CN109825538B - A kind of synthetic method of chiral 2-amino-1-butanol - Google Patents

Abstract

The invention discloses a method for synthesizing chiral 2-amino-1-butanol. The method comprises the following steps: using 1, 2-butanediol as a substrate, and generating 2-ketone-1-butanol through catalytic reaction of enzyme A and coenzyme thereof; 2-keto-1-butanol is used as a substrate, and chiral 2-amino-1-butanol is generated through catalytic reaction of enzyme B and coenzyme thereof; the enzyme A is selected from alcohol dehydrogenase, carbonyl reductase or a mutant of the two enzymes; the enzyme B is selected from amino acid dehydrogenase, transaminase or mutants of both enzymes. The invention provides a brand-new green biosynthesis route, which takes cheap 1, 2-butanediol as a raw material to synthesize chiral 2-amino-1-butanol, namely (S) -2-amino-1-butanol and (R) -2-amino-1-butanol, through multi-enzyme co-expression or cascade or step-by-step catalysis.

Description

A kind of synthetic method of chiral 2-amino-1-butanol

technical field

The invention belongs to the field of biotechnology, and relates to a method for synthesizing chiral 2-amino-1-butanol, in particular to a method for catalyzing and synthesizing chiral 2-amino-1-butanol by utilizing biological enzymes.

Background technique

In recent years, green chemical processes centered on the application of biocatalysts have received more and more attention, especially the cascade catalytic reactions that combine natural enzymes or new modified enzymes as multi-enzyme molecular machines are favored by researchers and industries (Fischereder et al. et al., ACS Catal., 2016, 6(1):23-30; France et al., ACS Catal., 2016, 6(6):3753-3759; Li et al., 2016, J Agr Food Chem ., 64(46):8927-8934.). The new pathway of cascade reaction constructed by multi-enzyme molecular machine can not only reduce the loss of intermediates, but also improve the conversion efficiency and greatly reduce the cost of process production (Schrittwieser et al., Curr Opin Chem Biol., 2011, 15(2) :249-256; Wheeldon et al., Nat Chem., 2016, 8(4):299-309.).

Chiral 2-amino-1-butanol, especially (S)-2-amino-1-butanol, has a wide range of uses in organic synthesis and pharmaceutical production, and can be used as an important intermediate to prepare optically active compounds . The structure is shown in Figure 1. (S)-2-Amino-1-butanol ((S)-2-Amino-1-butanol) is an important pharmaceutical intermediate. The current production methods include chemical methods, such as: using butyraldehyde, azodicarboxylate Benzyl ester and D-proline were used as raw materials to synthesize (S)-2-aminobutanol under the catalysis of NaBH ₄ , H ₂ and nickel (Kotkar & Sudalai, Tetrahedron: Asymmetry, 2006, 17(11): 1738-1742 ); also reported to use lithium aluminum hydride to reduce 2-aminobutyric acid to obtain the corresponding chiral amino alcohol product (Doherty & Shapira, J Org Chem, 1963, 28 (5): _1339-1342 ); using H and nickel under high pressure conditions Reduce L-2-aminobutyric acid to obtain (S)-2-aminobutanol (CN105481703A); or use NaHB ₄ to reduce the corresponding α-amino acid under H ₂ SO ₄ /THF conditions (Li et al., J Agr Food Chem, 2016, 64(46):8927-8934). Aminobutanol chemical resolution method, such as: bibenzoyl tartaric acid resolution method (Periasamy et al., Synthesis-Stuttgart, 2003 (13): 1965-1967); L-tartaric acid resolution method (Zhao et al., J Heterocyclic Chem, 2012, 49(4):943-946). There are also reports of enzymatic resolution, such as: in the case of amino group protection, the ester bond formed by the selective hydrolysis of hydroxyl groups by lipase obtains chiral monomers (Francalanci et al., J Org Chem, 1987, 52 (Francalanci et al., J Org Chem, 1987, 52( 23): 5079-5082); immobilized penicillin G acylase selectively hydrolyzes (S)-configuration in the racemic mixture of N-phenylacetyl-derived 2-aminobutanol to obtain (S)-2-aminobutanol , the ee value can reach 99% (Fadnavis et al., Tetrahedron: Asymmetry, 1999, 10(23): 4495-4500); the latest report achieves continuous splitting by acylating immobilized penicillin G on a fixed bed, and the conversion rate It reached 39.3% and the ee value was 98.2%.

The above-mentioned synthetic method of (S)-2-amino-1-butanol, the chemical method requires high temperature, high pressure and metal catalyst to obtain by hydrogenation reduction, the pollution is large, and the safety factor is low. The chemical resolution method also needs to use a large amount of acid, alkali and other chemical reagents. Although the enzymatic resolution method has mild reaction conditions and good stereoselectivity, the conversion rate of the resolution method is only 50%, and the yield is low.

SUMMARY OF THE INVENTION

In order to solve the above problems, the present invention provides a method for catalyzing and synthesizing chiral 2-amino-1-butanol by biological enzymes, which may include the following steps (Fig. 2):

(A) Using 1,2-butanediol as a substrate, 2-keto-1-butanol is generated by the catalytic reaction of enzyme A and its coenzyme;

(B) using the 2-keto-1-butanol generated in step (A) as a substrate, and generating chiral 2-amino-1-butanol through the catalytic reaction of enzyme B and its coenzyme;

The enzyme A may be selected from any of the following: alcohol dehydrogenase, carbonyl reductase, mutants of the alcohol dehydrogenase, mutants of the carbonyl reductase;

The enzyme B may be selected from any of the following: amino acid dehydrogenases, transaminases, mutants of said amino acid dehydrogenases, mutants of said transaminases.

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

Further, the alcohol dehydrogenase and the carbonyl reductase can be derived from any of the following microorganisms: Bacillus stearothermophilus (Geobacillus stearothermophilus), Lactobacillus kefiri (Lactobacillus kefiri), Lactobacillus brevis (Lactobacillus brevis), Bacillaceae, Thermoanaerobacter brockii, Leifsonia sp., Thermoanaerobacter wiegelii, Sporobolomyces salmonicolor.

Preferred are alcohol dehydrogenase LBADH derived from Lactobacillus brevis, alcohol dehydrogenase TbSADH derived from Thermoanaerobacter brockii, and Bacillaceae, Geobacillus stearothermophilus, Alcohol dehydrogenase derived from Lactobacillus kefiri, Leifsonia sp., Thermoanaerobacter wiegelii and carbonyl reductase derived from Sporobolomyces salmonicolor .

Further, the amino acid dehydrogenase can be derived from: Geobacillus stearothermophilus.

Further, the transaminase can be derived from any of the following microorganisms: Bacillus megaterium (Bacillus megaterium), Pseudomonas aeruginosa (P. aeruginosa), Aspergillus terreus (Aspergillus terreus), Aspergillus fumigatus (Aspergillus fumigatus), Fisher Neosartorya fischeri, Gibberella zeae, Mycobacterium vanbaalenii, or ATA-117 transaminase from Codexis.

Further, the alcohol dehydrogenase may specifically be any of the following (a1)-(a8):

(a1) alcohol dehydrogenase derived from Lactobacillus brevis, the amino acid sequence is SEQID No.2;

(a2) alcohol dehydrogenase derived from Bacillus stearothermophilus (Geobacillus stearothermophilus), the amino acid sequence is SEQ ID No.4;

(a3) alcohol dehydrogenase derived from thermoanaerobic bacteria (Thermoanaerobacter brockii), the amino acid sequence is SEQ ID No.6;

(a4) an alcohol dehydrogenase derived from Lactobacillus kefir, the amino acid sequence of which is SEQ ID No. 8;

(a5) alcohol dehydrogenase derived from Bacillaceae (Bacillaceae), the amino acid sequence is SEQ ID No.10;

(a6) alcohol dehydrogenase derived from Leifsonia sp., the amino acid sequence is SEQ ID No.12;

(a7) alcohol dehydrogenase derived from Thermoanaerobacter wiegelii, the amino acid sequence is SEQ ID No.14;

(a8) A fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the proteins defined in any of (a1)-(a7).

Further, the carbonyl reductase can specifically be the following (b1) or (b2):

(b1) carbonyl reductase derived from Sporobolomyces salmonicolor, the amino acid sequence is SEQ ID No. 16;

(b2) A fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in (b1).

Further, the amino acid dehydrogenase may specifically be any of the following (c1)-(c2):

(c1) Leucine dehydrogenase derived from Geobacillus stearothermophilus, SEQ ID No. 18;

(c2) A fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in (c1).

Further, the transaminase may specifically be any of the following (d1)-(d9):

(d1) ATA-117 transaminase of Codexis company, the amino acid sequence is SEQ ID No.20;

(d2) a transaminase derived from Aspergillus terreus, the amino acid sequence is SEQ ID No.22;

(d3) a transaminase derived from Aspergillus fumigatus, the amino acid sequence is SEQ ID No.24;

(d3) a transaminase derived from Neosartorya fischeri, the amino acid sequence is SEQID No.26;

(d5) a transaminase derived from Gibberella zeae, the amino acid sequence is SEQ ID No.28;

(d6) a transaminase derived from Mycobacterium vanbaalenii, the amino acid sequence is SEQID No.30;

(d7) a transaminase derived from Bacillus megaterium, the amino acid sequence is SEQID No.32;

(d8) a transaminase derived from Pseudomonas aeruginosa (P. aeruginosa), the amino acid sequence is SEQ ID No. 34;

(d9) A fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the proteins defined in any of (d1)-(d8).

Further, the mutant of the alcohol dehydrogenase may specifically be as follows (e1) or (e2):

(e1) compared with the alcohol dehydrogenase derived from Lactobacillus brevis shown in SEQ ID No.2, there is or only has at least one of the following mutations: I11V, G37D;

(e2) A fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in (e1).

Further, the mutant of the amino acid dehydrogenase may specifically be any of the following (f1)-(f2):

(f1) compared with the leucine dehydrogenase derived from Bacillus stearothermophilus (Geobacillus stearothermophilus) shown in SEQ ID No. 18, at least one of the following mutations is present or only present: K68X, N261X, wherein X represents 19 other amino acids (presented by recognized one-letter abbreviations including F, L, I, V, S, P, T, A, Y, H, Q, N, D, E, C, R, G, M, W). Preferably, the X is a polar amino acid (eg, S, T, Y, H, Q, N, D, E, C, R, etc.). Further preferably, the X is S, Y, L or C. Still further preferably, the mutant of the amino acid dehydrogenase is compared with the leucine dehydrogenase derived from Bacillus stearothermophilus (Geobacillus stearothermophilus) shown in SEQ ID No. 18, which has or only has the following mutations: K68S/N261L

Or K68Y/N261C.

(f2) A fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in (f1).

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

Further, in the method, both the enzyme A and the enzyme B can catalyze in the form of crude enzyme solution, lyophilized powder of crude enzyme solution, pure enzyme or whole cells.

Further, the crude enzyme liquid, the lyophilized powder of the crude enzyme liquid and the pure enzyme can be 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; The recombinant cell obtains the crude enzyme solution of the enzyme A and/or the enzyme B, the lyophilized powder of the crude enzyme solution 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 a host cell, and the obtained recombinant cell is the whole of the enzyme A and/or the enzyme B. cell.

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 expression of the enzyme after inducing and culturing. said recombinant cells of enzyme A and/or said enzyme B.

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

In one embodiment of the present invention, the recombinant vector is specifically a recombinant plasmid obtained by replacing the encoding gene of the enzyme A or the enzyme B with a small fragment between the restriction sites NdeI and XhoI of the pET22b vector.

In another embodiment of the present invention, the recombinant vector is that the gene encoding the enzyme A is inserted into the restriction enzyme restriction site EcoRI and Hind III of the pETDuet-1 vector, and the enzyme is inserted into the pETDuet-1 vector. The recombinant plasmid obtained by inserting the coding gene of B between the restriction sites NdeI and XhoI of the pETDuet-1 vector.

Further, the host cells can be prokaryotic cells or lower eukaryotic cells.

Further, the prokaryotic cells can be bacteria; the lower eukaryotic cells can be yeast cells.

In one embodiment of the present invention, the host cell is specifically Escherichia coli, more specifically E. coliBL21(DE3). Correspondingly, the induction culture is to add IPTG to the culture system to a final concentration of 0.1-0.5 mM (specifically, 0.1 mM), and induce and culture at 20-37° C. for 12-24 h (specifically, 16 h).

The sequence of the coding gene of the alcohol dehydrogenase derived from Lactobacillus brevis is SEQ ID No.1 or the fusion sequence obtained after connecting the tag coding sequence at its 5' end and/or 3' end or keep it Random or/and site-directed mutagenesis sequences that are functional and encode the same protein.

The sequence of the gene encoding alcohol dehydrogenase derived from Bacillus stearothermophilus (Geobacillus stearothermophilus) is SEQ ID No. 3 or a fusion sequence obtained by connecting a tag encoding sequence at its 5' end and/or 3' end Or random or/and site-directed mutagenesis sequences that retain function and encode the same protein.

