synthase, for example, in the region or SEQ ID NO: 82 to SEQ ID NO: 88 to 18 nt ~ 3 'terminus of the nucleotide sequence of SEQ ID NO: 14 18 nt ~ 3 'may be used a DNA having a region to the end, the 18 th in the nucleotide sequence of SEQ ID NO: 14 nucleotides to the 3' of Kano nucleotide sequence region to the end or SEQ ID NO: 82 through SEQ a protein having an 18 nt ~ 3 'hybridizes with DNA under stringent conditions with a region complementary to the nucleotide sequence to end, and pyridoxamine synthase activity of any nucleotide sequence of ID NO: 88 the DNA having a nucleotide sequence encoding It may be used. The nucleotide sequence of SEQ ID NO: 14, 17 nucleotides from the 5 'end has a upstream region, there is a start codon 18 nt to 20 nt. Thus, may be used range up to 18 nt ~ 3 'end of this nucleotide sequence as a gene region encoding the pyridoxamine synthase. Similarly, the nucleotide sequence of SEQ ID NO: 82 to SEQ ID NO: 88, either, 17 nucleotides from the 5 'end has a upstream region, there is a start codon 18 nt to 20 nt. Thus, may be used range up to 18 nt ~ 3 'end of these nucleotide sequences as a gene region encoding the pyridoxamine synthase.
　Alternatively, genes encoding the pyridoxamine synthase, for example, of any of the nucleotide sequences of the region or SEQ ID NO: 82 to SEQ ID NO: 88 to 18 nt ~ 3 'terminus of the nucleotide sequence of SEQ ID NO: 14 18 th 'it may be a DNA having a region up ends 18 nt ~ 3 in the nucleotide sequence of SEQ ID NO: 14' nucleotides to 3 or nucleotides in the region or SEQ ID NO: 82 to SEQ ID NO: 88 to the end 18 th nucleotides to the sequence 3 'region nucleotide substitutions relative to the terminal, deletion, insertion and 5 nucleotide sequence' terminus or 3 'terminus or one or more of the addition of additional nucleotides to both a nucleotide sequence was carried out, having a pyridoxamine synthase activity Protein or may be a DNA having a nucleotide sequence encoding. Nucleotide substitutions, deletions, for example of the order of addition of additional nucleotides to the N-terminus or C-terminus or both insertion and nucleotide sequences are as described above.
[0079]
　Alternatively, pyridoxamine synthase to '18 nt ~ 3 of any nucleotide sequence of the region or SEQ ID NO: 82 to SEQ ID NO: 88 to the end' terminal 18 nt ~ 3 in the nucleotide sequence of SEQ ID NO: 14 18 nt ~ 3 'region up end of the nucleotide sequence of the DNA, or SEQ ID NO: 14 having a region (Mesorhizobium loti of pyridoxamine - codon optimized nucleotide sequence of the gene coding for pyruvate transaminase), of SEQ ID NO: 82 18 nt ~ 3 'region up end of the nucleotide sequence (Mesorhizobium sp YR577 of pyridoxamine -. codon optimized nucleotide sequence of the gene coding for pyruvate transaminase), Region of up to 18 nt ~ 3 'terminus of the nucleotide sequence of SEQ ID NO: 83 (pyridoxamine of Pseudaminobacter salicylatoxidans - codon optimized nucleotide sequence of the gene coding for pyruvate transaminase), 18 th nucleotide of the nucleotide sequence of SEQ ID NO: 84 ~ 3 'to the end region (of Bauldia litoralis pyridoxamine - codon optimized nucleotide sequence of the gene coding for pyruvate transaminase), 18 th nucleotides 1-3 of the nucleotide sequence of SEQ ID NO: 85' to terminal region (the Skermanella stibiiresistens pyridoxamine - codon optimized nucleotide sequence of the gene coding for pyruvate transaminase) Region of up to 18 nt ~ 3 'terminus of the nucleotide sequence of SEQ ID NO: 86 (Rhizobium sp of AC44 / 96 pyridoxamine -. Codon optimized nucleotide sequence of the gene coding for pyruvate transaminase), the nucleotide sequence of SEQ ID NO: 87 18 nt ~ 3 'region up end (Erwinia toletana of pyridoxamine - pyruvate transaminase
[0080]
　Gene encoding the amino acid regenerating enzyme, for example, may be a DNA having the nucleotide sequence of SEQ ID NO: 11 (Shewanella sp. Ac10 gene of the nucleotide sequence encoding the alanine dehydrogenase), and the nucleotide sequence of SEQ ID NO: 11 a DNA which hybridizes with the DNA under stringent conditions with a nucleotide sequence complementary, and may be a DNA having a nucleotide sequence encoding a protein having alanine regenerating enzyme activity.
　Alternatively, genes encoding the amino acid regenerating enzyme, for example, may be a DNA having the nucleotide sequence of SEQ ID NO: 11, nucleotides to the nucleotide sequence of SEQ ID NO: 11 substitutions, deletions, insertion and nucleotide sequences 5 'terminus or 3' terminus or a nucleotide sequence which additional conducted one or more of the addition of nucleotides to both, even DNA having a nucleotide sequence encoding a protein having alanine regenerating enzyme activity good. Nucleotide substitutions, deletions, for example of the order of addition of additional nucleotides to the N-terminus or C-terminus or both insertion and nucleotide sequences are as described above.
