阅读说明：本技术 一种手性3-氨基-1-丁醇的合成方法 (Synthesis method of chiral 3-amino-1-butanol ) 是由 孙周通 赵强 刘保艳 刘贝贝 闫豪杰 于 2018-05-31 设计创作，主要内容包括：本发明公开了一种手性3-氨基-1-丁醇的合成方法。该方法包括如下步骤：以1,3-丁二醇为底物,经酶A及其辅酶催化反应生成3-酮-1-丁醇；以3-酮-1-丁醇为底物,经酶B及其辅酶催化反应生成手性3-氨基-1-丁醇；所述酶A选自醇脱氢酶或醇脱氢酶酶的突变体；所述酶B转氨酶或转氨酶的突变体。本发明提供了一条全新的绿色生物合成路线,以廉价的1,3-丁二醇为原料,通过多酶共表达或级联或分步催化合成手性3-氨基-1-丁醇,即(R)-3-氨基-1-丁醇和(S)-3-氨基-1-丁醇。(The invention discloses a method for synthesizing chiral 3-amino-1-butanol. The method comprises the following steps: using 1, 3-butanediol as a substrate, and generating 3-ketone-1-butanol through catalytic reaction of enzyme A and coenzyme thereof; using 3-ketone-1-butanol as a substrate, and generating chiral 3-amino-1-butanol through catalytic reaction of enzyme B and coenzyme thereof; the enzyme A is selected from alcohol dehydrogenase or a mutant of alcohol dehydrogenase enzyme; the enzyme B transaminase or a mutant of transaminase. The invention provides a brand-new green biosynthesis route, which takes cheap 1, 3-butanediol as a raw material to synthesize chiral 3-amino-1-butanol, namely (R) -3-amino-1-butanol and (S) -3-amino-1-butanol, through multi-enzyme co-expression or cascade or step-by-step catalysis.)

1. A method for synthesizing chiral 3-amino-1-butanol comprises the following steps:

(A) Using 1, 3-butanediol as a substrate, and generating 3-ketone-1-butanol through catalytic reaction of enzyme A and coenzyme thereof;

(B) Taking the 3-keto-1-butanol generated in the step (A) as a substrate, and generating chiral 3-amino-1-butanol through catalytic reaction of an enzyme B and a coenzyme thereof;

The enzyme A is selected from any one of the following: alcohol dehydrogenase, mutants of said alcohol dehydrogenase;

the enzyme B is selected from any one of the following: a transaminase, a mutant of said transaminase.

2. The method of claim 1, wherein:

The alcohol dehydrogenase is derived from any one of the following microorganisms: lactobacillus brevis, Thermoanaerobacter brevis, Lactobacillus kefir, Thermoanaerobacter virginica, enterococcus faecium, Enterobacter casseliflavus, Thermomyces carbonmonozae, Anemonobacterium alkylthermophilus, Mycobacteria, Methanosarcina methanolica, Clostridium beijerinckii, Clostridium unculturens, Clostridium sporogenes, Enterobacter devulcani, Clostridium dialosum, Clostridium spodum, Clostridium diumuliginosum, Clostridium bacteria, Candida parapsilosis;

And/or the transaminase is derived from any of the following microorganisms: bacillus megaterium, Pseudomonas aeruginosa, Violaceous bacillus, Aspergillus terreus, Fissistigma, Mycobacterium, Arthrobacter.

3. The method of claim 2, wherein:

The alcohol dehydrogenase is any one of the following (a1) - (a 20):

(a1) Alcohol dehydrogenase derived from Lactobacillus brevis (Lactobacillus brevis) and having an amino acid sequence of SEQ ID No. 2;

(a2) Alcohol dehydrogenase derived from Thermoanaerobacter brockii (Thermoanaerobacter brockii), the amino acid sequence of which is SEQ ID No. 4;

(a3) an alcohol dehydrogenase derived from Lactobacillus kefirs (Lactobacillus kefiri DSM 20587) having an amino acid sequence of SEQ ID No. 6;

(a4) An alcohol dehydrogenase derived from Thermoanaerobacter virginiae Rwielii 8.B1, having an amino acid sequence of SEQ ID No. 8;

(a5) Alcohol dehydrogenase derived from enterococcus alcoholierans (Oenococcus alcoholierans), and the amino acid sequence is SEQ ID No. 10;

(a6) Alcohol dehydrogenase derived from Desulfotomanum nigricans (Desulfotomaculum nigricans), the amino acid sequence of which is SEQ ID No. 12;

(a7) an alcohol dehydrogenase derived from Thermoascus carbox carboydivorans (Thermosinus carboxydivorans) having an amino acid sequence of SEQ ID No. 14;

(a8) Alcohol dehydrogenase derived from Thermoanaerobacterium alkanothermophilum (Thermoanaerobacter mathranii), and having an amino acid sequence of SEQ ID No. 16;

(a9) an alcohol dehydrogenase derived from a bacterium of the phylum Firmicutes (Firmicutes bacterium CAG:137) having the amino acid sequence of SEQ ID No. 18;

(a10) an alcohol dehydrogenase derived from Methanosarcina thermophila, having an amino acid sequence of SEQ ID No. 20;

(a11) An alcohol dehydrogenase derived from Clostridium beijerinckii (Clostridium beijerinckii) having an amino acid sequence of SEQ ID No. 22;

(a12) An alcohol dehydrogenase derived from uncultured Clostridium sp, having an amino acid sequence of SEQ ID No. 24;

(a13) An alcohol dehydrogenase derived from Clostridium sporogenes (Clostridium taeniosporum) having an amino acid sequence of SEQ ID No. 26;

(a14) An alcohol dehydrogenase derived from Enterobacter desulfatous (Desrotococcus putei), the amino acid sequence of which is SEQ ID No. 28;

(a15) An alcohol dehydrogenase derived from Clostridium difficile (Clostridium diolis) having the amino acid sequence of SEQ ID No. 30;

(a16) an alcohol dehydrogenase derived from Clostridium clostridia (Clostridium cochleariae) having the amino acid sequence of SEQ ID No. 32;

(a17) An alcohol dehydrogenase derived from Clostridium vaccinium uliginosum (Clostridium uliginosum), having an amino acid sequence of SEQ ID No. 34;

(a18) an alcohol dehydrogenase derived from a Clostridium bacterium (Clostridium sp-Y3), having the amino acid sequence of SEQ ID No. 36;