The sequence of the encoding gene of the alcohol dehydrogenase derived from thermoanaerobic bacillus (Thermoanaerobacter brockii) is SEQ ID No.5 or the fusion sequence obtained after connecting the tag encoding sequence at its 5' end and/or 3' end or Random or/and site-directed mutagenesis sequences that retain function and encode the same protein.

The sequence of the coding gene of the alcohol dehydrogenase derived from Lactobacillus kefiri is SEQ ID No. 7 or the fusion sequence obtained after connecting the tag coding sequence at its 5' end and/or 3' end or Random or/and site-directed mutagenesis sequences that retain function and encode the same protein.

The sequence of the gene encoding alcohol dehydrogenase derived from Bacillaceae (Bacillaceae) is SEQ ID No. 9 or a fusion sequence obtained after connecting a tag encoding sequence at its 5' end and/or 3' end or retains function and Random or/and site-directed mutagenesis sequences encoding the same protein.

The sequence of the coding gene of the alcohol dehydrogenase derived from the genus Leifsonia sp. is SEQ ID No. 11 or the fusion sequence obtained after connecting the tag coding sequence at its 5' end and/or 3' end or Random or/and site-directed mutagenesis sequences that retain function and encode the same protein.

The sequence of the coding gene of the alcohol dehydrogenase derived from Thermoanaerobacter wiegelii is SEQ ID No. 13 or the fusion obtained after connecting the tag coding sequence at its 5' end and/or 3' end Sequences or random or/and site-directed mutagenesis sequences that retain function and encode the same protein.

The sequence of the gene encoding carbonyl reductase derived from Sporobolomyces salmonicolor is SEQ ID No. 15 or a fusion sequence obtained by connecting a tag encoding sequence at its 5' end and/or 3' end or Random or/and site-directed mutagenesis sequences that retain function and encode the same protein.

The sequence of the encoding gene of the leucine dehydrogenase derived from Bacillus stearothermophilus (Geobacillus stearothermophilus) is SEQ ID No. 17 or obtained after connecting a tag encoding sequence at its 5' end and/or 3' end Fusion sequences or random or/and site-directed mutagenesis sequences that retain function and encode the same protein.

The sequence of the coding gene of the ATA-117 transaminase of the Codexis company is SEQ ID No. 19 or the fusion sequence obtained after connecting the tag coding sequence at its 5' end and/or 3' end, or a random sequence that retains the function and encodes the same protein. or/and site-directed mutagenesis sequences.

The sequence of the encoding gene of the transaminase derived from Aspergillus terreus is SEQ ID No. 21 or a fusion sequence obtained after connecting a tag encoding sequence at its 5' end and/or 3' end or retains function and encodes the same protein random or/and site-directed mutagenesis sequences.

The sequence of the encoding gene of the transaminase derived from Aspergillus fumigatus is SEQ ID No. 23 or a fusion sequence obtained after connecting a tag encoding sequence at its 5' end and/or 3' end or retains function and encodes Random or/and site-directed mutagenesis sequences of the same protein.

The sequence of the encoding gene of the transaminase derived from Neosartorya fischeri is SEQ ID No. 25 or the fusion sequence obtained after connecting the tag encoding sequence at its 5' end and/or 3' end or retains the Random or/and site-directed mutagenesis sequences that are functional and encode the same protein.

The sequence of the encoding gene of the transaminase derived from Gibberella zeae is SEQ ID No. 27 or the fusion sequence obtained after connecting the tag encoding sequence at its 5' end and/or 3' end or retains the function and encodes the same Random or/and site-directed mutagenesis sequences of proteins.

The sequence of the encoding gene of the transaminase derived from Mycobacterium is SEQ ID No. 29 or a fusion sequence obtained by connecting a tag encoding sequence at its 5' end and/or 3' end, or a random or random sequence that retains the function and encodes the same protein. / and site-directed mutagenesis sequences.

The sequence of the encoding gene of the transaminase derived from Bacillus megaterium is SEQ ID No. 31 or a fusion sequence obtained after connecting a tag encoding sequence at its 5' end and/or 3' end, or retains function and encodes Random or/and site-directed mutagenesis sequences of the same protein.

The sequence of the encoding gene of the transaminase derived from Pseudomonas aeruginosa (P. aeruginosa) is SEQ ID No. 33 or the fusion sequence obtained after connecting the tag encoding sequence at its 5' end and/or 3' end or retains Random or/and site-directed mutagenesis sequences that are functional and encode the same protein.

The sequence of the coding gene of the mutant alcohol dehydrogenase derived from Lactobacillus brevis is any one of the following (g1)-(g3): (g1) compared with SEQ ID No.1, there is Or only have at least one of the following mutations: A31G/T33G, G110A/C111T; (g2) a fusion sequence obtained by connecting a tag coding sequence at the 5' end and/or 3' end of the sequence defined in (g1); ( g3) Random or/and site-directed mutagenesis sequences that retain function compared to the sequences defined in (g1) or (g2) and encode the same protein.

The sequence of the coding gene of the leucine dehydrogenase mutant derived from Bacillus stearothermophilus (Geobacillus stearothermophilus) is any one of the following (h1)-(h3): (h1) and SEQ ID No. 17 In contrast, the presence or presence of at least one of the following mutations: A203G/A204C, A202T/A204T, A781C/A782T/C783G, A781T/A782G; (h2) at the 5' end of the sequence defined by (h1) and/or The fusion sequence obtained by linking the tag coding sequence at the 3' end; (h3) a random or/and site-directed mutagenesis sequence that retains function compared to the sequence defined by (h1) or (h2) and encodes the same protein.

Further, the (h1) is: compared with SEQ ID No. 17, there is or only any one of the following mutations: A203G/A204C/A781C/A782T/C783G, A202T/A204T/A781T/A782G.

In the present invention, for base substitutions, the following nomenclature is used: original base, position (ie, position in the W1 or W2 or W3 nucleotide sequence), substituted base. Correspondingly, the original G was replaced by A at the 31st position of SEQ ID No. 1, named "A31G". Variants containing multiple changes are separated by a slash character ("/").

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

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

When the enzyme A and the enzyme B are in the form of crude enzyme solution, lyophilized powder of crude enzyme solution or pure enzyme, catalysis occurs, in step (A), the catalytic reaction is buffered as shown in the following (k1) In step (B), the catalytic reaction is carried out in the buffer shown in any of the following (k2)-(k3); when the enzyme A and the enzyme B co-express the enzyme A When catalyzing with the whole cell form of the enzyme B, the catalytic reactions of step (A) and step (B) are both carried out in the buffer shown in the following (k1);

(k1) Phosphate buffer with a concentration of 50 to 100 mM and a pH of 6.5 to 8.0;

For example: phosphate buffer with a concentration of 100 mM and a pH of 8.0.

(k2) NH ₃ ·H ₂ O or NH ₄ Cl buffer with a concentration of 100mM to 2M and a pH of 8.0 to 9.6;

For example: NH ₃ ·H ₂ O or NH ₄ Cl buffer with a concentration of 1M and a pH of 8.7.

(k3) Phosphate buffer with a concentration of 50-100 mM, a concentration of isopropylamine of 250 mM-1 M, and a pH of 7.5-8.5;

Specifically, a phosphate buffer with a concentration of 100 mM, a concentration of isopropylamine of 500 mM, and a pH of 8.0.

When the enzyme A and the enzyme B are in the form of crude enzyme solution, lyophilized powder of the crude enzyme solution or pure enzyme, catalysis, in step (A) and step (B), the enzyme A and the The concentration of enzyme B in each reaction system can be 0.1-10 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, and the said The concentration of the whole cells (co-expressing the enzyme A and the enzyme B) in the reaction system was 100 g/L (the wet weight of the whole cells contained in the reaction system per liter was 100 g).

In the present invention, the coenzyme of the enzyme A is specifically oxidized cofactor II, namely NADP ⁺ , or oxidized cofactor I, namely NAD ⁺ . When the enzyme B is an amino acid dehydrogenase or a mutant of the amino acid dehydrogenase, the coenzyme of the enzyme B is specifically oxidized cofactor I, namely NAD ⁺ ; when the enzyme B is a transaminase or the In the case of a mutant of transaminase, the coenzyme of the enzyme B is specifically pyridoxal phosphate (PLP).

In the present invention, when the enzyme A and the enzyme B are in the form of crude enzyme solution, lyophilized powder of the crude enzyme solution or pure enzyme, the coenzymes of the enzyme A and the enzyme B are in their The concentration in each reaction system can be 0.5-3 mM (specifically, 1 mM).

When the enzyme A and the enzyme B are in the form of crude enzyme solution, lyophilized powder of crude enzyme solution or pure enzyme, catalyzing action occurs, in step (A), the reaction system of the catalyzed reaction contains 1 , 2-butanediol and the enzyme A and its coenzyme, it also contains acetone.

Specifically, in step (A), the reaction system of the catalytic reaction is composed of the following: a phosphate buffer with a concentration of 100 mM, a pH value of 8.0, 1,2-butanediol with a final concentration of 20 mM, and a final concentration of 1 mM Oxidized cofactor II, namely NADP ⁺ , or oxidized cofactor I, namely NAD ⁺ , acetone (v/v) with a final concentration of 5%, and a crude enzyme solution of the enzyme A with a final concentration of 10 g/L , lyophilized powder of crude enzyme liquid or pure enzyme.

When the enzyme A and the enzyme B are in the form of crude enzyme solution, lyophilized powder of the crude enzyme solution or pure enzyme, catalysis occurs, in step (B), when the enzyme B is an amino acid dehydrogenase or In the case of the amino acid dehydrogenase mutant, the reaction system of the catalytic reaction contains glucose dehydrogenase and glucose in addition to 2-keto-1-butanol and the enzyme B and its coenzyme. Further, the reaction system also contains calcium carbonate.

Specifically, in step (B), when the enzyme B is an amino acid dehydrogenase or a mutant of the amino acid dehydrogenase, the composition of the reaction system for the catalytic reaction may be as follows: the concentration is 1M, and the pH is 8.7 NH ₃ ·H ₂ O or NH ₄ Cl buffer; final concentration of 1 mM oxidized cofactor I, namely NAD ⁺ ; final concentration of 1 g/L glucose dehydrogenase; final concentration of 100 mM glucose; final concentration It is 10g/L of the crude enzyme solution of the enzyme B, the lyophilized powder of the crude enzyme solution or the pure enzyme; the final concentration is 5g/L of calcium carbonate. When the enzyme B is a transaminase or a mutant of the transaminase, the composition of the reaction system for the catalytic reaction may be as follows: the concentration of the isopropylamine is 50-100 mM, the concentration of isopropylamine is 500 mM, and the pH is 8.0 phosphate buffer solution ; Pyridoxal Phosphate (PLP) with a final concentration of 1 mM; Crude enzyme liquid, lyophilized powder of crude enzyme liquid or pure enzyme of the enzyme B with a final concentration of 10 g/L.

When the enzyme A and the enzyme B are catalyzed in the form of whole cells that co-express the enzyme A and the enzyme B, no coenzyme may be added to the reaction system. Also contains glucose.

Specifically, the composition of the reaction system can be as follows: phosphate buffer with a concentration of 100 mM and a pH value of 8.0; 1,2-butanediol with a final concentration of 50 mM; 100 mM glucose; The whole cells (that is, the wet weight of the whole cells contained in each liter of reaction system is 100 g).

In the method, the chiral 2-amino-1-butanol is (S)-2-amino-1-butanol and/or (R)-2-amino-1-butanol.

The aforementioned amino acid dehydrogenase and its mutants, as well as the ATA-117 transaminase derived from Codexis, the transaminase derived from Aspergillus terreus, the transaminase derived from Aspergillus fumigatus, and the transaminase derived from Feich Transaminase from Neosartorya fischeri, from Gibberella zeae, from Mycobacterium vanbaalenii for the synthesis of (S)-2-amino-1-butanol The aforementioned transaminase derived from Bacillus megaterium and transaminase derived from P. aeruginosa were used for the synthesis of (R)-2-amino-1-butanol.

The invention also provides an enzyme system and related products.

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

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

The application of the enzyme system or the related products in the synthesis of chiral 2-amino-1-butanol also belongs to the protection scope of the present invention.

In the method for synthesizing chiral 2-amino-1-butanol provided by the present invention, there is a cofactor regeneration system. The cofactor regeneration system is that glucose dehydrogenase catalyzes glucose oxidation, or alcohol dehydrogenase catalyzes acetone reduction or isopropanol oxidation to promote cofactor regeneration. In the method for synthesizing chiral 2-amino-1-butanol of the present invention, alcohol dehydrogenase or carbonyl reductase catalyzes the oxidation of 1,2-butanediol to 2-keto-1-butanol, NAD(P) ⁺ is reduced to NAD(P)H, at the same time, alcohol dehydrogenase catalyzes the reduction of acetone to isopropanol, NAD(P)H is re-oxidized to NAD(P) ⁺ , and the generated NAD(P) ⁺ re-engages in 1, Oxidation of 2-butanediol to 2-keto-1-butanol. Amino acid dehydrogenase and its mutants catalyze the reduction of 2-keto-1-butanol to 1,2-butanediol, NADH is oxidized to NAD ⁺ , and glucose dehydrogenase (GDH) catalyzes the oxidation of glucose to gluconic acid, NAD ⁺ is re-reduced to NADH, and the resulting NADH is re-involved in the oxidation of 2-keto-1-butanol to (S)-2-amino-1-butanol.