　Alternatively, amino acid regeneration enzymes, SEQ ID NO: 11 nucleotide DNA having the sequence or SEQ ID NO: 11 nucleotide sequence to example 80% or more, or 85%, or 90%, or 95% or more sequence identity to a nucleotide sequence having, or may be a DNA having a nucleotide sequence encoding a protein having alanine regenerating enzyme activity.
[0081]
　Alternatively, genes encoding the amino acid regenerating enzyme, for example, may be a DNA having the nucleotide sequence of SEQ ID NO: 12 (nucleotide sequence of the gene coding for glutamate dehydrogenase Escherichia coli), and the nucleotide sequence of SEQ ID NO: 12 a DNA which hybridizes with the DNA under stringent conditions with a nucleotide sequence complementary, and may be a DNA having a nucleotide sequence encoding a protein having a glutamate regenerating enzyme activity.
　Alternatively, genes encoding the amino acid regenerating enzyme, for example, may be a DNA having the nucleotide sequence of SEQ ID NO: 12, nucleotides to the nucleotide sequence of SEQ ID NO: 12 substitutions, deletions, insertion and nucleotide sequences 5 'terminus or 3' terminus or a nucleotide sequence of performing one or more of the addition of additional nucleotides to both, even DNA having a nucleotide sequence encoding a protein having a glutamate-regenerating enzyme activity good. Nucleotide substitutions, deletions, for example of the order of addition of additional nucleotides to the N-terminus or C-terminus or both insertion and nucleotide sequences are as described above.
　Alternatively, amino acid regenerating enzyme, the nucleotide sequence to example 80% or more DNA or SEQ ID NO: 12 having a nucleotide sequence of SEQ ID NO: 12, or 85%, or 90%, or 95% or more sequence identity to a nucleotide sequence having, or may be a DNA having a nucleotide sequence encoding a protein having a glutamate regenerating enzyme activity.
[0082]
　Gene encoding the amino acid regenerating enzyme, for example, may be a DNA having any nucleotide sequence of SEQ ID NO: 11 and SEQ ID NO: 67 to SEQ ID NO: 74, SEQ ID NO: 11 and SEQ ID NO: 67 to SEQ ID NO: 74 it may be a DNA having the nucleotide sequence hybridizes with DNA under stringent conditions, and encodes a protein having alanine regenerating enzyme activity having a nucleotide sequence complementary to any of the nucleotide sequences of.
　Alternatively, genes encoding the amino acid regenerating enzyme, for example, may be a DNA having any nucleotide sequence of SEQ ID NO: 11 and SEQ ID NO: 67 to SEQ ID NO: 74, SEQ ID NO: 11 and SEQ ID NO: 67 to SEQ nucleotide substitutions to any of the nucleotide sequences of No. 74, deletion, insertion and nucleotide sequences of the 5 'terminus or 3' terminus or one or more nucleotides was carried out the addition of additional nucleotides to both a sequence may be a DNA having a nucleotide sequence encoding a protein having alanine regenerating enzyme activity. Nucleotide substitutions, deletions, for example of the order of addition of additional nucleotides to the N-terminus or C-terminus or both insertion and nucleotide sequences are as described above.
　Alternatively, amino acid regeneration enzymes, DNA having any nucleotide sequence of SEQ ID NO: 11 and SEQ ID NO: 67 to SEQ ID NO: 74, or the nucleotide sequence of SEQ ID NO: 11, the alanine dehydrogenase nucleotide sequence (Aeromonas hydrophila of SEQ ID NO: 67 the nucleotide sequence of the gene encoding) the nucleotide sequence of SEQ ID NO: 68 (Rhizobium sp. nucleotide sequence of the gene encoding alanine dehydrogenase LPU83), the nucleotide sequence of the gene encoding alanine dehydrogenase nucleotide sequence (Pseudomonas mendocina of SEQ ID NO: 69 ), the nucleotide sequence of SEQ ID NO: 70 (Bradyrhizobium japonicum alanine dehydroepiandrosterone Gena One nucleotide sequence of the gene encoding one of zero), the nucleotide sequence of the gene encoding another one of the alanine dehydrogenase nucleotide sequence (Bradyrhizobium japonicum in SEQ ID NO: 71), the nucleotide sequence of SEQ ID NO: 72 ( gene nucleotide sequence encoding the alanine dehydrogenase Streptomyces aureofaciens), nucleotide sequences (Anabaena nucleotide sequence of the gene encoding alanine dehydrogenase cylindrica), and encoding the alanine dehydrogenase nucleotide sequence (Bacillus subtilis of SEQ ID NO: 74 SEQ ID NO: 73 for example 8 with respect to at least one of the nucleotide sequences of the gene) % Or more, or 85%, or 90% or more, or a nucleotide sequence having 95% or more sequence identity, or may be a DNA having a nucleotide sequence encoding a protein having alanine regenerating enzyme activity.