(a19) An alcohol dehydrogenase derived from Candida parapsilosis (Candida parapsilosis) having an amino acid sequence of SEQ ID No. 38;

(a20) A fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of a protein defined in any one of (a1) to (a 19);

and/or the transaminase is any one of (b1) - (b8) as follows:

(b1) transaminase derived from Bacillus megaterium (Bacillus megaterium) and having an amino acid sequence of SEQ ID No. 40;

(b2) a transaminase derived from pseudomonas aeruginosa (p. aeruginosa PAO2) having an amino acid sequence of SEQ ID No. 42;

(b3) A transaminase derived from Bacillus violaceum 2025, having the amino acid sequence of SEQ ID No. 44;

(b4) A transaminase derived from Aspergillus terreus (Aspergillus terreus) having the amino acid sequence of SEQ ID No. 46;

(b5) A transaminase derived from Fusarium fischer (Neosartorya fischeri) having an amino acid sequence of SEQ ID No. 48;

(b6) a transaminase derived from Mycobacterium (Mycobacterium vanbalenii) having an amino acid sequence of SEQ ID No. 50;

(b7) A transaminase derived from Arthrobacter sp.KNK168, having an amino acid sequence of SEQ ID No. 52;

(b8) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in any one of (b1) to (b 7).

4. The method of claim 1, wherein: the mutant of the alcohol dehydrogenase is (c1) - (c5) as follows:

(c1) compared with the alcohol dehydrogenase derived from Lactobacillus brevis shown in SEQ ID No.2, at least one of the following mutations is present or present: I11V, G37D;

(c2) Compared with the alcohol dehydrogenase derived from Thermoanaerobacterium as shown in SEQ ID No.4, at least one of the following mutations exists or exists only: a85G, I86A, W110A, G198D;

Further, the mutant of the alcohol dehydrogenase has the following mutations or only has the following mutations compared with the alcohol dehydrogenase derived from the high-temperature anaerobium as shown in SEQ ID No. 4: A85G/I86A or A85G/I86A/W110A or A85G/I86A/G198D;

(c3) Compared with the alcohol dehydrogenase derived from Lactobacillus kefir as shown in SEQ ID No.6, the following mutations are present or present: G37D;

(c4) in comparison with the alcohol dehydrogenase from Thermoanaerobacter virginiae shown in SEQ ID No.8, the following mutations are present or present: G198D;

(c5) A fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in any one of (C1) to (C4).

5. The method of any of claims 1 to 4, wherein: in the method, the enzyme A and the enzyme B are catalyzed in the form of crude enzyme solution, crude enzyme solution freeze-dried powder, pure enzyme or whole cells;

further, the crude enzyme solution freeze-dried powder and the pure enzyme are prepared according to the method comprising the following steps: expressing the enzyme A and/or the enzyme B in a host cell to obtain a recombinant cell; cracking the recombinant cells to obtain the crude enzyme solution, the crude enzyme solution freeze-dried powder or pure enzyme;

further, the whole cells are prepared according to a method comprising the following steps: expressing the enzyme A and/or the enzyme B in a host cell to obtain a recombinant cell, namely the whole cell;

Still further, the recombinant cell is prepared according to a method comprising the following steps: introducing a nucleic acid molecule capable of expressing the enzyme A and/or the enzyme B into the host cell, and obtaining the recombinant cell expressing the enzyme A and/or the enzyme B after induction culture;

Further, said "nucleic acid molecule capable of expressing said enzyme A and/or said enzyme B" is introduced into said host cell in the form of a recombinant vector; the recombinant vector is a bacterial plasmid, a bacteriophage, a yeast plasmid or a retrovirus packaging plasmid carrying the coding gene of the enzyme A and/or the enzyme B;

and/or the host cell is a prokaryotic cell or a lower eukaryotic cell;

Specifically, the prokaryotic cell is a bacterium; the lower eukaryotic cell is a yeast cell;

more specifically, the bacterium is escherichia coli.

6. The method of claim 5, wherein:

The sequence of the coding gene of the alcohol dehydrogenase from the Lactobacillus brevis is SEQ ID No.1 or a fusion sequence obtained after connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene or a random or/and site-directed mutagenesis sequence which has the function and codes the same protein;

the sequence of the coding gene of the alcohol dehydrogenase from the high-temperature anaerobic bacillus is SEQ ID No.3 or a fusion sequence obtained after the 5 'end and/or the 3' end of the coding gene is connected with a tag coding sequence or a random or/and site-directed mutagenesis sequence which has the function and codes the same protein;

the sequence of the coding gene of the alcohol dehydrogenase from the lactobacillus gasseri is SEQ ID No.5 or a fusion sequence obtained after connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene or a random or/and site-directed mutagenesis sequence which has the function and codes the same protein;

The sequence of the coding gene of the alcohol dehydrogenase from the anaerobic bacillus virginiae is SEQ ID No.7 or a fusion sequence obtained after connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene or a random or/and site-directed mutagenesis sequence which has the function and codes the same protein;

The sequence of the coding gene of the alcohol dehydrogenase from the wine coccus is SEQ ID No.9 or a fusion sequence obtained after connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene or a random or/and site-directed mutagenesis sequence which has the function and codes the same protein;

the sequence of the coding gene of the alcohol dehydrogenase from the black desulfurization enterobacter is SEQ ID No.11 or a fusion sequence obtained after the 5 'end and/or the 3' end of the coding gene are connected with a tag coding sequence or a random or/and site-directed mutagenesis sequence which has the function and codes the same protein;

the sequence of the coding gene of the alcohol dehydrogenase from the thermophilic antrodia carbon monoxide is SEQ ID No.13 or a fusion sequence obtained after the 5 'end and/or the 3' end of the coding gene is connected with a label coding sequence or a random or/and site-directed mutagenesis sequence which has the functions and codes the same protein;

The sequence of the coding gene of the alcohol dehydrogenase from the alkyl thermophilic anaerobe is SEQ ID No.15 or a fusion sequence obtained after the 5 'end and/or the 3' end of the coding gene is connected with a tag coding sequence or a random or/and site-directed mutagenesis sequence which has the function and codes the same protein;