The present invention provides a brand-new green biosynthesis route, which uses cheap 1,2-butanediol as raw material to synthesize chiral 2-amino-1-butanol through multi-enzyme co-expression or cascade or step-by-step catalysis, namely (S)-2-amino-1-butanol and/or (R)-2-amino-1-butanol.

Description of drawings

Figure 1 shows the structural formulas of (S)-2-amino-1-butanol and (R)-2-amino-1-butanol.

Fig. 2 is the reaction principle diagram of the biological preparation method of chiral 2-amino-1-butanol of the present invention. A: Alcohol dehydrogenase or carbonyl reductase coupled with amino acid dehydrogenase to prepare (S)-2-amino-1-butanol. B: Alcohol dehydrogenase or carbonyl reductase coupled transaminase to prepare (S)-2-amino-1-butanol or (R)-2-amino-1-butanol.

Figure 3 shows the identification results of 2-keto-1-butanol by gas chromatography (GC).

Figure 4 is a diagram showing the separation effect of 2-amino-1-butanol standard product after derivatization with o-phthalaldehyde. A: Mixed 2-amino-1-butanol; B: (S)-2-amino-1-butanol standard; C: (R)-2-amino-1-butanol standard.

Figure 5 is a liquid chromatogram of the reaction solution. A: The liquid chromatography result of the reaction solution of the negative control group; B: The liquid chromatography result of the negative control+(S)-2-amino-1-butanol standard product; C: The negative control+(R)-2-amino-1 -Butanol standard product liquid chromatography result; D: Experimental group 1 reaction liquid liquid chromatography result (the product is (S)-2-amino-1-butanol); E: Experimental group 2 reaction liquid liquid chromatography result ( The product is (R)-2-amino-1-butanol). Among them, the negative control reaction system only contains the expression host crude enzyme powder or enzyme solution or whole cells of the empty expression vector, and other components are the same as the experimental group.

Detailed ways

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

The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

Example 1. Preparation of (S)-2-amino-1-butanol by coupling alcohol dehydrogenase or carbonyl reductase to leucine dehydrogenase of Geobacillus stearothermophilus or its mutant

1. Preparation of engineering bacteria of recombinant alcohol dehydrogenase or its mutants, amino acid dehydrogenase or its mutants

The encoding genes of the related enzymes are separately synthesized by total gene synthesis (codon optimization is carried out using Escherichia coli as the host if necessary), and the synthesized genes are connected to various expression vectors to construct. The expression vectors are various conventional vectors in the field. The vector of the present invention is specifically pET22b(+), and the encoding gene of the related enzyme after the whole gene synthesis is inserted between the restriction sites NdeI and XhoI of pET22b(+), and the recombinant vector is obtained after the sequence is verified to be correct. . And use site-directed mutagenesis method to obtain its related gene mutants.

The above-mentioned sequence-verified correct recombinant expression vector is transformed into a suitable microbial host. The host microorganisms are various conventional host microorganisms in the field, as long as the above-mentioned recombinant expression vector can stably replicate by itself, and the carried alcohol dehydrogenase and amino acid dehydrogenase genes can be effectively expressed. Wherein, the preferred host microorganism is Escherichia coli, preferably Escherichia coli BL21 (DE3). The recombinant expression plasmid is transformed into E. coliBL21 (DE3) to obtain the genetically engineered strain of the present invention. The transformation method is a conventional transformation method in the field, preferably an electro-transformation method or a chemical transformation method.

The enzymes involved in this example and their mutants are detailed in Table 1.

Table 1 Enzymes involved in Example 1 and their mutants

Note: W1 represents alcohol dehydrogenase LBADH derived from Lactobacillus brevis; W2 represents alcohol dehydrogenase derived from Geobacillus stearothermophilus; W3 represents alcohol dehydrogenase derived from Thermoanaerobacter brockii ) alcohol dehydrogenase TbSADH; W4 represents alcohol dehydrogenase derived from Lactobacillus kefir; W5 represents alcohol dehydrogenase derived from Bacillaceae; W6 represents alcohol dehydrogenase derived from Lysella (Leifsonia sp.) alcohol dehydrogenase; W7 represents alcohol dehydrogenase derived from Thermoanaerobacter wiegelii; W8 represents carbonyl reductase SSCR derived from Sporobolomyces salmonicolor; W9 represents a leucine dehydrogenase derived from Geobacillus stearothermophilus. Wn-Mn represents a mutant of Wn (n is a natural number). The numbering of protein substitutions starts from the N-terminus of the wild-type amino acid sequence indicated by Wn (n is a natural number); the numbering of gene substitutions is from the 5' end of the wild-type nucleotide sequence indicated by Wn (n is a natural number). for starting. In the Tables, for amino acid substitutions, the following nomenclature is used: original amino acid, position (ie, position in the Wn amino acid sequence), substituted amino acid. Correspondingly, the original isoleucine (I) was replaced by valine (V) at position 11 of SEQ ID No. 2 and named "I11V". For base substitutions, the following nomenclature is used: original base, position (ie, position in the Wn nucleotide sequence), substituted base. Correspondingly, the original G was replaced by A at the 31st position of SEQ ID No. 1 and named "A31G". Variants containing multiple changes are separated by a slash character ("/").

2. Expression and crude enzyme preparation of recombinant alcohol dehydrogenase or its mutant, amino acid dehydrogenase or its mutant

The recombinant expression vector constructed in step 1 was transferred into Escherichia coli BL21 (DE3) competent cells, and cultured at 37°C for 12-16 hours. After the transformants grew out, a single transformant was inoculated into 5 mL of LB medium containing corresponding antibiotics. Incubate overnight (12-16h) at 37°C, 220rmp. Then inoculate it into TB medium at a ratio of 1% (volume percentage), cultivate at 37°C, 220rmp until the OD600 is about 0.6, add IPTG with a final concentration of 0.1mM, induce culture at 20-37°C for 16h, and then 4000rpm, The cells were collected by centrifugation at 4°C for 10 min, resuspended and washed once with 100 mM phosphate buffer with a pH value of 8.0, and then sonicated to prepare enzyme lyophilized powder.

3. Preparation of chiral 2-amino-1-butanol

The first reaction: phosphate buffer with a concentration of 100 mM, pH 8.0, 1,2-butanediol with a final concentration of 20 mM, and oxidized cofactor II with a final concentration of 1 mM, namely NADP, were sequentially added to the reaction system. ⁺ , or oxidized cofactor I, namely NAD ⁺ , acetone (v/v) with a final concentration of 5%, alcohol dehydrogenase or carbonyl reductase with a final concentration of 10 g/L lyophilized powder or enzyme liquid composition reaction system. After the reaction system was reacted at 30° C. for 24 h, the product was detected by gas chromatography (GC).

The detection conditions of gas chromatography (GC) are as follows: injection volume: 2 μL; chromatographic column: HP-5; split ratio: 20:1; split flow: 40 mL/min; temperature program: 40 °C, 5 minutes; 5 °C/min Raised to 60°C for 2 minutes; 30°C/min to 200°C for 2.333 minutes. Run time: 18 minutes.

The results refer to Figure 3, which proves that 2-keto-1-butanone is obtained in this step.

The second step reaction: the 2-keto-1-butanol generated in the previous step reaction is used as the substrate, and NH ₃ ·H ₂ O or NH ₄ Cl buffer solution with pH of 8.7 and final concentration of 1M is added to the reaction system in turn. , Oxidative cofactor I at a final concentration of 1 mM, namely NAD ⁺ , glucose dehydrogenase at a final concentration of 1 g/L, glucose at a final concentration of 100 mM, Bacillus stearothermophilus (Geobacillus stearothermophilus at a final concentration of 10 g/L) ) leucine dehydrogenase mutant freeze-dried powder or enzyme solution, and calcium carbonate with a final concentration of 5 g/L to form a reaction system. The reaction system was reacted at 30° C. for 24 h to obtain (S)-2-amino-1-butanol. The reaction solution was derivatized with o-phthalaldehyde and detected by liquid chromatography.

HPLC detection conditions are as follows: Agilent SB-Aq C18 column (4.6mm*250mm, 5um); detection wavelength 334nm; column temperature: 35°C; flow rate: 1 mL/min;

Table 2 Gradient elution procedure for HPLC

Note: (1) % in the table means volume percentage.

ee=(A _S -A _R )/(A _S +A _R )×100%; A _S : the peak area value of (S)-2-amino-1-butanol obtained by liquid chromatography analysis; A _R : The peak area values of (R)-2-amino-1-butanol obtained were analyzed by liquid chromatography.

Substrate conversion efficiency = C _turn /C _total × 100%; C _turn : the number of moles of substrate converted into (S) or (R)-2-amino-1-butanol in the reaction system; C _total : the reaction system Total moles of substrate.

HPLC detection results refer to Figure 4 and D in Figure 5, the substrate conversion efficiency is 45-55%, and the ee value is greater than 99%. The specific results are shown in Table 3.

Table 3 The results of preparing (S)-2-amino-1-butanol by coupling alcohol dehydrogenase or carbonyl reductase to leucine dehydrogenase of Geobacillus stearothermophilus or its mutants

Note: The meanings represented by Wn and Wn-Mn (n is a natural number) in the first-step reaction and the second-step reaction in the table are the same as those in table 1.

Example 2. Preparation of (S)-2-amino-1-butanol by alcohol dehydrogenase or carbonyl reductase coupled transaminase

1. Preparation of recombinant alcohol dehydrogenase or carbonyl reductase, transaminase engineering bacteria

Carry out with reference to Step 1 of Example 1.

The enzymes involved in this example and their mutants are detailed in Table 4.

Enzymes involved in Table 4 Example 2 and their mutants

Note: The meanings represented by W1-W8 are the same as in Table 1. W10 represents ATA-117 transaminase from Codexis; W11 represents transaminase derived from Aspergillus terreus; W12 represents transaminase derived from Aspergillus fumigatus; W13 represents transaminase derived from Neosartorya fischeri W14 represents the transaminase derived from Gibberella zeae; W15 represents the transaminase derived from Mycobacterium vanbaalenii. Wn-Mn represents a mutant of Wn (n is a natural number). The substitution numbers and specific nomenclature of proteins and genes are the same as in Table 1.

2. Expression and crude enzyme preparation of recombinant alcohol dehydrogenase or carbonyl reductase and transaminase

Carry out with reference to step 2 of Example 1.

3. Preparation of chiral 2-amino-1-butanol

The first reaction: phosphate buffer with a concentration of 100 mM, pH 8.0, 1,2-butanediol with a final concentration of 20 mM, and oxidized cofactor II with a final concentration of 1 mM, namely NADP, were sequentially added to the reaction system. ⁺ , or oxidized cofactor I, namely NAD ⁺ , acetone (v/v) with a final concentration of 5%, alcohol dehydrogenase or carbonyl reductase enzyme with a final concentration of 10 g/L lyophilized powder or enzyme solution Reaction system; after the reaction system was reacted at 30° C. for 24 hours, the product was detected by gas chromatography (GC). The detection conditions of gas chromatography (GC) are as shown in step 3 of Example 1. The results refer to Figure 3, which proves that 2-keto-1-butanone is obtained in this step.

The second step reaction: the 2-keto-1-butanol generated in the previous step reaction is used as the substrate, and the phosphate buffer solution with a concentration of 50-100 mM, a concentration of isopropylamine of 500 mM, and a pH value of 8.0 is sequentially added to the reaction system. , pyridoxal phosphate (PLP) at a final concentration of 1 mM, ATA-117 transaminase from Codexis at a final concentration of 10 g/L, or transaminase from Aspergillus terreus, or transaminase from Aspergillus fumigatus, or The transaminase of Neosartorya fischeri (Neosartorya fischeri), or the transaminase of Gibberella zeae, or the transaminase enzyme freeze-dried powder or enzyme liquid of Mycobacterium vanbaalenii constitutes a reaction system, and the reaction system is kept at 30 Under the condition of ℃, react for 24h to obtain (S)-2-amino-1-butanol. The reaction solution was derivatized with o-phthalaldehyde and detected by liquid chromatography. HPLC detection conditions are as shown in step 3 of Example 1.

The specific calculation method of ee value and substrate conversion efficiency is the same as that of step 3 of embodiment 1.

HPLC detection results refer to Figure 4 and D in Figure 5, the substrate conversion efficiency is 45-55%, and the ee value is greater than 99%. The specific results are shown in Table 5.