[0083]
　When expressing in a recombinant microorganism prokaryotic as a host, such as E. coli, the codons as described above in order to facilitate expression may be optimized. From this point of view, as a DNA containing a gene encoding the amino acid regenerating enzyme, for example, up to 18 nt ~ 3 'end of either the nucleotide sequence of SEQ ID NO: 15 and SEQ ID NO: 89 to SEQ ID NO: 96 it may be used a DNA having a region, DNA having 18 nt ~ 3 'region complementary to the nucleotide sequence to end of either of the nucleotide sequences of SEQ ID NO: 15 and SEQ ID NO: 89 to SEQ ID NO: 96 and hybridizes under stringent conditions, the protein may be used a DNA having a nucleotide sequence encoding a and having an alanine regenerating enzyme activity. The nucleotide sequence of SEQ ID NO: 15 and SEQ ID NO: 89 to SEQ ID NO: 96 are both 17 nucleotides from its 5 'end has a upstream region, there is a start codon 18 nt to 20 nt. Thus, may be used range up to 18 nt ~ 3 'end of these nucleotide sequences as a gene region encoding alanine regenerating enzyme.
　Alternatively, genes encoding the amino acid regenerating enzyme, for example, be a DNA having a region up to 18 nt ~ 3 'end of either the nucleotide sequence of SEQ ID NO: 15 and SEQ ID NO: 89 to SEQ ID NO: 96 It good, 18 nt to 3 of any nucleotide sequence of SEQ ID NO: 15 and SEQ ID NO: 89 to SEQ ID NO: 96 'substituted nucleotides to a region of up terminal, deletion, insertion and nucleotide sequence 5' a additional nucleotide nucleotide sequence was carried out one or more of the addition of on-terminus or 3 'terminus or both, may be a DNA having a nucleotide sequence encoding a protein having alanine regenerating enzyme activity. Nucleotide substitutions, deletions, for example of the order of addition of additional nucleotides to the N-terminus or C-terminus or both insertion and nucleotide sequences are as described above.
　Alternatively, amino acid regenerating enzyme is the nucleotide sequence of DNA or SEQ ID NO: 15, has an area of up to 18 nt ~ 3 'end of either the nucleotide sequence of SEQ ID NO: 15 and SEQ ID NO: 89 to SEQ ID NO: 96 18 of th nucleotides to the 3 'region of to the end (Shewanella sp. codon optimized nucleotide sequences encoding the amino acid sequence was the amino acid substitutions D198A to the amino acid sequence of alanine dehydrogenase AC10), the nucleotide sequence of SEQ ID NO: 89 18 nt ~ 3 'region up end (the alanine dehydrogenase of Aeromonas hydrophila NADP + corresponding amino acid substitutions gene codon optimized nucleotide sequences encoding the amino acid sequence was performed), of SEQ ID NO: 90 Region of up to 18 nt ~ 3 'end of the nucleotide sequence (Rhizobium sp. NADP alanine dehydrogenase LPU83 + Codon optimized nucleotide sequence of the gene encoding the amino acid sequence with a corresponding amino acid substitution), NADP to 18 nt ~ 3 'alanine dehydrogenase region (Pseudomonas mendocina until end of the nucleotide sequence of SEQ ID NO: 91 + corresponding amino acid codon-optimized nucleotide sequence of the gene encoding the amino acid sequence was substituted), NADP in one of the 18 th nucleotides to the 3 'region of to the end (Bradyrhizobium japonicum alanine dehydrogenase nucleotide sequence of SEQ ID NO: 92 + region from the corresponding amino acid substitutions gene codon optimized nucleotide sequences encoding the amino acid sequence was performed), 18 nt ~ 3 'terminus of the nucleotide sequence of SEQ ID NO: 93 ( Another one to NADP of alanine dehydrogenase Bradyrhizobium japonicum + corresponding amino acid substitutions gene codon optimized nucleotide sequences encoding the amino acid sequence was performed), 18 nt ~ 3 'terminus of the nucleotide sequence of SEQ ID NO: 94 region up (NADP alanine dehydrogenase Streptomyces aureofaciens + gene codon optimized nucleotide sequence encoding an amino acid sequence with a corresponding amino acid substitution), region up to 18 nt ~ 3 'terminus of the nucleotide sequence of SEQ ID NO: 95 (NADP to alanine dehydrogenase of Cylindrica Anabaena TasuCorresponding amino acid substitutions codon optimized nucleotide sequence of the gene encoding the amino acid sequence was performed), and NADP to 18 nt ~ 3 'alanine dehydrogenase region (Bacillus subtilis until end of the nucleotide sequence of SEQ ID NO: 96 + corresponding amino acid having at least one for example 80% or more of the codon optimized nucleotide sequence) of the gene encoding the amino acid sequence was substituted, or 85%, or 90%, or 95% or more sequence identity to a nucleotide sequence may be a DNA having a nucleotide sequence encoding a protein having alanine regenerating enzyme activity.