The sequence of the coding gene of the alcohol dehydrogenase from the firmicutes bacteria is SEQ ID No.17 or a fusion sequence obtained after connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene or a random or/and site-directed mutagenesis sequence which has the function and codes the same protein;

The sequence of the coding gene of the alcohol dehydrogenase from the methanosarcina sarcina is SEQ ID No.19 or a fusion sequence obtained after connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene or a random or/and site-directed mutagenesis sequence which has the function and codes the same protein;

The sequence of the coding gene of the alcohol dehydrogenase derived from clostridium beijerinckii is SEQ ID No.21 or a fusion sequence obtained by connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene or a random or/and site-directed mutagenesis sequence which has the functions and codes the same protein;

The sequence of the coding gene of the alcohol dehydrogenase from the uncultured clostridium is SEQ ID No.23 or a fusion sequence obtained after connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene or a random or/and site-directed mutagenesis sequence which has the functions and codes the same protein;

the sequence of the coding gene of the alcohol dehydrogenase from the clostridium filamentous spore is SEQ ID No.25 or a fusion sequence obtained by connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene or a random or/and site-directed mutagenesis sequence which has the functions and codes the same protein;

The sequence of the coding gene of the alcohol dehydrogenase from the desulfurization enterobacter is SEQ ID No.27 or a fusion sequence obtained after the 5 'end and/or the 3' end of the coding gene are connected with a tag coding sequence or a random or/and site-directed mutagenesis sequence which has the function and codes the same protein;

the sequence of the coding gene of the alcohol dehydrogenase from clostridium dialicum is SEQ ID No.29 or a fusion sequence obtained by connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene or a random or/and site-directed mutagenesis sequence which has the functions and codes the same protein;

The sequence of the coding gene of the alcohol dehydrogenase from the clostridium spongoides is SEQ ID No.31 or a fusion sequence obtained after connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene or a random or/and site-directed mutagenesis sequence which has the functions and codes the same protein;

the sequence of the coding gene of the alcohol dehydrogenase derived from clostridium vaccinium is SEQ ID No.33 or a fusion sequence obtained by connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene or a random or/and site-directed mutagenesis sequence which has the function and codes the same protein;

the sequence of the coding gene of the alcohol dehydrogenase from clostridium bacteria is SEQ ID No.35 or a fusion sequence obtained after connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene or a random or/and site-directed mutagenesis sequence which has the function and codes the same protein;

the sequence of the coding gene of the alcohol dehydrogenase derived from the candida parapsilosis is SEQ ID No.37 or a fusion sequence obtained after connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene or a random or/and site-directed mutagenesis sequence which has the function and codes the same protein;

The sequence of the coding gene of the transaminase derived from the bacillus megatherium is SEQ ID No.39 or a fusion sequence obtained by connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene or a random or/and site-directed mutagenesis sequence which has the functions and codes the same protein;

the sequence of the coding gene of the transaminase derived from the pseudomonas aeruginosa is SEQ ID No.41 or a fusion sequence obtained by connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene or a random or/and site-directed mutagenesis sequence which has the functions and codes the same protein;

the sequence of the coding gene of the transaminase derived from the violaceous bacillus is SEQ ID No.43 or a fusion sequence obtained by connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene or a random or/and site-directed mutagenesis sequence which has the functions and codes the same protein;

The sequence of the coding gene of the aminotransferase from the aspergillus terreus is SEQ ID No.45 or a fusion sequence obtained after connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene or a random or/and site-directed mutagenesis sequence which has the function and codes the same protein;

the sequence of the transaminase coding gene from Fischer-Tropsch is SEQ ID No.47 or a fusion sequence obtained by connecting a tag coding sequence at the 5 'end and/or the 3' end of the transaminase coding gene or a random or/and site-directed mutagenesis sequence which retains the function and codes the same protein;

the sequence of the coding gene of the transaminase derived from the mycobacteria is SEQ ID No.49 or a fusion sequence obtained by connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene or a random or/and site-directed mutagenesis sequence which has the functions and codes the same protein;

the sequence of the coding gene of the transaminase derived from the arthrobacter is SEQ ID No.51 or a fusion sequence obtained by connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene or a random or/and site-directed mutagenesis sequence which has the functions and codes the same protein;

The sequence of the coding gene of the mutant of the alcohol dehydrogenase derived from the lactobacillus brevis is any one of the following (d1) to (d 3): (d1) compared to SEQ ID No.1, at least one of the following mutations is present or present only: A31G/T33G, G110A/C111T; (d2) a fusion sequence obtained by ligating a tag-encoding sequence to the 5 'end and/or 3' end of the sequence defined in (d 1); (d3) random or/and site-directed mutagenesis sequences which retain a function compared to the sequences defined in (d1) or (d2) and which encode the same protein;

The sequence of the encoding gene of the mutant of alcohol dehydrogenase derived from Thermoanaerobacterium is any one of the following (e1) to (e 3): (e1) compared to SEQ ID No.3, at least one of the following mutations is present or present only: C254G/T255C, A256G/T257C/T258G, T328G/G329C, G593A/C594T; (e2) a fusion sequence obtained by ligating a tag-encoding sequence to the 5 'end and/or 3' end of the sequence defined in (e 1); (e3) random or/and site-directed mutagenesis sequences which retain a function compared to the sequences defined in (e1) or (e2) and which encode the same protein;

Further, the (e1) is: compared to SEQ ID No.3, any of the following mutations is present or only present: C254G/T255C/A256G/T257C/T258G, C254G/T255C/A256G/T257C/T258G/T328G/G329C, C254G/T255C/A256G/T257C/T258G/G593A/C594T;

The sequence of the coding gene of the mutant of the alcohol dehydrogenase derived from lactobacillus gasseri is any one of the following (f1) - (f 3): (f1) in comparison with SEQ ID No.5, the following mutations are present or only present: G110A/C111T; (f2) a fusion sequence obtained by ligating a tag-encoding sequence to the 5 'end and/or 3' end of the sequence defined in (f 1); (f3) (ii) a random or/and site-directed mutagenesis sequence retaining a function compared to the sequence defined in (f1) or (f2) and encoding the same protein;

the sequence of the coding gene of the mutant of the alcohol dehydrogenase derived from the thermoanaerobacterium virginiae is any one of the following (g1) to (g 3): (g1) compared to SEQ ID No.7, at least one of the following mutations is present or present only: G593A/C594T; (g2) a fusion sequence obtained by ligating a tag-encoding sequence to the 5 'end and/or 3' end of the sequence defined in (g 1); (g3) random or/and site-directed mutagenesis sequences which retain the function and encode the same protein as the sequences defined in (g1) or (g 2).