Table 5 Results of the preparation of (S)-2-amino-1-butanol by alcohol dehydrogenase or its mutant coupled with transaminase

Note: The meanings represented by Wn and Wn-Mn (n is a natural number) in the first-step reaction and the second-step reaction in the table are the same as those in table 4.

Example 3. Preparation of (R)-2-amino-1-butanol by alcohol dehydrogenase or carbonyl reductase coupled transaminase

1. Preparation of recombinant alcohol dehydrogenase and transaminase engineering bacteria

Carry out with reference to Step 1 of Example 1.

The enzymes involved in this example and their mutants are detailed in Table 6.

Enzymes involved in Table 6 Example 3 and their mutants

Note: The meanings represented by W1-W8 are the same as in Table 1. W16 represents a transaminase derived from Bacillus megaterium; W17 represents a transaminase derived from P. aeruginosa. Wn-Mn represents a mutant of Wn (n is a natural number). The substitution numbers and specific nomenclature of proteins and genes are the same as in Table 1.

2. Expression and crude enzyme preparation of recombinant alcohol dehydrogenase and transaminase

Carry out with reference to step 2 of Example 1.

3. Preparation of chiral 2-amino-1-butanol

The first reaction: phosphate buffer with a concentration of 100 mM and a pH of 8.0, 1,2-butanediol with a final concentration of 20 mM, and oxidized cofactor II with a final concentration of 1 mM are added to the reaction system in turn, namely NADP ⁺ , or oxidized cofactor I, namely NAD ⁺ , acetone (v/v) with a final concentration of 5%, alcohol dehydrogenase or carbonyl reductase with a final concentration of 10 g/L lyophilized powder or enzyme solution Reaction system; after the reaction system was reacted at 30° C. for 24 hours, the product was detected by gas chromatography (GC). The detection conditions of gas chromatography (GC) are as shown in step 3 of Example 1. The results refer to Figure 3, which proves that 2-keto-1-butanone is obtained in this step.

The second step reaction: the 2-keto-1-butanol generated in the previous step reaction is used as the substrate, and the phosphate buffer solution with a concentration of 100 mM, a concentration of isopropylamine of 500 mM, and a pH value of 8.0 is added to the reaction system in turn. Pyridoxal phosphate (PLP) at a concentration of 1mM, transaminase from Bacillus megaterium at a final concentration of 10g/L, or transaminase from P. aeruginosa (P.aeruginosa PAO2) lyophilized powder or enzyme solution A reaction system was formed, and the reaction system was reacted at 30° C. for 24 h to obtain (S)-2-amino-1-butanol. The reaction solution was derivatized with o-phthalaldehyde and detected by liquid chromatography. HPLC detection conditions are as shown in step 3 of Example 1.

HPLC detection results refer to E in Figure 4 and Figure 5, the substrate conversion efficiency is 45-50%, and the ee value is greater than 99%. The specific results are shown in Table 7.

Table 7 Results of preparing (R)-2-amino-1-butanol by alcohol dehydrogenase coupled transaminase

Note: The meanings represented by Wn and Wn-Mn (n is a natural number) in the first-step reaction and the second-step reaction in the table are the same as those in Table 6.

Example 4. Co-expression of enzyme A and enzyme B Whole cell preparation of (S)-2-amino-1-butanol and (R)-2-amino-1-butanol

1. Preparation of co-expression engineering bacteria of enzyme A and enzyme B

The encoding genes of the relevant enzymes are separately synthesized by total gene synthesis (codon optimization is carried out using Escherichia coli as the host as required), and the synthesized genes are connected to various expression vectors to construct. The expression vectors are various conventional vectors in the field. The vector of the present invention is specifically pETDuet-1. The DNA fragment of enzyme A after full gene synthesis is inserted between the restriction sites EcoRI and HindIII of pETDuet-1, and the DNA fragment of enzyme B after full gene synthesis is inserted into pETDuet- 1 between the enzyme cleavage sites NdeI and XhoI. The recombinant vector was transferred into Escherichia coli DH5α competent cells; positive transformants were picked and sequenced and identified, and the correct recombinant expression vector was obtained.

The above-mentioned sequence-verified correct recombinant expression vector is transformed into a suitable microbial host. The host microorganisms are various conventional host microorganisms in the field, as long as the above-mentioned recombinant expression vector can stably replicate by itself, and the carried alcohol dehydrogenase and amino acid dehydrogenase genes can be effectively expressed. Wherein the host microorganism is preferably: Escherichia coli (Escherichia coli), preferably Escherichia coli BL21 (DE3), the aforementioned recombinant expression plasmid is transformed into E. coli BL21 (DE3), the genetic engineering of the present invention can be obtained strains. The transformation method is a conventional transformation method in the field, preferably an electro-transformation method or a chemical transformation method.

The enzymes involved in this example and their mutants are detailed in Table 8.

Table 8 Enzymes involved in Example 4 and their mutants

Note: The meanings represented by W1-W17 are the same as in Tables 1, 4, and 6. Wn-Mn represents a mutant of Wn (n is a natural number). The substitution numbers and specific nomenclature of proteins and genes are the same as in Table 1.

2. Co-expression of enzyme A and enzyme B

The recombinant expression vector constructed in step 1 was transferred into Escherichia coli BL21 (DE3) competent cells, and cultured at 37°C for 12-16 hours. After the transformants grew out, a single transformant was inoculated into 5 mL of LB medium containing corresponding antibiotics. Incubate overnight (12-16h) at 37°C, 220rmp. Then inoculate it into TB medium at a ratio of 1% (volume percentage), cultivate at 37°C, 220rmp until the OD600 is about 0.6, add IPTG with a final concentration of 0.1mM, induce culture at 20-37°C for 16h, and then 4000rpm, Cells were collected by centrifugation at 4°C for 10 min.

3. Preparation of chiral 2-amino-1-butanol

Phosphate buffer with a concentration of 100 mM and a pH of 8.0 was added to the reaction system in turn, 1,2-butanediol with a final concentration of 50 mM, 100 mM glucose, and a final concentration of 100 g/L, which can co-express enzyme A and enzyme Whole cells of B (wet bacterial weight) constitute the reaction system. After the reaction system was reacted at 30° C. for 24 h, either (R) or (S)-2-amino-1-butanol was obtained. The fermentation broth was derivatized with o-phthalaldehyde and detected by liquid chromatography. HPLC detection conditions are as shown in step 3 of Example 1.

The specific calculation method of ee value and substrate conversion efficiency is the same as that of step 3 of embodiment 1.

HPLC detection results refer to D-E in Figure 4 and Figure 5, the substrate conversion efficiency is 15-30%, and the ee value is greater than 99%. The specific results are shown in Table 9.

Table 9 Results of co-expression of enzymes A and B in whole cell catalytic preparation of chiral 2-amino-1-butanol

Note: The meanings represented by Wn and Wn-Mn (n is a natural number) in the table are the same as those in Table 8.

<110> Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences

<120> A kind of synthetic method of chiral 2-amino-1-butanol

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ggtacccgtc ttggaatcca acgtatgaag aataaaggac tcggagcatc aatcatcaat 420

atgtcatcta tcgaaggttt tgttggtgat ccaactctgg gtgcatacaa cgcttcaaaa 480

ggtgctgtca gaattatgtc taaatcagct gccttggatt gcgctttgaa ggactacgat 540

gttcgggtta acactgttca tccaggttat atcaagacac cattggttga cgatcttgaa 600

ggggcagaag aaatgatgtc acagcggacc aagacaccaa tgggtcatat cggtgaacct 660

aacgatatcg cttggatctg tgtttacctg gcatctgacg aatctaaatt tgccactggt 720

gcagaattcg ttgtcgatgg tggatacact gctcaataa 759

<210> 8

<211> 252

<212> PRT

<213> Lactobacillus kefiri

<400> 8

Met Thr Asp Arg Leu Lys Gly Lys Val Ala Ile Val Thr Gly Gly Thr

1 5 10 15

Leu Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala

20 25 30

Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala

35 40 45

Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Ala

50 55 60

Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Ala

65 70 75 80

Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Ala Val Ser

85 90 95

Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser

100 105 110

Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg

115 120 125

Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile

130 135 140

Glu Gly Phe Val Gly Asp Pro Thr Leu Gly Ala Tyr Asn Ala Ser Lys

145 150 155 160

Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu

165 170 175

Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Tyr Ile Lys

180 185 190

Thr Pro Leu Val Asp Asp Leu Glu Gly Ala Glu Glu Met Met Ser Gln

195 200 205

Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala

210 215 220

Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly

225 230 235 240

Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln

245 250

<210> 9

<211> 1023

<212> DNA

<213> Bacillaceae

<400> 9

atgaaagctg cagtagtaga gcaatttaag gaaccattaa aaattaaaga agtggaaaag 60

ccatccattt catatggcga agtattagtc cgcattaaag catgcggtgt atgccatacg 120

gacttgcacg ccgctcatgg cgattggcca gtaaaaccaa aacttccttt aatccctggc 180

catgaaggag tcggaattgt tgaagaagtc ggtccggggg taacccattt aaaagtggga 240

gaccgcgttg gaattccttg gttatattct gcttgcggcc attgcgaata ttgtttaagc 300

ggacaagaga cattatgtga acatcaagaa aacgccggct actcagtcga cgggggttat 360

gcagaatatt gcagagctgc ggcagactat gtggtgaaaa ttcctgacaa cttgtcgttt 420

gaagaagctg ctcctatttt ctgcgccgga gttactactt ataaagcgtt aaaagtcaca 480

ggtacaaaac cgggagaatg ggtagcgatc tatggcatcg gcggccttgg acatgttgcc 540

gtccagtatg cgaaagcgat ggggcttcat gttgttgcag tggatatcgg cgatgagaaa 600

ctggaacttg caaaagagct tggcgccgat cttgttgtaa atcctgcaaa agaaaatgcg 660

gcacaattta tgaaagagaa agtcggcgga gtacacgcgg ctgttgtgac agctgtatct 720

aaacctgctt ttcaatctgc gtacaattct atccgcagag gcggcacgtg cgtgcttgtc 780

ggattaccgc cggaagaaat gcctattcca atctttgata cggtattaaa cggaattaaa 840

attatcggtt ccattgtcgg cacgcggaaa gacttgcaag aagcgcttca gttcgctgca 900

gaaggtaaag taaaaaccat tattgaagtg caacctcttg aaaaaattaa cgaagtattt 960

gacagaatgc taaaaggaga aattaacgga cgggttgttt taacgttaga aaataataat 1020

taa 1023

<210> 10

<211> 340

<212> PRT

<213> Bacillaceae

<400> 10

Met Lys Ala Ala Val Val Glu Gln Phe Lys Glu Pro Leu Lys Ile Lys

1 5 10 15

Glu Val Glu Lys Pro Ser Ile Ser Tyr Gly Glu Val Leu Val Arg Ile

20 25 30

Lys Ala Cys Gly Val Cys His Thr Asp Leu His Ala Ala His Gly Asp

35 40 45

Trp Pro Val Lys Pro Lys Leu Pro Leu Ile Pro Gly His Glu Gly Val

50 55 60

Gly Ile Val Glu Glu Val Gly Pro Gly Val Thr His Leu Lys Val Gly

65 70 75 80

Asp Arg Val Gly Ile Pro Trp Leu Tyr Ser Ala Cys Gly His Cys Glu

85 90 95

Tyr Cys Leu Ser Gly Gln Glu Thr Leu Cys Glu His Gln Glu Asn Ala

100 105 110

Gly Tyr Ser Val Asp Gly Gly Tyr Ala Glu Tyr Cys Arg Ala Ala Ala

115 120 125

Asp Tyr Val Val Lys Ile Pro Asp Asn Leu Ser Phe Glu Glu Ala Ala

130 135 140

Pro Ile Phe Cys Ala Gly Val Thr Thr Tyr Lys Ala Leu Lys Val Thr

145 150 155 160

Gly Thr Lys Pro Gly Glu Trp Val Ala Ile Tyr Gly Ile Gly Gly Leu

165 170 175

Gly His Val Ala Val Gln Tyr Ala Lys Ala Met Gly Leu His Val Val

180 185 190

Ala Val Asp Ile Gly Asp Glu Lys Leu Glu Leu Ala Lys Glu Leu Gly

195 200 205

Ala Asp Leu Val Val Asn Pro Ala Lys Glu Asn Ala Ala Gln Phe Met

210 215 220

Lys Glu Lys Val Gly Gly Val His Ala Ala Val Val Thr Ala Val Ser

225 230 235 240

Lys Pro Ala Phe Gln Ser Ala Tyr Asn Ser Ile Arg Arg Gly Gly Thr

245 250 255

Cys Val Leu Val Gly Leu Pro Pro Glu Glu Met Pro Ile Pro Ile Phe

260 265 270

Asp Thr Val Leu Asn Gly Ile Lys Ile Ile Gly Ser Ile Val Gly Thr

275 280 285

Arg Lys Asp Leu Gln Glu Ala Leu Gln Phe Ala Ala Glu Gly Lys Val

290 295 300

Lys Thr Ile Ile Glu Val Gln Pro Leu Glu Lys Ile Asn Glu Val Phe

305 310 315 320

Asp Arg Met Leu Lys Gly Glu Ile Asn Gly Arg Val Val Leu Thr Leu

325 330 335

Glu Asn Asn Asn

340

<210> 11

<211> 747

<212> DNA

<213> Leifsonia sp.