[0084]
　
　in recombinant the microorganism according to the present disclosure, the gene encoding pyridoxine dehydrogenase, pyridoxamine synthase genes encoding, and each of the gene encoding the amino acid regenerating enzyme having an enzyme activity to play an amino acid the pyridoxamine synthase consumes may be a gene inherent in bacterial cells, inherent in bacterial cells there may be a gene with enhanced expression may be a gene that has been introduced from the outside of cells to intracellular. However, the gene encoding pyridoxine dehydrogenase, the gene encoding the pyridoxamine synthase, and at least two extracellular of the genes encoding the amino acid regenerating enzyme having an enzyme activity to play an amino acid the pyridoxamine synthase consumes Although inherent in or bacteria is introduced gene is a gene whose expression is enhanced.
　In other words, the gene encoding pyridoxine dehydrogenase, the gene encoding the pyridoxamine synthase, and the pyridoxamine each gene synthase encodes an amino acid regenerating enzyme having an enzyme activity for reproducing the amino acid consumption before recombination host may be a gene inherent in the genome of the microorganism, but inherent in the genome of the host microorganism may be a gene with enhanced expression by operating the substitution or the like of the promoter, a vector such as a plasmid it may be a gene that has been introduced from the extracellular to the intracellular used. In a first aspect, exhibit pyridoxamine production capacity by combination of the three enzymes, playing a gene encoding pyridoxine dehydrogenase, the gene encoding the pyridoxamine synthase, and amino acids the pyridoxamine synthase consumes for at least two of the genes encoding the amino acid regenerating enzyme having an enzymatic activity, respectively, to enhance the expression of the gene endogenous to the host bacterium by introducing or from extracellular host microorganism, or substitution or the like of the promoter by, increased expression of the gene, and further enhance the pyridoxamine production capacity by combination of the three enzymes. Reinforcing wherein gene encoding pyridoxine dehydrogenase, the gene encoding the pyridoxamine synthase, and for each of the gene encoding the amino acid regenerating enzyme having an enzyme activity to play an amino acid the pyridoxamine synthase consumes the introduction or expression to the case of performing the introduction of the extracellular host microorganism of the strengthening of the gene expression by substitution or the like of the promoter, may be carried out only one, both may be performed.
[0085]
　Gene encoding pyridoxine dehydrogenase, the gene encoding the pyridoxamine synthase, and for at least two of the genes encoding the amino acid regenerating enzyme having an enzyme activity to play an amino acid the pyridoxamine synthase consumed, genes such as the without elevated expression, sufficient production capacity for high production of pyridoxamine, or a salt thereof can not be obtained. Here, the terms amino acid regenerating enzyme referred, to refer to an amino acid regenerating enzyme having an enzyme activity to play a particular amino acid species pyridoxamine synthase consumed, one pair for both the pyridoxamine synthase amino acid-regenerating enzyme it is possible to capture. Gene encoding pyridoxine dehydrogenase, the gene encoding the pyridoxamine synthase, and for at least two of the genes encoding the amino acid regenerating enzyme having an enzyme activity to play an amino acid the pyridoxamine synthase consumed, genes such as the since the increase in the expression is carried out, in the cells of the recombinant microorganism described above pairs exist, and for at least one of said pair would increase in gene expression is performed.
[0086]
　Target of at least one of introducing and expressing enhanced genes inherent in bacterial cells from extracellular to a gene encoding pyridoxine dehydrogenase, the gene encoding the pyridoxamine synthase, and the pyridoxamine synthase consumed amino be only two of the gene encoding the amino acid regenerating enzyme having an enzyme activity to play may be. Gene encoding pyridoxine dehydrogenase genes encoding pyridoxamine synthase, and for two of the gene encoding the amino acid regenerating enzyme having an enzyme activity for reproducing the pyridoxamine synthase consumes amino, respectively, from the extracellular when performing at least one of the strengthening introduction and gene inherent in bacterial expression, the gene encoding the gene and pyridoxamine synthase encoding pyridoxine dehydrogenase, respectively, the introduction and cells from extracellular it may be performed at least one of strengthening the expressed gene to endogenous, for genes encoding the gene and amino acid regenerating enzyme encoding pyridoxine dehydrogenase, respectively, the expression of genes endogenous to the introduction and cells from extracellular strengthening It out may be performed at least one, for genes encoding the gene and amino acid regenerating enzyme encoding pyridoxamine synthase, respectively, at least one of enhanced expression of genes underlying the introduction and cells from extracellular it may be carried out.
[0087]
　From the viewpoint of pyridoxamine or production efficiency in the production of a salt thereof, a gene encoding pyridoxine dehydrogenase, the gene encoding the pyridoxamine synthase, and all of the gene encoding the amino acid regenerating enzyme, respectively, from the extracellular it is preferable to perform at least one of enhanced expression of genes underlying the introduction and bacteria. The gene encoding at least amino acids regenerating enzyme, it is preferable from the viewpoint of production efficiency of pyridoxamine or a salt thereof to perform at least one of enhanced expression of genes underlying the introduction and cells from extracellular. Incidentally, introduction of the enzyme gene from extracellular is necessarily not limited to introduction to compensate for the enzyme gene that is not present in the host microorganism, performed for the purpose of increasing the expression also gene endogenous to the host microorganism it may be. The gene that is not present in the host microorganism can be readily ascertained using enzyme database, eg KEGG and BRENDA.