7. the method of any of claims 1 to 6, wherein: in the step (A) and the step (B), the temperature of the catalytic reaction is 25-37 ℃; the time of the catalytic reaction is 4-48 h;

And/or when the enzyme A and the enzyme B are catalyzed in the form of crude enzyme liquid, crude enzyme liquid freeze-dried powder or pure enzyme, in the step (A) and the step (B), the concentration of the enzyme A and the concentration of the enzyme B in each reaction system are both 0.1 g/L-10 g/L; when the enzyme A and the enzyme B are catalyzed in the form of whole cells co-expressing the enzyme A and the enzyme B, the wet weight of the whole cells contained in each liter of the reaction system is 100 g;

and/or when the enzyme A and the enzyme B are catalyzed in the form of crude enzyme liquid, crude enzyme liquid freeze-dried powder or pure enzyme, the catalytic reaction is carried out in a buffer solution shown as the following (h1) in the step (A); in the step (B), the catalytic reaction is carried out in a buffer solution as shown in the following (h 2); when the enzyme A and the enzyme B are catalytically effected in the form of whole cells co-expressing the enzyme A and the enzyme B, the catalytic reactions of step (A) and step (B) are each carried out in a buffer as shown in (h1) below; (h1) a phosphate buffer solution having a concentration of 50 to 100mM and a pH of 6.5 to 8.0; (h2) a phosphate buffer solution having a concentration of 50 to 100mM and a pH of 7.5 to 8.5.

8. The method of any of claims 1 to 7, wherein: when the enzyme A and the enzyme B are subjected to catalytic action in the form of crude enzyme liquid, crude enzyme liquid freeze-dried powder or pure enzyme, in the step (A), a reaction system of the catalytic reaction contains acetone besides 1, 3-butanediol, the enzyme A and coenzyme thereof;

and/or when the enzyme A and the enzyme B are catalyzed in the form of crude enzyme liquid, crude enzyme liquid freeze-dried powder or pure enzyme, in the step (B), when the enzyme B is transaminase, the reaction system of the catalytic reaction contains isopropylamine or alanine besides 3-keto-1-butanol, the enzyme B and coenzyme thereof, namely pyridoxal phosphate;

And/or the chiral 3-amino-1-butanol is (R) -3-amino-1-butanol and/or (S) -3-amino-1-butanol.

9. An enzyme system or a related product thereof, characterized in that: the enzyme system comprising the enzyme A and the enzyme B of any one of claims 1 to 8; the related products are nucleic acid molecules capable of expressing each enzyme in the enzyme system, or expression cassettes, recombinant vectors, recombinant bacteria or transgenic cell lines containing the nucleic acid molecules.

10. use of the enzyme system of claim 9 or a related product thereof in the synthesis of chiral 3-amino-1-butanol.

Technical Field

the invention belongs to the technical field of biology, relates to a method for synthesizing chiral 3-amino-1-butanol, and particularly relates to a method for synthesizing chiral 3-amino-1-butanol by using catalysis of a biological enzyme.

background

Compounds containing chiral amino groups have very wide application in the pharmaceutical field, and can be used for synthesizing various compounds with biological activity, so that the development of a cheap and effective method for synthesizing chiral amino compounds has very wide demand in industrial production.

₄(R) -3-amino-1-butanol (structural formula is shown as A in figure 1) is an important chiral drug intermediate containing a chiral amino group, has very wide application in The fields of organic synthesis and pharmacy, (R) -3-amino-1-butanol can be used for synthesizing an antitumor drug 4-methyl cyclophosphamide and an anti-AIDS drug Dolutegravir (sold in The United states in 2013 under The trade name of Tivicay), can also be derived into beta-lactam for synthesizing penem antibiotics, at present, (R) -3-amino-1-butanol is mainly synthesized by a chemical method, The first method is a method which takes chiral (R) -alanine as a raw material, is changed into beta-amino acid ester by diazomethane growth after amino protection, is reduced to obtain a target product (Gertzmann et al, Tetrahedron, 1995, 51(33), 9031-90-44) after amino protection, has The defects of high chiral purity (R) -alanine, is obtained by using Gertzmann et al, is not enough for a diazo-amino acid ester, is not suitable for a large-ethyl ester production process, is obtained by a large-ethyl ester-chloride-ethyl ester-synthesizing an important synthetic method which is difficult to obtain a large-ethyl ester-synthesizing process, has a large-ethyl ester-synthesizing process because of a large-ethyl ester-synthesizing process, and a large-ethyl ester-amino-aldehyde-carboxylic acid ester-carboxylic acid ester-carboxylic acid.

the 1, 3-butanediol is an important chemical raw material and has the characteristics of easy acquisition, low price and the like.

Disclosure of Invention

The technical problem to be solved by the invention is to synthesize chiral 3-amino-1-butanol.

in order to solve the above technical problems, the present invention firstly provides a method for synthesizing chiral 3-amino-1-butanol by using biological enzyme catalysis, which comprises the following steps (the reaction principle is shown in fig. 2):

(A) using 1, 3-butanediol as a substrate, and generating 3-ketone-1-butanol through catalytic reaction of enzyme A and coenzyme thereof;

(B) Taking the 3-keto-1-butanol generated in the step (A) as a substrate, and generating chiral 3-amino-1-butanol through catalytic reaction of an enzyme B and a coenzyme thereof;

The enzyme A is selected from any one of the following: alcohol dehydrogenase, mutants of said alcohol dehydrogenase;

the enzyme B is selected from any one of the following: a transaminase, a mutant of said transaminase.

The method provided by the invention is realized by a method of multi-enzyme co-expression or cascade or step-by-step catalysis.