<400> 11

atggctcagt acgacgtcgc cgaccggtcc gcgatcgtga ccggaggcgg ctcgggcatc 60

gggcgcgccg tggcgctcac tctcgcggcg agcggcgcag ccgtcctcgt caccgacctg 120

aacgaggagc acgcgcaggc cgtcgtggcc gagatcgagg ccgcgggcgg taaggccgcc 180

gcgctcgcgg gcgacgtgac cgaccccgcg ttcggcgagg cgagcgtcgc cggggcgaac 240

gctctcgcgc ccctcaagat cgcggtcaac aacgcgggca tcggcggcga ggccgccacg 300

gtcggcgact actcgctcga cagctggcgc acggtgatcg aggtcaacct caacgccgtg 360

ttctacggga tgcagccgca gctgaaggcc atggccgcca acggcggcgg tgcgatcgtc 420

aacatggcgt ccatcctggg aagcgtcggc ttcgccaact cgtcggccta cgtcacggcc 480

aagcacgcgc tgctcggtct cacccagaac gccgcgctcg agtacgccgc cgacaaggtg 540

cgcgtcgtcg cggtcggccc cggcttcatc cgcaccccgc tcgtggaggc caacctctcc 600

gccgacgcgc tggcgttcct cgagggcaag cacgccctcg gccgcctggg cgagccggaa 660

gaggtcgcct cgctggtcgc gttcctcgcc tccgacgccg cgagcttcat caccggcagc 720

taccacctgg tggacggcgg ctacacc 747

<210> 12

<211> 249

<212> PRT

<213> Leifsonia sp.