[0088]
　If enhancing expression of the gene by replacing the promoter of the gene endogenous to the host microorganism with a different promoter, enhanced expression of genes in host microorganism as such alternative promoters (promoter newly introduced) it is not particularly as long as it (can be enhanced than before promoter replacement) limit, or a inducible promoter in constitutive promoter. Promoter replacement can be carried out using general genetic recombination techniques. The arrangement of a promoter endogenous to the host microorganism, unless, may be left partially or completely adversely affect as a practical problem for expression by the newly introduced promoter.
　When the host microorganism is, for example, prokaryotes, Examples of usable promoters Promoters newly introduced, trp promoter, lac promoter derived from E. coli, and GAPDH promoter, PL promoter and PR promoter derived from lambda phage, Bacillus subtilis-derived gluconic acid synthetase promoter (gnt), alkali protease promoter (apr), neutral protease promoter (npr), alpha-amylase promoter (amy) and the like. Furthermore, uniquely modified or designed promoter sequence as tac promoter may also be utilized.
[0089]
　When the host microorganism is, for example, filamentous fungi, examples of usable promoters Promoters newly introduced, cellobiohydrolase (cbh) promoter, endoglucanase (egl) promoter, xylanase III (XYN3) promoter, U6 promoter, alpha-amylase (amy) promoter, and the like.
[0090]
　When the host microorganism is, for example, yeast, examples of usable promoters Promoters newly introduced, alcohol dehydrogenase (ADH1) promoter, phosphoglycerate kinase (PGK1) promoter, peptide chain elongation factor (TEF) promoter , include glycerol 3-phosphate dehydrogenase (GPD) promoter, galactokinase (GAL1) promoter, metallothionein (CUP1) promoter, repressible acid phosphatase (PHO5) promoter and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter It is. Incidentally, from the sequence of the promoter is not limited to the yeast as a host microorganism. Such as cytomegalovirus (CMV) promoter, may be used as the promoter of the foreign.
[0091]
　When introducing the genes from extracellular host microorganism (foreign (heterogenous) gene), examples of the method to introduce genes into bacterial cells (intracellular), to express the enzyme encoded by the gene if it is possible, not particularly limited, transformed with a plasmid carrying the enzyme gene introduction into the genome of the gene, and can be combinations thereof. When introducing genes the expression vector incorporating the gene may be introduced in the cells. The expression vector is not particularly limited as long as it incorporates the nucleotide sequence of the gene, a plasmid vector shown from the viewpoint of enhancing the transformation efficiency and translation efficiency, a structure as shown below and more preferably, which is a phage vector.
[0092]
　Expression vector comprises a nucleotide sequence of the gene is not particularly limited as long as it can transform the host microorganism. If necessary, in addition to said nucleotide sequence, the nucleotide sequence constituting the other areas (hereinafter, simply referred to as "other regions".) May be contained. Other regions, for example, a recombinant microorganism obtained by transformation, and the control area required to produce the desired enzymes, and the like region required for autonomous replication.
　Further, from the viewpoint of facilitating the selection of the recombinant microorganism may further contain a nucleotide sequence encoding a selection gene can be a selectable marker.
　The control area needed to produce the desired enzyme, the promoter sequence (including an operator sequence to control transcription.), Ribosome binding sequence (SD sequence) include transcription termination sequences and the like.

WE claims

Have a gene that encodes a gene encoding pyridoxine dehydrogenase gene encodes pyridoxamine synthase having an enzyme activity to synthesize pyridoxamine from pyridoxal, and an amino acid regenerating enzyme having an enzyme activity to play an amino acid wherein the pyridoxamine synthase consumes and,
　the gene encoding the pyridoxine dehydrogenase, wherein at least two of gene gene and encodes the amino acid regenerating enzyme encoding pyridoxamine synthase inherent in or bacterial cells a gene introduced from the outside of the cells There is a gene whose expression has been strengthening its recombinant microorganism.
[Requested item 2]
　The pyridoxamine synthase, pyridoxamine - pyruvic transaminase, pyridoxamine - oxaloacetic transaminase, aspartate transaminase, or pyridoxamine phosphate transaminase recombinant microorganism of claim 1.
[Requested item 3]
　The pyridoxine dehydrogenase includes at least one of the partial amino acid sequence of the following (a) and the partial amino acid sequence (b), and has a pyridoxine dehydrogenase activity, recombinant microorganism according to claim 1 or claim 2. (A)
NX 1 X 2 EX 3 YG (SEQ ID NO: 97) 　　(wherein, X 1 represents V, C, I, A, M, S, G or 　　L, X 2 represents G or A , 　　X 3 represents F or L) (b) X 4 X 5 X 6 KGX 7 (SEQ ID NO: 　　98) (X 4 represents I, V, F or 　　L, X 5 is S, T, N, represents C or 　　M, X 6

Represents C, V, A, I, W or
　　F, X 7 represents G, A, S or C)
[Requested item 4]
　The pyridoxine dehydrogenase is represented by enzyme number EC1.1.1.65, recombinant microorganism as claimed in any one of claims 1 to 3.
[Requested item 5]
　The pyridoxine gene encoding Shin dehydrogenase is derived from Saccharomyces cerevisiae, the recombinant microorganism as claimed in any one of claims 1 to 4.