Further, the alcohol dehydrogenase may be derived from any of the following microorganisms: lactobacillus brevis, Thermoanaerobacter brevis, Lactobacillus kefir, Thermoanaerobacter virginica, enterococcus faecium, Enterobacter casseliflavus, Thermomyces carbonmonozae, Anemonobacterium alkylthermophilus, Mycobacteria, Methanosarcina methanolica, Clostridium beijerinckii, Clostridium unculturens, Clostridium sporogenes, Enterobacter devulcani, Clostridium dialosum, Clostridium spodum, Clostridium diumuliginosum, Clostridium bacteria, Candida parapsilosis;

further, the transaminase is derived from any one of the following microorganisms: bacillus megaterium, Pseudomonas aeruginosa, Violaceous bacillus, Aspergillus terreus, Fissistigma, Mycobacterium, Arthrobacter.

Further, the alcohol dehydrogenase may specifically be any one of the following (a1) - (a 20):

(a1) Alcohol dehydrogenase derived from Lactobacillus brevis (Lactobacillus brevis) and having an amino acid sequence of SEQ ID No. 2;

(a2) alcohol dehydrogenase derived from Thermoanaerobacter brockii (Thermoanaerobacter brockii), the amino acid sequence of which is SEQ ID No. 4;

(a3) an alcohol dehydrogenase derived from Lactobacillus kefirs (Lactobacillus kefiri DSM 20587) having an amino acid sequence of SEQ ID No. 6;

(a4) An alcohol dehydrogenase derived from Thermoanaerobacter virginiae Rwielii 8.B1, having an amino acid sequence of SEQ ID No. 8;

(a5) alcohol dehydrogenase derived from enterococcus alcoholierans (Oenococcus alcoholierans), and the amino acid sequence is SEQ ID No. 10;

(a6) alcohol dehydrogenase derived from Desulfotomanum nigricans (Desulfotomaculum nigricans), the amino acid sequence of which is SEQ ID No. 12;

(a7) An alcohol dehydrogenase derived from Thermoascus carbox carboydivorans (Thermosinus carboxydivorans) having an amino acid sequence of SEQ ID No. 14;

(a8) Alcohol dehydrogenase derived from Thermoanaerobacterium alkanothermophilum (Thermoanaerobacter mathranii), and having an amino acid sequence of SEQ ID No. 16;

(a9) an alcohol dehydrogenase derived from a bacterium of the phylum Firmicutes (Firmicutes bacterium CAG:137) having the amino acid sequence of SEQ ID No. 18;

(a10) an alcohol dehydrogenase derived from Methanosarcina thermophila, having an amino acid sequence of SEQ ID No. 20;

(a11) an alcohol dehydrogenase derived from Clostridium beijerinckii (Clostridium beijerinckii) having an amino acid sequence of SEQ ID No. 22;

(a12) an alcohol dehydrogenase derived from uncultured Clostridium sp, having an amino acid sequence of SEQ ID No. 24;

(a13) An alcohol dehydrogenase derived from Clostridium sporogenes (Clostridium taeniosporum) having an amino acid sequence of SEQ ID No. 26;

(a14) an alcohol dehydrogenase derived from Enterobacter desulfatous (Desrotococcus putei), the amino acid sequence of which is SEQ ID No. 28;

(a15) an alcohol dehydrogenase derived from Clostridium difficile (Clostridium diolis) having the amino acid sequence of SEQ ID No. 30;

(a16) An alcohol dehydrogenase derived from Clostridium clostridia (Clostridium cochleariae) having the amino acid sequence of SEQ ID No. 32;

(a17) An alcohol dehydrogenase derived from Clostridium vaccinium uliginosum (Clostridium uliginosum), having an amino acid sequence of SEQ ID No. 34;

(a18) an alcohol dehydrogenase derived from a Clostridium bacterium (Clostridium sp-Y3), having the amino acid sequence of SEQ ID No. 36;

(a19) An alcohol dehydrogenase derived from Candida parapsilosis (Candida parapsilosis) having an amino acid sequence of SEQ ID No. 38;

(a20) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in any one of (a1) to (a 19).

further, the transaminase may specifically be any one of the following (b1) - (b 8):

(b1) Transaminase derived from Bacillus megaterium (Bacillus megaterium) and having an amino acid sequence of SEQ ID No. 40;

(b2) a transaminase derived from pseudomonas aeruginosa (p. aeruginosa PAO2) having an amino acid sequence of SEQ ID No. 42;

(b3) A transaminase derived from Bacillus violaceum 2025, having the amino acid sequence of SEQ ID No. 44;

(b4) A transaminase derived from Aspergillus terreus (Aspergillus terreus) having the amino acid sequence of SEQ ID No. 46;

(b5) a transaminase derived from Fusarium fischer (Neosartorya fischeri) having an amino acid sequence of SEQ ID No. 48;

(b6) A transaminase derived from Mycobacterium (Mycobacterium vanbalenii) having an amino acid sequence of SEQ ID No. 50;

(b7) a transaminase derived from Arthrobacter sp.KNK168, having an amino acid sequence of SEQ ID No. 52;

(b8) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in any one of (b1) to (b 7).

further, the mutant of alcohol dehydrogenase may specifically be the following (c1) - (c 5):

(c1) Compared with the alcohol dehydrogenase derived from Lactobacillus brevis shown in SEQ ID No.2, at least one of the following mutations is present or present: I11V, G37D;

(c2) Compared with the alcohol dehydrogenase derived from Thermoanaerobacterium as shown in SEQ ID No.4, at least one of the following mutations exists or exists only: a85G, I86A, W110A, G198D;

further, the mutant of the alcohol dehydrogenase has the following mutations or only has the following mutations compared with the alcohol dehydrogenase derived from the high-temperature anaerobium as shown in SEQ ID No. 4: A85G/I86A or A85G/I86A/W110A or A85G/I86A/G198D;

(c3) compared with the alcohol dehydrogenase derived from Lactobacillus kefir as shown in SEQ ID No.6, the following mutations are present or present: G37D;

(c4) in comparison with the alcohol dehydrogenase from Thermoanaerobacter virginiae shown in SEQ ID No.8, the following mutations are present or present: G198D;

(c5) A fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in any one of (C1) to (C4).

in the present invention, for amino acid substitutions, the following nomenclature is used: original amino acid, position, substituted amino acid. For example, the substitution of valine (V) for isoleucine (I) originally present at position 11 of SEQ ID No.2 is designated as "I11V". Variants containing multiple changes are separated by a slash symbol ("/").

further, in the method, the enzyme A and the enzyme B can be catalyzed in the form of crude enzyme liquid, crude enzyme liquid freeze-dried powder, pure enzyme or whole cells.