<400> 12

Met Ala Gln Tyr Asp Val Ala Asp Arg Ser Ala Ile Val Thr Gly Gly

1 5 10 15

Gly Ser Gly Ile Gly Arg Ala Val Ala Leu Thr Leu Ala Ala Ser Gly

20 25 30

Ala Ala Val Leu Val Thr Asp Leu Asn Glu Glu His Ala Gln Ala Val

35 40 45

Val Ala Glu Ile Glu Ala Ala Gly Gly Lys Ala Ala Ala Leu Ala Gly

50 55 60

Asp Val Thr Asp Pro Ala Phe Gly Glu Ala Ser Val Ala Gly Ala Asn

65 70 75 80

Ala Leu Ala Pro Leu Lys Ile Ala Val Asn Asn Ala Gly Ile Gly Gly

85 90 95

Glu Ala Ala Thr Val Gly Asp Tyr Ser Leu Asp Ser Trp Arg Thr Val

100 105 110

Ile Glu Val Asn Leu Asn Ala Val Phe Tyr Gly Met Gln Pro Gln Leu

115 120 125

Lys Ala Met Ala Ala Asn Gly Gly Gly Ala Ile Val Asn Met Ala Ser

130 135 140

Ile Leu Gly Ser Val Gly Phe Ala Asn Ser Ser Ala Tyr Val Thr Ala

145 150 155 160

Lys His Ala Leu Leu Gly Leu Thr Gln Asn Ala Ala Leu Glu Tyr Ala

165 170 175

Ala Asp Lys Val Arg Val Val Ala Val Gly Pro Gly Phe Ile Arg Thr

180 185 190

Pro Leu Val Glu Ala Asn Leu Ser Ala Asp Ala Leu Ala Phe Leu Glu

195 200 205

Gly Lys His Ala Leu Gly Arg Leu Gly Glu Pro Glu Glu Val Ala Ser

210 215 220

Leu Val Ala Phe Leu Ala Ser Asp Ala Ala Ser Phe Ile Thr Gly Ser

225 230 235 240

Tyr His Leu Val Asp Gly Gly Tyr Thr

245

<210> 13

<211> 1059

<212> DNA

<213> Thermoanaerobacter wiegelii

<400> 13

atgaaaggtt ttgcaatgct cagtatcggt aaggttggct ggattgaggt agaaaagcct 60

aatccaggac cctttgatgc tatcgtaaga cccctagctg tggccccttg ctcttcggac 120

attcacactg tttttgaagg aggccttggt gaacttcaca acgcagtgct aggtcacgaa 180

gctgtaggtg aagtagtcga agtcggtagt gaagtaaaag actttaaacc tggtgataag 240

gtggtcattc ctgctatcac tcctgattgg agaacgttag atgttcaacg tggttatcat 300

cagcagtccg gaggtatgct tgctggttac aagttcacag cccagaaacc tggtgtgttc 360

gccgagtaca tctacgttaa cgatgcagac atgaatcttg ctcatttacc tgacggcatc 420

tctttagaag cggccgtcat gatcacagat atgatgacta ccggttttca cggagccgaa 480

ctggcagaaa tagaattagg tgcaacagta gcggttttgg gtattggtcc agtaggtctt 540

atggcagtcg ctggtgccaa attgcggggt gctggaagaa ttattgcagt aggcagtaga 600

cctgtttgtg tagatgctgc aaaatactat ggagctactg atattgtaaa ctataaaaat 660

ggtcctatcg acagtcagat tatggattta acgaaaggca aaggtgttga tgctgccatc 720

atcgctggag gaaatgttga catcatggct acagcagtta agattgttaa acctggtggc 780

accattgcta atgtaaatta ctttggcgaa ggagatgttt tgcctgttcc tcgtcttgaa 840

tggggttgcg gcatggctca taaagctata aaaggcggtt tatgccctgg tggacgtcta 900

agaatggaaa gactgattga ccttgttttt tataagcgtg tcgatccttc caaactcgtc 960

actcatgttt ttcaaggatt tgataatatt gaaaaagctc taatgctgat gaaagataaa 1020

ccaaaggacc taatcaaacc tgttgtaata ttagcataa 1059

<210> 14

<211> 352

<212> PRT

<213> Thermoanaerobacter wiegelii

<400> 14

Met Lys Gly Phe Ala Met Leu Ser Ile Gly Lys Val Gly Trp Ile Glu

1 5 10 15

Val Glu Lys Pro Asn Pro Gly Pro Phe Asp Ala Ile Val Arg Pro Leu

20 25 30

Ala Val Ala Pro Cys Ser Ser Asp Ile His Thr Val Phe Glu Gly Gly

35 40 45

Leu Gly Glu Leu His Asn Ala Val Leu Gly His Glu Ala Val Gly Glu

50 55 60

Val Val Glu Val Gly Ser Glu Val Lys Asp Phe Lys Pro Gly Asp Lys

65 70 75 80

Val Val Ile Pro Ala Ile Thr Pro Asp Trp Arg Thr Leu Asp Val Gln

85 90 95

Arg Gly Tyr His Gln Gln Ser Gly Gly Met Leu Ala Gly Tyr Lys Phe

100 105 110

Thr Ala Gln Lys Pro Gly Val Phe Ala Glu Tyr Ile Tyr Val Asn Asp

115 120 125

Ala Asp Met Asn Leu Ala His Leu Pro Asp Gly Ile Ser Leu Glu Ala

130 135 140

Ala Val Met Ile Thr Asp Met Met Thr Thr Gly Phe His Gly Ala Glu

145 150 155 160

Leu Ala Glu Ile Glu Leu Gly Ala Thr Val Ala Val Leu Gly Ile Gly

165 170 175

Pro Val Gly Leu Met Ala Val Ala Gly Ala Lys Leu Arg Gly Ala Gly

180 185 190

Arg Ile Ile Ala Val Gly Ser Arg Pro Val Cys Val Asp Ala Ala Lys

195 200 205

Tyr Tyr Gly Ala Thr Asp Ile Val Asn Tyr Lys Asn Gly Pro Ile Asp

210 215 220

Ser Gln Ile Met Asp Leu Thr Lys Gly Lys Gly Val Asp Ala Ala Ile

225 230 235 240

Ile Ala Gly Gly Asn Val Asp Ile Met Ala Thr Ala Val Lys Ile Val

245 250 255

Lys Pro Gly Gly Thr Ile Ala Asn Val Asn Tyr Phe Gly Glu Gly Asp

260 265 270

Val Leu Pro Val Pro Arg Leu Glu Trp Gly Cys Gly Met Ala His Lys

275 280 285

Ala Ile Lys Gly Gly Leu Cys Pro Gly Gly Arg Leu Arg Met Glu Arg

290 295 300

Leu Ile Asp Leu Val Phe Tyr Lys Arg Val Asp Pro Ser Lys Leu Val

305 310 315 320

Thr His Val Phe Gln Gly Phe Asp Asn Ile Glu Lys Ala Leu Met Leu

325 330 335

Met Lys Asp Lys Pro Lys Asp Leu Ile Lys Pro Val Val Ile Leu Ala

340 345 350

<210> 15

<211> 1035

<212> DNA

<213> Artificial sequences

<220>

<223>

<400> 15

atggcgaaga tcgacaacgc ggtgctgccg gaaggtagcc tggtgctggt taccggtgcg 60

aacggtttcg tggcgagcca cgtggttgag cagctgctgg aacacggtta caaggttcgt 120

ggcaccgcgc gtagcgcgag caaactggcg aacctgcaaa agcgttggga cgcgaaatac 180

ccgggtcgtt ttgagaccgc ggtggttgaa gacatgctga agcagggcgc gtatgatgaa 240

gtgatcaagg gtgcggcggg cgttgcgcac attgcgagcg tggttagctt cagcaacaag 300

tatgatgagg tggttacccc ggcgatcggt ggcaccctga acgcgctgcg tgctgcggcg 360

gcgaccccga gcgtgaaacg ttttgttctg accagcagca ccgtgagcgc gctgatcccg 420

aagccgaacg ttgagggtat ttacctggat gagaagagct ggaacctgga gagcattgac 480

aaggcgaaaa ccctgccgga aagcgatccg caaaagagcc tgtgggtgta tgcggcgagc 540

aaaaccgagg cggaactggc ggcgtggaag ttcatggacg agaacaaacc gcactttacc 600

ctgaacgcgg ttctgccgaa ctacaccatc ggcaccattt tcgatccgga aacccagagc 660

ggtagcacca gcggctggat gatgagcctg tttaacggcg aggtgtctcc ggcgctggcg 720

ctgatgccgc cgcagtacta tgtgagcgcg gttgacatcg gtctgctgca cctgggttgc 780

ctggtgctgc cgcaaattga gcgtcgtcgt gtttacggca ccgcgggcac cttcgattgg 840

aacaccgttc tggcgacctt tcgtaagctg tatccgagca aaaccttccc ggcggacttt 900

ccggatcagg gtcaagacct gagcaagttc gataccgcgc cgagcctgga aattctgaaa 960

agcctgggtc gtccgggttg gcgtagcatc gaggaaagca ttaaagacct ggttggcagc 1020

gagaccgcgc actaa 1035

<210> 16

<211> 344

<212> PRT

<213> Sporobolomyces salmonicolor

<400> 16

Met Ala Lys Ile Asp Asn Ala Val Leu Pro Glu Gly Ser Leu Val Leu

1 5 10 15

Val Thr Gly Ala Asn Gly Phe Val Ala Ser His Val Val Glu Gln Leu

20 25 30

Leu Glu His Gly Tyr Lys Val Arg Gly Thr Ala Arg Ser Ala Ser Lys

35 40 45

Leu Ala Asn Leu Gln Lys Arg Trp Asp Ala Lys Tyr Pro Gly Arg Phe

50 55 60

Glu Thr Ala Val Val Glu Asp Met Leu Lys Gln Gly Ala Tyr Asp Glu

65 70 75 80

Val Ile Lys Gly Ala Ala Gly Val Ala His Ile Ala Ser Val Val Ser

85 90 95

Phe Ser Asn Lys Tyr Asp Glu Val Val Thr Pro Ala Ile Gly Gly Thr

100 105 110

Leu Asn Ala Leu Arg Ala Ala Ala Ala Thr Pro Ser Val Lys Arg Phe

115 120 125

Val Leu Thr Ser Ser Thr Val Ser Ala Leu Ile Pro Lys Pro Asn Val

130 135 140

Glu Gly Ile Tyr Leu Asp Glu Lys Ser Trp Asn Leu Glu Ser Ile Asp

145 150 155 160

Lys Ala Lys Thr Leu Pro Glu Ser Asp Pro Gln Lys Ser Leu Trp Val

165 170 175

Tyr Ala Ala Ser Lys Thr Glu Ala Glu Leu Ala Ala Trp Lys Phe Met

180 185 190

Asp Glu Asn Lys Pro His Phe Thr Leu Asn Ala Val Leu Pro Asn Tyr

195 200 205

Thr Ile Gly Thr Ile Phe Asp Pro Glu Thr Gln Ser Gly Ser Thr Ser

210 215 220

Gly Trp Met Met Ser Leu Phe Asn Gly Glu Val Ser Pro Ala Leu Ala

225 230 235 240

Leu Met Pro Pro Gln Tyr Tyr Val Ser Ala Val Asp Ile Gly Leu Leu

245 250 255

His Leu Gly Cys Leu Val Leu Pro Gln Ile Glu Arg Arg Arg Val Tyr

260 265 270

Gly Thr Ala Gly Thr Phe Asp Trp Asn Thr Val Leu Ala Thr Phe Arg

275 280 285

Lys Leu Tyr Pro Ser Lys Thr Phe Pro Ala Asp Phe Pro Asp Gln Gly

290 295 300

Gln Asp Leu Ser Lys Phe Asp Thr Ala Pro Ser Leu Glu Ile Leu Lys

305 310 315 320

Ser Leu Gly Arg Pro Gly Trp Arg Ser Ile Glu Glu Ser Ile Lys Asp

325 330 335

Leu Val Gly Ser Glu Thr Ala His

340

<210> 17

<211> 1104

<212> DNA

<213> Artificial sequences

<220>

<223>

<400> 17

atggaactgt ttaagtatat ggaaacctat gattatgaac aagtgctgtt ttgccaggac 60

aaggaaagcg gtctgaaggc gattattgcc attcatgata ccacgctggg tccggcactg 120

ggcggtaccc gtatgtggat gtataacagc gaagaagaag cgctggaaga tgccctgcgt 180

ctggcacgcg gtatgaccta caaaaacgca gcagcaggtc tgaatctggg cggtggcaag 240

acggtgatta tcggtgatcc gcgcaaagac aagaacgaag ctatgtttcg tgcgttcggc 300

cgctttatcc agggtctgaa tggccgttat attaccgcgg aagatgtggg taccacggtt 360

gccgatatgg acattatcta tcaagaaacc gactacgtga cgggcatttc accggaattt 420

ggtagctctg gtaacccgtc gccggcaacg gcctatggtg tttaccgtgg catgaaagct 480

gcggccaagg aagcatttgg tagtgattcc ctggaaggca aagtggttgc agttcagggt 540

gtcggcaatg tggcttatca cctgtgccgc catctgcacg aagaaggcgc caaactgatt 600

gtgaccgata tcaacaagga agtcgtggca cgtgctgttg aagaatttgg tgcgaaagcc 660

gtcgatccga atgacatcta cggcgtggaa tgcgatattt ttgcgccgtg tgccctgggt 720

ggcattatca acgaccagac catcccgcaa ctgaaagcga aggttattgc aggtagtgct 780

aacaatcagc tgaaagaacc gcgtcatggc gatattatcc acgaaatggg catcgtctat 840

gccccggact acgtgattaa cgcaggtggc gttatcaatg tcgctgatga actgtatggc 900

tacaatcgtg aacgcgcgat gaaaaagatt gaacaaatct atgacaacat cgaaaaagtt 960

ttcgcaatcg ctaagcgtga taatattccg acctacgtcg cagctgaccg tatggccgaa 1020

gaacgcattg aaacgatgcg taaagcccgt tcccagttcc tgcaaaatgg tcatcatatt 1080

ctgagccgcc gtcgtgcccg ctaa 1104

<210> 18

<211> 367

<212> PRT

<213> Geobacillus stearothermophilus

<400> 18

Met Glu Leu Phe Lys Tyr Met Glu Thr Tyr Asp Tyr Glu Gln Val Leu

1 5 10 15

Phe Cys Gln Asp Lys Glu Ser Gly Leu Lys Ala Ile Ile Ala Ile His

20 25 30

Asp Thr Thr Leu Gly Pro Ala Leu Gly Gly Thr Arg Met Trp Met Tyr

35 40 45

Asn Ser Glu Glu Glu Ala Leu Glu Asp Ala Leu Arg Leu Ala Arg Gly

50 55 60

Met Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys

65 70 75 80

Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Asn Glu Ala Met Phe

85 90 95

Arg Ala Phe Gly Arg Phe Ile Gln Gly Leu Asn Gly Arg Tyr Ile Thr

100 105 110

Ala Glu Asp Val Gly Thr Thr Val Ala Asp Met Asp Ile Ile Tyr Gln

115 120 125

Glu Thr Asp Tyr Val Thr Gly Ile Ser Pro Glu Phe Gly Ser Ser Gly

130 135 140

Asn Pro Ser Pro Ala Thr Ala Tyr Gly Val Tyr Arg Gly Met Lys Ala

145 150 155 160

Ala Ala Lys Glu Ala Phe Gly Ser Asp Ser Leu Glu Gly Lys Val Val

165 170 175

Ala Val Gln Gly Val Gly Asn Val Ala Tyr His Leu Cys Arg His Leu

180 185 190

His Glu Glu Gly Ala Lys Leu Ile Val Thr Asp Ile Asn Lys Glu Val

195 200 205

Val Ala Arg Ala Val Glu Glu Phe Gly Ala Lys Ala Val Asp Pro Asn

210 215 220

Asp Ile Tyr Gly Val Glu Cys Asp Ile Phe Ala Pro Cys Ala Leu Gly

225 230 235 240

Gly Ile Ile Asn Asp Gln Thr Ile Pro Gln Leu Lys Ala Lys Val Ile

245 250 255

Ala Gly Ser Ala Asn Asn Gln Leu Lys Glu Pro Arg His Gly Asp Ile

260 265 270

Ile His Glu Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala

275 280 285