[Requested item 6]
　The gene encoding pyridoxine dehydrogenase,
　or having any of the nucleotide sequences of SEQ ID NO: 7 and SEQ ID NO: 53 to SEQ ID NO: 59,
　SEQ ID NO: 13 and SEQ ID NO: 75-81 18 th in either of the nucleotide sequences of nucleotides to the 3 'having a region to the end,
　SEQ ID NO: 7 and SEQ ID NO: 53 DNA having a nucleotide sequence complementary to any of the nucleotide sequences of to SEQ ID NO: 59 or SEQ ID NO: 13 and SEQ ID NO: 75 ~ 81 nucleotide sequence encoding a protein having any of the 18 th nucleotides and hybridizes with the DNA under stringent conditions with a region complementary to the nucleotide sequence from the 3 'terminus of the nucleotide sequence, and pyridoxine dehydrogenase activity with a The recombinant microorganism as claimed in any one of claims 1 to 5.
[Requested item 7]
　The pyridoxine gene encoding Shin dehydrogenase is derived from Schizosaccharomyces pombe, recombinant microorganism as claimed in any one of claims 1 to 4.
[Requested item 8]
Gene encoding the pyridoxine dehydrogenase, or having the nucleotide sequence of SEQ ID NO: 8, a DNA which hybridizes with the DNA under stringent conditions with a nucleotide sequence complementary to a nucleotide sequence of SEQ ID NO: 8, a and pyridoxine dehydrogenase activity having a nucleotide sequence encoding a protein having recombinant microorganism as claimed in any one of claims 1 to 4 and claim 7.
[Requested item 9]
　The gene encoding pyridoxine dehydrogenase, SEQ ID NO: 1, any of the amino acid sequence of SEQ ID NO: 2 and SEQ ID NO: 22 to SEQ ID NO: 28, or SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 22 to 28 amino acid sequence of having a nucleotide sequence encoding the amino acid sequence, which has at least one 80% or more sequence identity to have and pyridoxine dehydrogenase activity, recombinant microorganism as claimed in any one of claims 1 to 8 .
[Requested item 10]
　The pyridoxamine synthase partial amino acid sequence of the following (c), the partial amino acid sequence of (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) out includes at least one, and having an enzymatic activity to synthesize pyridoxamine from pyridoxal recombinant microorganism as claimed in any one of claims 1 to 9. (C)
X 8 X 9 X 10 X 11 X 12 X 13 (SEQ ID NO: 　　99) (X 8 represents L, M, I or 　　V, X 9 represents H or 　　Q, X 10 is, G represents C or 　　a, X 11 represents E or 　　D, X 12 represents P or 　　a, X 13 represents V, I, L or a)

(D) X 14 X 15 TPSGTX 16 X 17 (SEQ ID NO:
　　100) (X 14 represents H or
　　S, X 15 represents D or
　　E, X 16 represents I, V or
　　L, X 17 represents N or
T) (e) X 18 DX 19 VSX 20 X 21 (SEQ ID NO: 　　101) (X 18 is, V, represents I or 　　a, X 19 is, a, represents T or S, 　　X 20 represents S, a or 　　G, X

21 is F, W, or an
V) (f) X 22 X 23 X 24 KCX 25 GX 26 X 27 P (SEQ ID NO: 　　102) (X 22 represents G or 　　S, X 23 is, P, S or 　　a, X 24 represents N, G, S, a or 　　Q, X 25 represents L or 　　M, X 26 represents a, S, C or 　　G, X 27 is P, T, represents an S or a) (g) X 28 X 29 X 30 X 31

SX 32 GX 33 X 34 (SEQ ID NO:
103) (X 28 represents G or
　　D, X 29 represents V or
　　I, X 30 is V, T, A, S, M, I or L
　　represents, X 31 represents F, M, L, I or
　　V, X 32 represents S, G, a, T, I, L or
　　H, X 33 represents R, M or Q,
　　X 34 is, G, R, a, D , H or
K) (h) X 35 X 36 RX 37 X 38 HMGX 39 X 40A (SEQ ID NO:
　　104) (X 35 represents L or
　　V, X 36 represents T, I, V or
　　L, X 37 represents I, V or
　　L, X 38 is, G or S the
　　stands, X 39 is, P, represents a or
　　R, X 40 represents T, V or S)
[Requested item 11]
　The pyridoxamine synthase is expressed in enzyme number EC2.6.1.30, recombinant microorganism as claimed in any one of claims 1 to 10.
[Requested item 12]
　The gene encoding the pyridoxamine synthase is derived from Mesorhizobium loti, recombinant microorganism according to any one of claims 1 to 11.
[Requested item 13]
　Gene encoding the pyridoxamine synthase,
　or having any of the nucleotide sequences of SEQ ID NO: 9 and SEQ ID NO: 60 to SEQ ID NO: 66,
　the region of up to 18 nt ~ 3 'terminus of the nucleotide sequence of SEQ ID NO: 14 or 18 nt ~ 3 in any of the nucleotide sequence of SEQ ID NO: 82 to SEQ ID NO: 88 'or with a region of up to end,
　and any nucleotide sequence of SEQ ID NO: 9 and SEQ ID NO: 60 to SEQ ID NO: 66 18 nt ~ 3 in any of the nucleotide sequences of the 18 th nucleotides to the 3 'region up-or SEQ ID NO: 82 to SEQ ID NO: 88 in the nucleotide sequence of the DNA or SEQ ID NO: 14, having a complementary nucleotide sequence complementary nucleotides a region of 'to the end A DNA which hybridizes with the DNA under stringent conditions with the sequence, and has a nucleotide sequence encoding a protein with the enzymatic activity to synthesize pyridoxamine from pyridoxal, claimed in any one of claims 1 to 12 the recombinant microorganism.