Further, the crude enzyme solution freeze-dried powder and the pure enzyme are prepared according to the method comprising the following steps: expressing the enzyme A and/or the enzyme B in a host cell to obtain a recombinant cell; and cracking the recombinant cells to obtain the crude enzyme solution, the crude enzyme solution freeze-dried powder or the pure enzyme. The whole cell can be prepared according to a method comprising the following steps: expressing the enzyme A and/or the enzyme B in host cells, wherein the obtained recombinant cells are the whole cells of the enzyme A and/or the enzyme B;

still further, the recombinant cell can be specifically prepared according to a method comprising the following steps: introducing a nucleic acid molecule capable of expressing the enzyme A and/or the enzyme B into the host cell, and obtaining the recombinant cell expressing the enzyme A and/or the enzyme B after induction culture.

Further, said "nucleic acid molecule capable of expressing said enzyme A and/or said enzyme B" is introduced into said host cell in the form of a recombinant vector. Wherein, the recombinant vector can be bacterial plasmid (such as expression vector based on T7 promoter expressed in bacteria, such as pET-28a, etc.), bacteriophage, yeast plasmid (such as YEp series vector, etc.) or retrovirus packaging plasmid carrying the coding gene of the enzyme A and/or the enzyme B.

in one embodiment of the invention, the recombinant vector is specifically a recombinant plasmid obtained by replacing a small fragment between enzyme cutting sites Nde I and Xho I of pET-22B vector with the gene coding for the enzyme A or the enzyme B.

In another embodiment of the invention, the recombinant vector is a recombinant plasmid obtained by inserting the coding gene of the enzyme A into the restriction sites EcoRI and HindIII of the pETDuet-1 vector and inserting the coding gene of the enzyme B into the restriction sites Nde I and Xho I of the pETDuet-1 vector.

Further, the host cell may be a prokaryotic cell or a lower eukaryotic cell.

further, the prokaryotic cell may specifically be a bacterium. The lower eukaryotic cell may specifically be a yeast cell.

In one embodiment of the invention, the host cell is specifically e.coli, more specifically e.coli BL21(DE 3). Correspondingly, the induction culture is to add IPTG to the culture system to a final concentration of 0.1-0.5mM (specifically 0.1mM), and induce culture at 20-37 ℃ for 12-24h (specifically 16 h).

The sequence of the coding gene of the alcohol dehydrogenase from the lactobacillus brevis is SEQ ID No.1 or a fusion sequence obtained after connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene or a random or/and site-directed mutagenesis sequence which has the function and codes the same protein.

The sequence of the coding gene of the alcohol dehydrogenase from the high-temperature anaerobic bacillus is SEQ ID No.3 or a fusion sequence obtained after the 5 'end and/or the 3' end of the coding gene is connected with a tag coding sequence or a random or/and site-directed mutagenesis sequence which has the function and codes the same protein.

The sequence of the coding gene of the alcohol dehydrogenase from the lactobacillus gasseri is SEQ ID No.5 or a fusion sequence obtained after the 5 'end and/or the 3' end of the coding gene is connected with a tag coding sequence or a random or/and site-directed mutagenesis sequence which has the function and codes the same protein.

The sequence of the coding gene of the alcohol dehydrogenase derived from the thermoanaerobacterium virginiae is SEQ ID No.7 or a fusion sequence obtained by connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene or a random or/and site-directed mutagenesis sequence which has the functions and codes the same protein.

The sequence of the coding gene of the alcohol dehydrogenase from the wine coccus is SEQ ID No.9 or a fusion sequence obtained after connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene or a random or/and site-directed mutagenesis sequence which has the function and codes the same protein.

The sequence of the coding gene of the alcohol dehydrogenase from the xylaria nigricans is SEQ ID No.11 or a fusion sequence obtained after the 5 'end and/or the 3' end of the coding gene is connected with a tag coding sequence or a random or/and site-directed mutagenesis sequence which has the functions and codes the same protein.

The sequence of the coding gene of the alcohol dehydrogenase from the thermophilic antrodia carbonmonoxide is SEQ ID No.13 or a fusion sequence obtained after the 5 'end and/or the 3' end of the coding gene is connected with a label coding sequence or a random or/and site-directed mutagenesis sequence which has the function and codes the same protein.

The sequence of the coding gene of the alcohol dehydrogenase from the thermophilic anaerobacterium is SEQ ID No.15 or a fusion sequence obtained after the 5 'end and/or the 3' end of the coding gene are connected with a tag coding sequence or a random or/and site-directed mutagenesis sequence which has the functions and codes the same protein.

the sequence of the coding gene of the alcohol dehydrogenase from the firmicutes bacteria is SEQ ID No.17 or a fusion sequence obtained after connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene or a random or/and site-directed mutagenesis sequence which has the functions and codes the same protein.

The sequence of the coding gene of the alcohol dehydrogenase from the methanosarcina sarcina is SEQ ID No.19 or a fusion sequence obtained after connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene or a random or/and site-directed mutagenesis sequence which has the functions and codes the same protein.

The sequence of the coding gene of the alcohol dehydrogenase derived from clostridium beijerinckii is SEQ ID No.21 or a fusion sequence obtained by connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene or a random or/and site-directed mutagenesis sequence which has the functions and codes the same protein.

The sequence of the coding gene of the alcohol dehydrogenase from the uncultured clostridium is SEQ ID No.23 or a fusion sequence obtained after connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene or a random or/and site-directed mutagenesis sequence which has the functions and codes the same protein.

The sequence of the coding gene of the alcohol dehydrogenase derived from the clostridium filamentous spore is SEQ ID No.25 or a fusion sequence obtained after connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene or a random or/and site-directed mutagenesis sequence which has the functions and codes the same protein.

The sequence of the coding gene of the alcohol dehydrogenase from the desulfurization enterobacter is SEQ ID No.27 or a fusion sequence obtained after the 5 'end and/or the 3' end of the coding gene are connected with a tag coding sequence or a random or/and site-directed mutagenesis sequence which has the function and codes the same protein.

The sequence of the coding gene of the alcohol dehydrogenase from clostridium dialicum is SEQ ID No.29 or a fusion sequence obtained by connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene or a random or/and site-directed mutagenesis sequence which has the functions and codes the same protein.