Gly Gly Val Ile Asn Val Ala Asp Glu Leu Tyr Gly Tyr Asn Arg Glu

290 295 300

Arg Ala Met Lys Lys Ile Glu Gln Ile Tyr Asp Asn Ile Glu Lys Val

305 310 315 320

Phe Ala Ile Ala Lys Arg Asp Asn Ile Pro Thr Tyr Val Ala Ala Asp

325 330 335

Arg Met Ala Glu Glu Arg Ile Glu Thr Met Arg Lys Ala Arg Ser Gln

340 345 350

Phe Leu Gln Asn Gly His His His Ile Leu Ser Arg Arg Arg Ala Arg

355 360 365

<210> 19

<211> 993

<212> DNA

<213> Artificial sequences

<220>

<223>

<400> 19

atggcatttt ctgcagatac cagcgaaatt gtttataccc atgataccgg tctggattat 60

attacctata gcgattatga actggaccct gccaatccgc tggccggcgg tgctgcttgg 120

attgaaggtg cctttgtgcc gccgagtgaa gcacgcatta gtatttttga tcagggttat 180

ctgcatagtg atgtgaccta taccgtgttt catgtttgga atggtaatgc ctttcgtctg 240

gatgatcata ttgaacgtct gtttagcaat gccgaaagca tgcgcattat tccgccgctg 300

acccaggatg aagttaaaga aattgcactg gaactggttg caaaaaccga actgcgtgaa 360

gcatttgtta gtgttagcat tacccgtggc tatagcagca ccccgggtga acgtgatatt 420

accaaacatc gcccgcaggt ttatatgtat gcagttccgt atcagtggat tgttccgttt 480

gatcgcattc gtgatggtgt gcatgcaatg gttgcacaga gtgttcgtcg caccccgcgt 540

agcagcattg atccgcaggt taaaaatttt cagtggggcg atctgattcg cgccgttcag 600

gaaacccatg atcgcggctt tgaagcaccg ctgctgctgg atggcgatgg tctgctggcc 660

gaaggtagcg gctttaatgt ggtggttatt aaggatggcg ttgttcgcag cccgggtcgt 720

gcagcactgc cgggtattac ccgcaaaacc gtgctggaaa ttgcagaaag cctgggccat 780

gaagccattc tggcagatat taccctggca gaactgctgg atgcagatga agttctgggt 840

tgtaccaccg ccggtggcgt gtggccgttt gttagtgtgg atggtaatcc gattagcgat 900

ggcgtgccgg gtccggttac ccagagtatt attcgccgtt attgggaact gaatgtggaa 960

agcagcagtc tgctgacccc ggttcagtat taa 993

<210> 20

<211> 330

<212> PRT

<213> ATA-117

<400> 20

Met Ala Phe Ser Ala Asp Thr Ser Glu Ile Val Tyr Thr His Asp Thr

1 5 10 15

Gly Leu Asp Tyr Ile Thr Tyr Ser Asp Tyr Glu Leu Asp Pro Ala Asn

20 25 30

Pro Leu Ala Gly Gly Ala Ala Trp Ile Glu Gly Ala Phe Val Pro Pro

35 40 45

Ser Glu Ala Arg Ile Ser Ile Phe Asp Gln Gly Tyr Leu His Ser Asp

50 55 60

Val Thr Tyr Thr Val Phe His Val Trp Asn Gly Asn Ala Phe Arg Leu

65 70 75 80

Asp Asp His Ile Glu Arg Leu Phe Ser Asn Ala Glu Ser Met Arg Ile

85 90 95

Ile Pro Pro Leu Thr Gln Asp Glu Val Lys Glu Ile Ala Leu Glu Leu

100 105 110

Val Ala Lys Thr Glu Leu Arg Glu Ala Phe Val Ser Val Ser Ile Thr

115 120 125

Arg Gly Tyr Ser Ser Thr Pro Gly Glu Arg Asp Ile Thr Lys His Arg

130 135 140

Pro Gln Val Tyr Met Tyr Ala Val Pro Tyr Gln Trp Ile Val Pro Phe

145 150 155 160

Asp Arg Ile Arg Asp Gly Val His Ala Met Val Ala Gln Ser Val Arg

165 170 175

Arg Thr Pro Arg Ser Ser Ile Asp Pro Gln Val Lys Asn Phe Gln Trp

180 185 190

Gly Asp Leu Ile Arg Ala Val Gln Glu Thr His Asp Arg Gly Phe Glu

195 200 205

Ala Pro Leu Leu Leu Asp Gly Asp Gly Leu Leu Ala Glu Gly Ser Gly

210 215 220

Phe Asn Val Val Val Ile Lys Asp Gly Val Val Arg Ser Pro Gly Arg

225 230 235 240

Ala Ala Leu Pro Gly Ile Thr Arg Lys Thr Val Leu Glu Ile Ala Glu

245 250 255

Ser Leu Gly His Glu Ala Ile Leu Ala Asp Ile Thr Leu Ala Glu Leu

260 265 270

Leu Asp Ala Asp Glu Val Leu Gly Cys Thr Thr Ala Gly Gly Val Trp

275 280 285

Pro Phe Val Ser Val Asp Gly Asn Pro Ile Ser Asp Gly Val Pro Gly

290 295 300

Pro Val Thr Gln Ser Ile Ile Arg Arg Tyr Trp Glu Leu Asn Val Glu

305 310 315 320

Ser Ser Ser Leu Leu Thr Pro Val Gln Tyr

325 330

<210> 21

<211> 975

<212> DNA

<213> Artificial sequences

<220>

<223>

<400> 21

atggctagta tggataaggt gttcgccggc tatgccgcac gtcaagcaat tctggaaagt 60

accgaaacca ccaatccgtt tgcaaaaggt attgcctggg ttgaaggtga actggtgccg 120

ttagccgaag cacgtattcc gctgctggat cagggcttta tgcatagtga tctgacctat 180

gatgtgccga gtgtttggga tggtcgcttt ttccgcctgg atgatcatat tacccgcctg 240

gaagcaagct gcaccaaact gcgtctgcgc ttaccgctgc ctcgtgacca ggtgaaacag 300

attctggtgg aaatggttgc aaagagcggt attcgcgatg cctttgtgga actgattgtt 360

acccgcggcc tgaaaggtgt gcgcggtacg cgtcctgaag atattgtgaa taatctgtac 420

atgttcgtgc agccgtatgt ttgggttatg gaaccggata tgcagcgtgt gggtggcagt 480

gcagtggttg caagaaccgt gcgtcgcgtt cctcctggtg caattgatcc gaccgttaaa 540

aatctgcagt ggggtgacct ggttcgcggt atgtttgaag ccgccgatcg tggtgccacc 600

tatccttttc tgaccgatgg tgacgcacat ctgaccgaag gcagcggttt taatattgtg 660

ctggttaaag acggcgtgct gtataccccg gatcgcggtg tgttacaggg cgtgaccaga 720

aaaagtgtta ttaatgcagc cgaggccttt ggcattgaag tgcgtgtgga atttgtgccg 780

gtggaactgg cctatcgctg cgacgagatt tttatgtgca ccaccgccgg tggtattatg 840

ccgattacca ccctggatgg tatgccggtg aatggcggtc agattggccc tattaccaaa 900

aagatttggg acggttactg ggccatgcat tatgatgcag catatagctt tgagatcgac 960

tataacgagc gtaat 975

<210> 22

<211> 325

<212> PRT

<213> Aspergillus terreus

<400> 22

Met Ala Ser Met Asp Lys Val Phe Ala Gly Tyr Ala Ala Arg Gln Ala

1 5 10 15

Ile Leu Glu Ser Thr Glu Thr Thr Asn Pro Phe Ala Lys Gly Ile Ala

20 25 30

Trp Val Glu Gly Glu Leu Val Pro Leu Ala Glu Ala Arg Ile Pro Leu

35 40 45

Leu Asp Gln Gly Phe Met His Ser Asp Leu Thr Tyr Asp Val Pro Ser

50 55 60

Val Trp Asp Gly Arg Phe Phe Arg Leu Asp Asp His Ile Thr Arg Leu

65 70 75 80

Glu Ala Ser Cys Thr Lys Leu Arg Leu Arg Leu Pro Leu Pro Arg Asp

85 90 95

Gln Val Lys Gln Ile Leu Val Glu Met Val Ala Lys Ser Gly Ile Arg

100 105 110

Asp Ala Phe Val Glu Leu Ile Val Thr Arg Gly Leu Lys Gly Val Arg

115 120 125

Gly Thr Arg Pro Glu Asp Ile Val Asn Asn Leu Tyr Met Phe Val Gln

130 135 140

Pro Tyr Val Trp Val Met Glu Pro Asp Met Gln Arg Val Gly Gly Ser

145 150 155 160

Ala Val Val Ala Arg Thr Val Arg Arg Val Pro Pro Gly Ala Ile Asp

165 170 175

Pro Thr Val Lys Asn Leu Gln Trp Gly Asp Leu Val Arg Gly Met Phe

180 185 190

Glu Ala Ala Asp Arg Gly Ala Thr Tyr Pro Phe Leu Thr Asp Gly Asp

195 200 205

Ala His Leu Thr Glu Gly Ser Gly Phe Asn Ile Val Leu Val Lys Asp

210 215 220

Gly Val Leu Tyr Thr Pro Asp Arg Gly Val Leu Gln Gly Val Thr Arg

225 230 235 240

Lys Ser Val Ile Asn Ala Ala Glu Ala Phe Gly Ile Glu Val Arg Val

245 250 255

Glu Phe Val Pro Val Glu Leu Ala Tyr Arg Cys Asp Glu Ile Phe Met

260 265 270

Cys Thr Thr Ala Gly Gly Ile Met Pro Ile Thr Thr Leu Asp Gly Met

275 280 285

Pro Val Asn Gly Gly Gln Ile Gly Pro Ile Thr Lys Lys Ile Trp Asp

290 295 300

Gly Tyr Trp Ala Met His Tyr Asp Ala Ala Tyr Ser Phe Glu Ile Asp

305 310 315 320

Tyr Asn Glu Arg Asn

325

<210> 23

<211> 969

<212> DNA

<213> Artificial sequences

<220>

<223>

<400> 23

atggcaagta tggataaggt gttcagtggt tattacgccc gccagaaact gctggaacgt 60

agcgataatc cgtttagcaa aggtattgca tacgtggaag gcaaactggt tctgccgagc 120

gatgcacgta ttccgctgtt agatgaaggt tttatgcaca gtgacctgac ctatgatgtt 180

attagcgtgt gggatggtcg ctttttccgc ctggatgatc atctgcagcg tattctggaa 240

agttgtgata aaatgcgcct gaaattcccg ctggccctgt caagtgttaa aaatattctg 300

gcagagatgg tggccaaaag tggcattcgt gatgcatttg ttgaggttat tgtgacccgc 360

ggtctgaccg gtgtgagagg tagcaaaccg gaagatttgt ataacaacaa catctacctg 420

ctggtgctgc cgtatatttg ggttatggca ccggaaaatc agctgcatgg tggtgaagcc 480

attattaccc gcaccgtgcg ccgtacaccg cctggtgcat tcgaccctac aattaagaat 540

ctgcagtggg gtgacctgac caaaggtctg tttgaagcaa tggatcgcgg cgccacctat 600

ccttttctga ccgatggtga caccaatctg accgaaggta gcggttttaa tatcgttctg 660

gttaagaacg gcatcatcta taccccggat cgtggtgtgc tgcgtggtat tacccgcaaa 720

agtgtgattg atgttgcacg tgcaaacagt attgacattc gtctggaagt tgtgccggtt 780

gaacaggcct atcatagcga tgaaattttc atgtgcacca ccgccggtgg cattatgcct 840

attaccctgc tggatggcca gccggttaat gatggtcagg tgggtccgat taccaaaaag 900

atttgggatg gttactggga aatgcattac aatccggcat atagcttccc ggtggattat 960

ggtagtggt 969

<210> 24

<211> 323

<212> PRT

<213> Aspergillus fumigatus

<400> 24

Met Ala Ser Met Asp Lys Val Phe Ser Gly Tyr Tyr Ala Arg Gln Lys

1 5 10 15

Leu Leu Glu Arg Ser Asp Asn Pro Phe Ser Lys Gly Ile Ala Tyr Val

20 25 30

Glu Gly Lys Leu Val Leu Pro Ser Asp Ala Arg Ile Pro Leu Leu Asp

35 40 45

Glu Gly Phe Met His Ser Asp Leu Thr Tyr Asp Val Ile Ser Val Trp

50 55 60

Asp Gly Arg Phe Phe Arg Leu Asp Asp His Leu Gln Arg Ile Leu Glu

65 70 75 80

Ser Cys Asp Lys Met Arg Leu Lys Phe Pro Leu Ala Leu Ser Ser Val

85 90 95

Lys Asn Ile Leu Ala Glu Met Val Ala Lys Ser Gly Ile Arg Asp Ala

100 105 110

Phe Val Glu Val Ile Val Thr Arg Gly Leu Thr Gly Val Arg Gly Ser

115 120 125

Lys Pro Glu Asp Leu Tyr Asn Asn Asn Ile Tyr Leu Leu Val Leu Pro

130 135 140

Tyr Ile Trp Val Met Ala Pro Glu Asn Gln Leu His Gly Gly Glu Ala

145 150 155 160

Ile Ile Thr Arg Thr Val Arg Arg Thr Pro Pro Gly Ala Phe Asp Pro

165 170 175

Thr Ile Lys Asn Leu Gln Trp Gly Asp Leu Thr Lys Gly Leu Phe Glu

180 185 190

Ala Met Asp Arg Gly Ala Thr Tyr Pro Phe Leu Thr Asp Gly Asp Thr

195 200 205

Asn Leu Thr Glu Gly Ser Gly Phe Asn Ile Val Leu Val Lys Asn Gly

210 215 220

Ile Ile Tyr Thr Pro Asp Arg Gly Val Leu Arg Gly Ile Thr Arg Lys

225 230 235 240

Ser Val Ile Asp Val Ala Arg Ala Asn Ser Ile Asp Ile Arg Leu Glu

245 250 255

Val Val Pro Val Glu Gln Ala Tyr His Ser Asp Glu Ile Phe Met Cys

260 265 270

Thr Thr Ala Gly Gly Ile Met Pro Ile Thr Leu Leu Asp Gly Gln Pro

275 280 285

Val Asn Asp Gly Gln Val Gly Pro Ile Thr Lys Lys Ile Trp Asp Gly

290 295 300

Tyr Trp Glu Met His Tyr Asn Pro Ala Tyr Ser Phe Pro Val Asp Tyr

305 310 315 320

Gly Ser Gly

<210> 25

<211> 969

<212> DNA

<213> Artificial sequences

<220>

<223>

<400> 25

atggctagta tggataaggt gttcagcggc tatcatgccc gccagaaact gctggaacgt 60

agtgataatc cgtttagtaa gggcattgcc tatgtggaag gtaaactggt gctgccgagt 120

gatgcccgta ttcctctgct ggatgaaggc tttatgcatg gtgacctgac ctatgatgtt 180

accaccgtgt gggatggtcg ctttttccgt ctggatgatc acatgcagcg tattctggaa 240

agctgcgata aaatgcgtct gaaattcccg ctggccccga gtacagttaa aaatattctg 300

gcagagatgg tggcaaagag cggcattcgc gatgcctttg ttgaagtgat tgttacccgt 360

ggtctgaccg gtgttcgtgg tagtaaaccg gaagatttgt ataacaacaa catctacctg 420

ctggtgctgc cttatgtgtg ggttatggca ccggaaaatc agctgctggg cggttcagca 480

attattaccc gcaccgtgcg ccgtacccct cctggtgcat tcgaccctac aattaagaat 540

ctgcagtggg gcgatctgac caaaggctta tttgaagcaa tggatcgcgg cgccacctat 600

ccttttctga ccgatggtga caccaatctg accgaaggta gcggctttaa tattgttctg 660

gtgaaaaacg gcatcatcta caccccggat cgcggtgttc tgcgtggtat tacccgcaaa 720

agtgttattg atgtggcccg cgcaaataat attgatattc gtctggaggt ggtgccggtt 780

gaacaggttt atcatagtga tgaaatcttc atgtgcacca ccgccggcgg tattatgcct 840

attaccctgc tggatggtca gccggttaat gatggtcagg ttggcccgat taccaaaaag 900

atttgggatg gctattggga aatgcattac aatccggcat acagctttcc ggttgattat 960