[Requested item 14]
　The pyridoxamine gene encoding synthase, SEQ ID NO: 3 and SEQ ID NO: 29 to any of the amino acid sequence, or SEQ ID NO: 3 and at least one 80 of the amino acid sequence of SEQ ID NO: 29 to SEQ ID NO: 35 of SEQ ID NO: 35 % with a nucleotide sequence encoding the amino acid sequence, having an enzyme activity to synthesize sequence pyridoxamine from identity to have and pyridoxal above recombinant microorganism as claimed in any one of claims 1 to 13 .
[Requested item 15]
　Wherein the amino acid regenerating enzyme is alanine dehydrogenase, recombinant microorganism as claimed in any one of claims 10 to claim 14.
[Requested item 16]
　The alanine dehydrogenase, pyruvate and NH 3 and NADPH during the reaction to produce L- alanine is available as a coenzyme, recombinant microorganism of claim 15.
[Requested item 17]
　The alanine dehydrogenase partial amino acid sequence of the following (i), the partial amino acid sequence (j), wherein at least one of the partial amino acid sequence (k) and the partial amino acid sequence (l), and pyruvate and NH 3 from L- the recombinant microorganism of claim 15 or claim 16 having the activity to form alanine. (I)
EX 41 KX 42 X 43 EX 44 RX 45 X 46 (SEQ ID NO: 　　105) (X 41 represents I, T, S, F, N or 　　V, X 42 is N, M, A, V , L, represents T or 　　D, X 43 is, H, N, represents L or 　　Q, X 44 is, Y, represents N or 　　F, X 45 is, V, or an 　　I, X 46

Is, G or an
A) (j) X 47 X 48 X 49 KVKEPX 50 (SEQ ID NO: 　　106) (X 47 represents M or 　　L, X 48 is, I, represents L or 　　V, X 49 is represents V, L, I or 　　M, X 50 is Q, L, V represents N or I) (k) LX 51 TYLHLA (SEQ ID NO: 　　107) (X 51 represents F or Y) ( l) X 52 DX 53 AX 54 DQGG (SEQ ID NO: 　　108) (X 52 represents V or a,

　　X 53 represents V or
　　I, X 54 represents I or V)
[Requested item 18]
　Gene encoding the amino acid regeneration enzyme is Shewanella sp. From AC10, recombinant microorganism according to any one of claims 15 to claim 17.
[Requested item 19]
　Gene encoding the amino acid regeneration enzyme,
　or has any nucleotide sequence of SEQ ID NO: 11 and SEQ ID NO: 67 to SEQ ID NO: 74,
　any nucleotide sequence of SEQ ID NO: 15 and SEQ ID NO: 89 to SEQ ID NO: 96 18 nt ~ 3 'having a region to the end, in
　DNA having a nucleotide sequence complementary to any of the nucleotide sequences of SEQ ID NO: 11 and SEQ ID NO: 67 to SEQ ID NO: 74, or SEQ ID NO: 15 and SEQ number 89 to a DNA which hybridizes with the DNA under stringent conditions with a 18 nt ~ 3 'region complementary to the nucleotide sequence to end in any of the nucleotide sequence of SEQ ID NO: 96, and alanine regenerating enzyme activity have the nucleotide sequence encoding a protein having a That recombinant microorganism according to any one of claims 15 to claim 18.
[Requested item 20]
　Gene encoding the amino acid regeneration enzyme, SEQ ID NO: 5 and SEQ ID NO: 45 to any of the amino acid sequence, or SEQ ID NO: 5 and SEQ ID NO: 45 to at least one 80 of the amino acid sequence of SEQ ID NO: 52 of SEQ ID NO: 52 % with a nucleotide sequence encoding the amino acid sequence, having the above sequence identity have and alanine regenerating enzyme activity, recombinant microorganism as claimed in any one of claims 15 to claim 19.
[Requested item 21]
　The pyridoxamine synthase is expressed in enzyme number EC2.6.1.31 or EC2.6.1.1, recombinant microorganism as claimed in any one of claims 1 to 9.
[Requested item 22]
　The pyridoxamine gene encoding synthase is derived from Escherichia coli, recombinant microorganism as claimed in any one of claims 1 to 9 and claim 21.
[Requested item 23]
　Gene encoding the pyridoxamine synthase, or having the nucleotide sequence of SEQ ID NO: 10, a DNA which hybridizes with the DNA under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 10, and pyridoxamine from pyridoxal having a nucleotide sequence encoding a protein having an enzymatic activity to synthesize, claims 1 to 9, the recombinant microorganism according to any one of claims 21 and claim 22.