The sequence of the coding gene of the alcohol dehydrogenase from the clostridium spongoides is SEQ ID No.31 or a fusion sequence obtained after connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene or a random or/and site-directed mutagenesis sequence which has the functions and codes the same protein.

the sequence of the coding gene of the alcohol dehydrogenase derived from Clostridium vaccinium is SEQ ID No.33 or a fusion sequence obtained by connecting a tag coding sequence to the 5 'end and/or the 3' end of the gene or a random or/and site-directed mutagenesis sequence which retains the function and codes the same protein.

the sequence of the coding gene of the alcohol dehydrogenase from clostridium bacteria is SEQ ID No.35 or a fusion sequence obtained after connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene or a random or/and site-directed mutagenesis sequence which has the functions and codes the same protein.

The sequence of the coding gene of the alcohol dehydrogenase derived from the candida parapsilosis is SEQ ID No.37 or a fusion sequence obtained after connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene or a random or/and site-directed mutagenesis sequence which has the function and codes the same protein.

the sequence of the coding gene of the transaminase derived from the bacillus megatherium is SEQ ID No.39 or a fusion sequence obtained by connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene or a random or/and site-directed mutagenesis sequence which has the functions and codes the same protein.

The sequence of the coding gene of the transaminase derived from the pseudomonas aeruginosa is SEQ ID No.41 or a fusion sequence obtained by connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene or a random or/and site-directed mutagenesis sequence which has the functions and codes the same protein.

The sequence of the coding gene of the transaminase derived from the violaceous bacillus is SEQ ID No.43 or a fusion sequence obtained by connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene or a random or/and site-directed mutagenesis sequence which has the functions and codes the same protein.

the sequence of the coding gene of the aminotransferase from the aspergillus terreus is SEQ ID No.45 or a fusion sequence obtained after connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene or a random or/and site-directed mutagenesis sequence which has the functions and codes the same protein.

The sequence of the coding gene of the transaminase derived from the Fischer-Tropsch bacterium is SEQ ID No.47 or a fusion sequence obtained by connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene or a random or/and site-directed mutagenesis sequence which has functions and codes the same protein.

the sequence of the coding gene of the transaminase derived from the mycobacteria is SEQ ID No.49 or a fusion sequence obtained by connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene or a random or/and site-directed mutagenesis sequence which has the functions and codes the same protein.

The sequence of the coding gene of the transaminase derived from the arthrobacter is SEQ ID No.51 or a fusion sequence obtained by connecting a tag coding sequence at the 5 'end and/or the 3' end of the coding gene or a random or/and site-directed mutagenesis sequence which has the functions and codes the same protein.

The sequence of the coding gene of the mutant of the alcohol dehydrogenase derived from the lactobacillus brevis is any one of the following (d1) to (d 3): (d1) compared to SEQ ID No.1, at least one of the following mutations is present or present only: A31G/T33G, G110A/C111T; (d2) a fusion sequence obtained by ligating a tag-encoding sequence to the 5 'end and/or 3' end of the sequence defined in (d 1); (d3) random or/and site-directed mutagenesis sequences which retain a function compared to the sequences defined in (d1) or (d2) and which encode the same protein.

the sequence of the encoding gene of the mutant of alcohol dehydrogenase derived from Thermoanaerobacterium is any one of the following (e1) to (e 3): (e1) compared to SEQ ID No.3, at least one of the following mutations is present or present only: C254G/T255C, A256G/T257C/T258G, T328G/G329C, G593A/C594T; (e2) a fusion sequence obtained by ligating a tag-encoding sequence to the 5 'end and/or 3' end of the sequence defined in (e 1); (e3) random or/and site-directed mutagenesis sequences which retain function and encode the same protein as compared to the sequences defined in (e1) or (e 2).

Further, the (e1) is: compared to SEQ ID No.3, any of the following mutations is present or only present: C254G/T255C/A256G/T257C/T258G, C254G/T255C/A256G/T257C/T258G/T328G/G329C, C254G/T255C/A256G/T257C/T258G/G593A/C594T.

the sequence of the coding gene of the mutant of the alcohol dehydrogenase derived from lactobacillus gasseri is any one of the following (f1) - (f 3): (f1) in comparison with SEQ ID No.5, the following mutations are present or only present: G110A/C111T; (f2) a fusion sequence obtained by ligating a tag-encoding sequence to the 5 'end and/or 3' end of the sequence defined in (f 1); (f3) (iii) a random or/and site-directed mutagenesis sequence which retains a function compared to the sequence defined in (f1) or (f2) and encodes the same protein.

the sequence of the coding gene of the mutant of the alcohol dehydrogenase derived from the thermoanaerobacterium virginiae is any one of the following (g1) to (g 3): (g1) compared to SEQ ID No.7, at least one of the following mutations is present or present only: G593A/C594T; (g2) a fusion sequence obtained by ligating a tag-encoding sequence to the 5 'end and/or 3' end of the sequence defined in (g 1); (g3) random or/and site-directed mutagenesis sequences which retain the function and encode the same protein as the sequences defined in (g1) or (g 2).

In the present invention, for the base substitution, the following nomenclature is used: the original base, position (i.e. position in the nucleotide sequence of W1 or W2 or W3 or W4), substituted base. Accordingly, substitution of the original G with A at position 31 of SEQ ID No.1 was designated "A31G". Variants containing multiple changes are separated by a slash symbol ("/").

In the step (A) and the step (B), the temperature of the catalytic reaction can be 25-37 ℃, such as 30-37 ℃, and specifically such as 30 ℃ or 37 ℃.

in the step (A) and the step (B), the time of the catalytic reaction can be 4-48 h, such as 24 h.

When the enzyme A and the enzyme B are catalyzed in the form of crude enzyme solution, crude enzyme solution freeze-dried powder or pure enzyme, the catalytic reaction in the step (A) can be carried out in a buffer solution as shown in the following (h 1); in the step (B), the catalytic reaction may be carried out in a buffer as shown below (h 2); when the enzyme A and the enzyme B are catalytically effected in the form of whole cells co-expressing the enzyme A and the enzyme B, the catalytic reactions of both the step (A) and the step (B) can be carried out in a buffer as shown below (h 1). (h1) A phosphate buffer solution having a concentration of 50 to 100mM and a pH of 6.5 to 8.0; the method specifically comprises the following steps: phosphate buffer at a concentration of 100mM, pH 8.0. (h2) A phosphate buffer solution having a concentration of 50 to 100mM and a pH of 7.5 to 8.5; the method specifically comprises the following steps: phosphate buffer solution with the concentration of 100mM and the pH value of 7.5-8.5.

when the enzyme A and the enzyme B are catalyzed by crude enzyme liquid, crude enzyme liquid freeze-dried powder or pure enzyme, in the step (A) and the step (B), the concentration of the enzyme A and the concentration of the enzyme B in respective reaction systems can be 0.1 g/L-10 g/L, such as 10 g/L. When the enzyme A and the enzyme B are catalyzed in the form of whole cells co-expressing the enzyme A and the enzyme B, the step (A) and the step (B) are completed in one reaction system in which the concentration of the whole cells (co-expressing the enzyme A and the enzyme B) is 100g/L (100 g of wet weight of the whole cells is contained per liter of the reaction system).

in the present invention, the coenzyme of the enzyme a may be specifically oxidized coenzyme I (i.e., NAD ⁺) or oxidized coenzyme II (i.e., NADP ⁺) — when the enzyme B is a transaminase or a mutant of the transaminase, the coenzyme of the enzyme B is specifically pyridoxal phosphate (PLP).

In the invention, when the enzyme A and the enzyme B are catalyzed by crude enzyme liquid, crude enzyme liquid freeze-dried powder or pure enzyme, the concentrations of the coenzymes of the enzyme A and the enzyme B in respective reaction systems can be 0.1-3 mM (specifically 1 mM).

When the enzyme A and the enzyme B are catalyzed by crude enzyme liquid, crude enzyme liquid freeze-dried powder or pure enzyme, in the step (A), a reaction system of the catalytic reaction contains acetone besides 1, 3-butanediol, the enzyme A and coenzyme thereof.

Specifically, in the step (A), the reaction system of the catalytic reaction is composed of phosphate buffer with a concentration of 100mM and a pH value of 8.0, 1, 3-butanediol with a final concentration of 20mM, oxidized coenzyme I (namely NAD ⁺) or oxidized coenzyme II (namely NADP ⁺) with a final concentration of 1mM, acetone (v/v) with a final concentration of 5%, crude enzyme solution of the enzyme A with a final concentration of 10g/L, crude enzyme solution freeze-dried powder or pure enzyme.

When the enzyme A and the enzyme B are catalyzed in the form of crude enzyme liquid, crude enzyme liquid freeze-dried powder or pure enzyme, in the step (B), when the enzyme B is transaminase, the reaction system of the catalytic reaction contains isopropylamine or alanine besides 3-keto-1-butanol, the enzyme B and coenzyme thereof, namely pyridoxal phosphate;

Specifically, in step (B), when the enzyme B is a transaminase or a mutant of the transaminase, the composition of the reaction system for catalyzing the reaction may be as follows: 50-100 mM of phosphate buffer solution with the pH value of 8.0, 500mM of isopropylamine (or alanine) with the final concentration, 1mM of pyridoxal phosphate (PLP), 10g/L of crude enzyme solution, freeze-dried powder of the crude enzyme solution or pure enzyme of the enzyme B with the final concentration.

When the enzyme a and the enzyme B are catalyzed in the form of whole cells co-expressing the enzyme a and the enzyme B, no coenzyme may be added to the reaction system, and further, the reaction system may further contain glucose.

Specifically, the composition of the reaction system may be as follows: phosphate buffer at a concentration of 100mM and pH 8.0; 1, 3-butanediol at a final concentration of 50 mM; 100mM glucose; the whole cells were contained at a final concentration of 100g/L (i.e., 100g of wet weight per liter of the reaction system).

In the method, the chiral 3-amino-1-butanol is (R) -3-amino-1-butanol and/or (S) -3-amino-1-butanol.

hereinbefore, transaminase derived from Fischer-Tropsch bacteria, transaminase derived from mycobacteria, transaminase derived from Aspergillus terreus or transaminase derived from Arthrobacter can be used for the synthesis of (R) -3-amino-1-butanol. Transaminase derived from Bacillus megaterium, transaminase derived from Pseudomonas aeruginosa, and transaminase derived from Bacillus violaceus can be used for synthesizing (S) -3-amino-1-butanol.

the invention also provides an enzyme system and a related product thereof.

The enzyme system provided by the invention comprises the enzyme A and the enzyme B. Of course, the respective coenzymes of the enzyme A and the enzyme B may be included.

the related product can be a nucleic acid molecule capable of expressing each enzyme in the enzyme system, or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule.

The use of the enzyme system or the related product in the synthesis of chiral 3-amino-1-butanol also falls within the scope of the present invention.

in the method for synthesizing chiral 3-amino-1-butanol, alcohol dehydrogenase catalyzes the oxidation of 1, 3-butanediol into 3-keto-1-butanol, NAD (P) ⁺ is reduced into NAD (P) H, meanwhile, the alcohol dehydrogenase catalyzes the reduction of acetone into isopropanol, NAD (P) H is re-oxidized into NAD (P) ⁺, and the generated NAD (P) ⁺ participates in the oxidation of 1, 3-butanediol into 3-keto-1-butanol again.

The invention provides a brand-new green biosynthesis route, which takes cheap 1, 3-butanediol as a raw material to synthesize chiral 3-amino-1-butanol, namely (R) -3-amino-1-butanol and/or (S) -3-amino-1-butanol, through multi-enzyme co-expression or cascade or step-by-step catalysis.

drawings

FIG. 1 shows the structural formulae of (R) -3-amino-1-butanol and (S) -3-amino-1-butanol.

FIG. 2 is a reaction scheme for preparing (R) -3-amino-1-butanol or (S) -3-amino-1-butanol by coupling alcohol dehydrogenase with transaminase.

FIG. 3 shows the results of Gas Chromatography (GC) identification of 3-keto-1-butanol.

FIG. 4 shows the results of liquid chromatography on 3-amino-1-butanol standard. A is a mixed spinning type 3-amino-1-butanol standard product; b is (R) -3-amino-1-butanol standard substance.

FIG. 5 is a liquid chromatogram of the reaction solution.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

materials, reagents and the like used in the following examples are commercially available unless otherwise specified.