[Requested item 24]
　Gene encoding the pyridoxamine synthase, the amino acid sequence of SEQ ID NO: 4, or amino acid sequence and amino acid sequence having and enzyme activity to synthesize pyridoxamine from pyridoxal has 80% or more sequence identity with SEQ ID NO: 4, the having a nucleotide sequence encoding recombinant microorganism as claimed in any one of claims 1 to 9 and claims 21 to claim 23.
[Requested item 25]
Wherein the amino acid regenerating enzyme is glutamate dehydrogenase, recombinant microorganism as claimed in any one of claims 21 to claim 24.
[Requested item 26]
　Gene encoding the amino acid regenerating enzyme is derived from Escherichia coli, recombinant microorganism of claim 25.
[Requested item 27]
　Gene encoding the amino acid regeneration enzyme, or has the nucleotide sequence of SEQ ID NO: 12, a DNA which hybridizes with the DNA under stringent conditions with a nucleotide sequence complementary to a nucleotide sequence of SEQ ID NO: 12, and glutamic acid regenerating enzyme having a nucleotide sequence encoding a protein having activity, recombinant microorganism of claim 25 or claim 26.
[Requested item 28]
　The nucleotide sequence of the gene encoding the amino acid regenerating enzyme encodes the amino acid sequence of SEQ ID NO: 6, or amino acid sequence and amino acid sequence having and glutamate regenerating enzyme activity has 80% or more sequence identity with SEQ ID NO: 6, the the a recombinant microorganism according to any one of claims 25 to claim 27.
[Requested item 29]
　A recombinant E. coli, the recombinant microorganism as claimed in any one of claims 1 to 28.
[Requested item 30]
　And treated product of the culture or the recombinant microorganism or the culture of a recombinant microorganism or the recombinant microorganism according to any one of claims 1 to 29 is brought into contact with pyridoxine or a salt thereof, pyridoxamine or to produce a salt thereof, pyridoxamine or a salt thereof.
[Requested item 31]
　Processed product of said recombinant microorganism or culture of the recombinant microorganism or the recombinant microorganism or the culture comprises the pyridoxine dehydrogenase, the pyridoxamine synthase, and the amino acid regenerating enzyme, prepared according to claim 30 Method.
[Requested item 32]
　Treated product of the recombinant microorganism of the workpiece or culture of the recombinant microorganism, heat treatment, cooling treatment, mechanical disruption of the cells, sonication, freeze-thaw treatment, drying treatment, pressurization or depressurization treatment, osmotic pressure treatment, cell autolysis, detergent treatment, an enzyme treatment, the cell separation process, treated by the process comprising one or more selected from the group consisting of purification treatment and extraction process, according to claim 30 or claim the method according to 31.
[Requested item 33]
　The following (1) to (7) Recombinant at least one of the polynucleotide described is introduced into a microorganism:
(1) a polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 1,
(2) SEQ ID NO: 1 in the amino acid sequence, 1 to 50 amino acids are deleted, a polynucleotide encoding a protein consisting of the additional or substituted amino acid sequence, the protein synthesis of pyridoxine or a salt thereof or pyridoxal or its salt those having activity, polynucleotides,
(3) a polynucleotide encoding a protein comprising the amino acid sequence having an amino acid sequence at least 60% sequence identity with SEQ ID NO: 1, wherein the protein is pyridoxine or a salt thereof or pyridoxal or case of the salt Those having activity, polynucleotide,
(4) a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 13,
in the nucleotide sequence of (5) SEQ ID NO: 13, 1 to 150 nucleotides are deleted, added or substituted a polynucleotide consisting of in which the nucleotide sequence, wherein the polynucleotide is a polynucleotide encoding a protein having the synthetic activity of pyridoxine or a salt thereof or pyridoxal or its salt,
(6) a nucleotide sequence of SEQ ID NO: 13 with at least 60% a polynucleotide comprising a nucleotide sequence having a sequence identity, the polynucleotide a polynucleotide encoding a protein having an activity of synthesizing the pyridoxine or a salt thereof or pyridoxal or its salt,
(7) a polynucleotide hybridizing with a polynucleotide under stringent conditions comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 13, the polynucleotide pyridoxine or a salt thereof, or pyridoxal or synthetic activity of the salt polynucleotide encoding a protein having.
[Requested item 34]
　The recombinant microorganism is a recombinant bacterium, a recombinant microorganism of claim 33.
[Requested item 35]
　The recombinant microorganism is a recombinant Escherichia coli The recombinant microorganism of claim 34.
[Requested item 36]
　It said protein is an enzyme classified in EC number 1.1.1.65, recombinant microorganism according to any one of claims 33 to claim 35.
[Requested item 37]
　It said protein is a pyridoxine dehydrogenase from Saccharomyces cerevisiae, the recombinant microorganism according to any one of claims 33 to claim 36.
[Requested item 38]
　A recombinant microorganism according to any one of claims 33 to claim 37, comprising contacting a pyridoxine or a salt thereof, pyridoxal or a salt thereof.
[Requested item 39]
　And treated product of any culture obtained by culturing the recombinant microorganisms described (1) or cultures of claims 33 to claim 37, comprising contacting a pyridoxine or a salt thereof, pyridoxal or its method for producing a salt.