ggtagcggc 969

<210> 26

<211> 323

<212> PRT

<213> Neosartorya fischeri

<400> 26

Met Ala Ser Met Asp Lys Val Phe Ser Gly Tyr His Ala Arg Gln Lys

1 5 10 15

Leu Leu Glu Arg Ser Asp Asn Pro Phe Ser Lys Gly Ile Ala Tyr Val

20 25 30

Glu Gly Lys Leu Val Leu Pro Ser Asp Ala Arg Ile Pro Leu Leu Asp

35 40 45

Glu Gly Phe Met His Gly Asp Leu Thr Tyr Asp Val Thr Thr Val Trp

50 55 60

Asp Gly Arg Phe Phe Arg Leu Asp Asp His Met Gln Arg Ile Leu Glu

65 70 75 80

Ser Cys Asp Lys Met Arg Leu Lys Phe Pro Leu Ala Pro Ser Thr Val

85 90 95

Lys Asn Ile Leu Ala Glu Met Val Ala Lys Ser Gly Ile Arg Asp Ala

100 105 110

Phe Val Glu Val Ile Val Thr Arg Gly Leu Thr Gly Val Arg Gly Ser

115 120 125

Lys Pro Glu Asp Leu Tyr Asn Asn Asn Ile Tyr Leu Leu Val Leu Pro

130 135 140

Tyr Val Trp Val Met Ala Pro Glu Asn Gln Leu Leu Gly Gly Ser Ala

145 150 155 160

Ile Ile Thr Arg Thr Val Arg Arg Thr Pro Pro Gly Ala Phe Asp Pro

165 170 175

Thr Ile Lys Asn Leu Gln Trp Gly Asp Leu Thr Lys Gly Leu Phe Glu

180 185 190

Ala Met Asp Arg Gly Ala Thr Tyr Pro Phe Leu Thr Asp Gly Asp Thr

195 200 205

Asn Leu Thr Glu Gly Ser Gly Phe Asn Ile Val Leu Val Lys Asn Gly

210 215 220

Ile Ile Tyr Thr Pro Asp Arg Gly Val Leu Arg Gly Ile Thr Arg Lys

225 230 235 240

Ser Val Ile Asp Val Ala Arg Ala Asn Asn Ile Asp Ile Arg Leu Glu

245 250 255

Val Val Pro Val Glu Gln Val Tyr His Ser Asp Glu Ile Phe Met Cys

260 265 270

Thr Thr Ala Gly Gly Ile Met Pro Ile Thr Leu Leu Asp Gly Gln Pro

275 280 285

Val Asn Asp Gly Gln Val Gly Pro Ile Thr Lys Lys Ile Trp Asp Gly

290 295 300

Tyr Trp Glu Met His Tyr Asn Pro Ala Tyr Ser Phe Pro Val Asp Tyr

305 310 315 320

Gly Ser Gly

<210> 27

<211> 975

<212> DNA

<213> Artificial sequences

<220>

<223>

<400> 27

atgagcacaa tggataagat ttttgcaggc catgcacagc gccaggcaac attagtggcc 60

agtgataata ttttcgcgaa cggcattgca tggattcagg gtgaactggt tccgctgaat 120

gaagcacgta ttccgctgat ggatcagggc tttatgcatg gtgacctgac ctatgatgtt 180

ccggcagttt gggatggccg ctttttccgt ctggatgatc atctggatcg cctggaagca 240

agtgttaaaa agatgcgtat gcagttcccg attccgcgtg atgaaattcg catgaccctg 300

ctggatatgc tggcaaaaag tggcattaag gatgcctttg tggaactgat tgtgacccgc 360

ggtctgaaac cggtgcgtga ggcaaaaccg ggtgaagttc tgaataatca tctgtatctg 420

atcgtgcagc cgtatgtttg ggttatgagc ccggaagccc agtatgttgg cggtaatgcc 480

gtgattgcac gcaccgttcg tcgtattccg ccgggtagca tggaccctac aattaagaat 540

ctgcagtgga gcgatttcac ccgcggcatg tttgaagcct atgatcgcgg cgcccagtat 600

ccttttctga ccgatggcga taccaatatt accgaaggta gcggttttaa cgtggttttt 660

gttaagaaca acgtgatcta caccccgaat cgtggtgttc tgcagggtat tacccgtaaa 720

agtgttattg acgccgcaaa atggtgtggt catgaagtgc gtgtggaata tgttccggtt 780

gaaatggcct atgaagcaga tgaaatcttc atgtgcacca ccgcaggcgg cattatgcct 840

attaccacaa tggatggtaa accggtgaaa gatggtaaag tgggtccggt taccaaagca 900

atttgggatc gttattgggc catgcattgg gaagatgaat tttcattcaa gatcgactac 960

cagaagctga aactg 975

<210> 28

<211> 325

<212> PRT

<213> Gibberella zeae

<400> 28

Met Ser Thr Met Asp Lys Ile Phe Ala Gly His Ala Gln Arg Gln Ala

1 5 10 15

Thr Leu Val Ala Ser Asp Asn Ile Phe Ala Asn Gly Ile Ala Trp Ile

20 25 30

Gln Gly Glu Leu Val Pro Leu Asn Glu Ala Arg Ile Pro Leu Met Asp

35 40 45

Gln Gly Phe Met His Gly Asp Leu Thr Tyr Asp Val Pro Ala Val Trp

50 55 60

Asp Gly Arg Phe Phe Arg Leu Asp Asp His Leu Asp Arg Leu Glu Ala

65 70 75 80

Ser Val Lys Lys Met Arg Met Gln Phe Pro Ile Pro Arg Asp Glu Ile

85 90 95

Arg Met Thr Leu Leu Asp Met Leu Ala Lys Ser Gly Ile Lys Asp Ala

100 105 110

Phe Val Glu Leu Ile Val Thr Arg Gly Leu Lys Pro Val Arg Glu Ala

115 120 125

Lys Pro Gly Glu Val Leu Asn Asn His Leu Tyr Leu Ile Val Gln Pro

130 135 140

Tyr Val Trp Val Met Ser Pro Glu Ala Gln Tyr Val Gly Gly Asn Ala

145 150 155 160

Val Ile Ala Arg Thr Val Arg Arg Ile Pro Pro Gly Ser Met Asp Pro

165 170 175

Thr Ile Lys Asn Leu Gln Trp Ser Asp Phe Thr Arg Gly Met Phe Glu

180 185 190

Ala Tyr Asp Arg Gly Ala Gln Tyr Pro Phe Leu Thr Asp Gly Asp Thr

195 200 205

Asn Ile Thr Glu Gly Ser Gly Phe Asn Val Val Phe Val Lys Asn Asn

210 215 220

Val Ile Tyr Thr Pro Asn Arg Gly Val Leu Gln Gly Ile Thr Arg Lys

225 230 235 240

Ser Val Ile Asp Ala Ala Lys Trp Cys Gly His Glu Val Arg Val Glu

245 250 255

Tyr Val Pro Val Glu Met Ala Tyr Glu Ala Asp Glu Ile Phe Met Cys

260 265 270

Thr Thr Ala Gly Gly Ile Met Pro Ile Thr Thr Met Asp Gly Lys Pro

275 280 285

Val Lys Asp Gly Lys Val Gly Pro Val Thr Lys Ala Ile Trp Asp Arg

290 295 300

Tyr Trp Ala Met His Trp Glu Asp Glu Phe Ser Phe Lys Ile Asp Tyr

305 310 315 320

Gln Lys Leu Lys Leu

325

<210> 29

<211> 1011

<212> DNA

<213> Artificial sequences

<220>

<223>

<400> 29

atgggtatcg acaccggtac aagcaatctg gtggccgtgg aaccgggtgc aattagagaa 60

gataccccgg ccggtagcgt gattcagtat agcgattatg aaatcgacta cagcagcccg 120

tttgcaggtg gtgtggcttg gattgaaggc gaatatctgc cggccgaaga tgccaaaatt 180

agcatttttg acaccggttt cggccatagc gatctgacct ataccgttgc acatgtttgg 240

catggcaata ttttccgcct gggcgatcat ctggatcgtc tgttagatgg cgcacgtaaa 300

ctgcgtctgg atagtggcta taccaaagat gaactggcag atattaccaa gaagtgcgtg 360

agcctgagcc agctgcgtga atcatttgtg aatctgacca ttacccgcgg ttatggtaaa 420

cgcaaaggtg aaaaagacct gagtaagctg acccatcagg tgtatatcta tgccattccg 480

tatctgtggg cctttccgcc tgccgagcaa atttttggca ccaccgccgt tgtgccgcgt 540

cacgtgcgtc gtgcaggtcg taacacagtt gatccgacca ttaagaatta ccagtggggt 600

gacctgaccg cagccagctt cgaggcaaaa gatcgcggtg ctcgcaccgc aattctgatg 660

gatgccgata attgtgtggc agaaggcccg ggttttaatg tgtgcattgt taaagacggc 720

aagctggcaa gcccgagtcg taatgcactg cctggtatta cccgtaaaac cgtgtttgaa 780

atcgccggtg caatgggcat tgaagccgca ttacgtgatg ttaccagtca tgaactgtac 840

gatgcagatg aaatcatggc agttaccacc gccggcggtg ttacacctat taataccctg 900

gatggcgttc cgattggtga cggtgaaccg ggtcctgtta ccgttgctat tcgtgatcgc 960

ttttgggcac tgatggatga accgggtccg ttaattgaag ccattcagta t 1011

<210> 30

<211> 337

<212> PRT

<213> Mycobacterium vanbaalenii

<400>30

Met Gly Ile Asp Thr Gly Thr Ser Asn Leu Val Ala Val Glu Pro Gly

1 5 10 15

Ala Ile Arg Glu Asp Thr Pro Ala Gly Ser Val Ile Gln Tyr Ser Asp

20 25 30

Tyr Glu Ile Asp Tyr Ser Ser Pro Phe Ala Gly Gly Val Ala Trp Ile

35 40 45

Glu Gly Glu Tyr Leu Pro Ala Glu Asp Ala Lys Ile Ser Ile Phe Asp

50 55 60

Thr Gly Phe Gly His Ser Asp Leu Thr Tyr Thr Val Ala His Val Trp

65 70 75 80

His Gly Asn Ile Phe Arg Leu Gly Asp His Leu Asp Arg Leu Leu Asp

85 90 95

Gly Ala Arg Lys Leu Arg Leu Asp Ser Gly Tyr Thr Lys Asp Glu Leu

100 105 110

Ala Asp Ile Thr Lys Lys Cys Val Ser Leu Ser Gln Leu Arg Glu Ser

115 120 125

Phe Val Asn Leu Thr Ile Thr Arg Gly Tyr Gly Lys Arg Lys Gly Glu

130 135 140

Lys Asp Leu Ser Lys Leu Thr His Gln Val Tyr Ile Tyr Ala Ile Pro

145 150 155 160

Tyr Leu Trp Ala Phe Pro Pro Ala Glu Gln Ile Phe Gly Thr Thr Ala

165 170 175

Val Val Pro Arg His Val Arg Arg Ala Gly Arg Asn Thr Val Asp Pro

180 185 190

Thr Ile Lys Asn Tyr Gln Trp Gly Asp Leu Thr Ala Ala Ser Phe Glu

195 200 205

Ala Lys Asp Arg Gly Ala Arg Thr Ala Ile Leu Met Asp Ala Asp Asn

210 215 220

Cys Val Ala Glu Gly Pro Gly Phe Asn Val Cys Ile Val Lys Asp Gly

225 230 235 240

Lys Leu Ala Ser Pro Ser Arg Asn Ala Leu Pro Gly Ile Thr Arg Lys

245 250 255

Thr Val Phe Glu Ile Ala Gly Ala Met Gly Ile Glu Ala Ala Leu Arg

260 265 270

Asp Val Thr Ser His Glu Leu Tyr Asp Ala Asp Glu Ile Met Ala Val

275 280 285

Thr Thr Ala Gly Gly Val Thr Pro Ile Asn Thr Leu Asp Gly Val Pro

290 295 300

Ile Gly Asp Gly Glu Pro Gly Pro Val Thr Val Ala Ile Arg Asp Arg

305 310 315 320

Phe Trp Ala Leu Met Asp Glu Pro Gly Pro Leu Ile Glu Ala Ile Gln

325 330 335

Tyr

<210> 31

<211> 1428

<212> DNA

<213> Artificial sequences

<220>

<223>

<400> 31

atgagcctga ccgtgcaaaa aattaactgg gaacaggtta aggagtggga tcgtaaatat 60

ctgatgcgta cctttagcac ccagaatgaa tatcagccgg ttccgattga aagtaccgaa 120

ggcgattatc tgatcatgcc ggatggtaca cgcctgctgg atttctttaa tcagctgtat 180

tgcgtgaacc tgggtcagaa aaatcagaaa gttaacgcag ccatcaagga agcactggat 240

cgctatggct ttgtttggga tacctatgcc accgattata aagccaaagc agcaaaaatc 300

atcatcgagg atattctggg tgacgaagat tggccgggca aagtgcgttt tgtgagtacc 360

ggcagcgaag ccgtggaaac agctttaaat attgcacgcc tgtacaccaa tcgcccgctg 420

gtggtgacac gtgaacatga ttatcatggc tggaccggcg gcgcagcaac cgtgacccgt 480

ctgcgtagct atcgtagcgg tctggtgggt gaaaatagcg aaagttttag tgcccagatc 540

ccgggcagta gctataatag cgcagtgctg atggccccga gccctaacat gtttcaggat 600

agcgatggta atctgctgaa agatgaaaac ggcgaactgc tgagcgttaa atatacccgc 660

cgcatgattg aaaactacgg tccggaacag gtggcagcag ttattaccga agttagccag 720

ggtgccggta gtgctatgcc tccttatgaa tatatcccgc agattcgcaa aatgaccaaa 780

gaactgggcg tgctgtggat taatgatgaa gtgctgaccg gttttggccg caccggtaaa 840

tggtttggtt atcagcatta cggtgtgcag ccggatatta ttacaatggg taaaggtctg 900

agcagcagca gtctgccggc tggtgcagtg ttagtgagca aagaaattgc agcattcatg 960

gataagcacc gttgggaaag cgtgagtacc tatgccggtc atccggttgc aatggctgcc 1020

gtgtgtgcaa atctggaagt gatgatggaa gaaaacttcg ttgagcaggc aaaagatagt 1080

ggtgaatata tccgtagcaa gctggaactg ctgcaggaaa aacataaaag catcggtaac 1140

ttcgacggct atggcctgct gtggattgtt gatattgtta atgccaagac caagaccccg 1200

tatgttaaac tggatcgcaa ttttacccac ggtatgaatc cgaatcagat tccgacccag 1260

attattatga agaaggccct ggaaaagggc gtgctgattg gtggtgtgat gccgaatacc 1320

atgcgcattg gtgcaagcct gaatgtgagt cgcggcgata ttgataaagc aatggatgca 1380

ctggactacg ccctggatta tctggaaagt ggtgaatggc agcagagc 1428

<210> 32

<211> 476

<212> PRT

<213> Bacillus megaterium

<400> 32

Met Ser Leu Thr Val Gln Lys Ile Asn Trp Glu Gln Val Lys Glu Trp

1 5 10 15

Asp Arg Lys Tyr Leu Met Arg Thr Phe Ser Thr Gln Asn Glu Tyr Gln

20 25 30

Pro Val Pro Ile Glu Ser Thr Glu Gly Asp Tyr Leu Ile Met Pro Asp

35 40 45

Gly Thr Arg Leu Leu Asp Phe Phe Asn Gln Leu Tyr Cys Val Asn Leu

50 55 60

Gly Gln Lys Asn Gln Lys Val Asn Ala Ala Ile Lys Glu Ala Leu Asp

65 70 75 80

Arg Tyr Gly Phe Val Trp Asp Thr Tyr Ala Thr Asp Tyr Lys Ala Lys

85 90 95

Ala Ala Lys Ile Ile Ile Glu Asp Ile Leu Gly Asp Glu Asp Trp Pro

100 105 110

Gly Lys Val Arg Phe Val Ser Thr Gly Ser Glu Ala Val Glu Thr Ala

115 120 125

Leu Asn Ile Ala Arg Leu Tyr Thr Asn Arg Pro Leu Val Val Thr Arg

130 135 140

Glu His Asp Tyr His Gly Trp Thr Gly Gly Ala Ala Thr Val Thr Arg

145 150 155 160

Leu Arg Ser Tyr Arg Ser Gly Leu Val Gly Glu Asn Ser Glu Ser Phe

165 170 175

Ser Ala Gln Ile Pro Gly Ser Ser Tyr Asn Ser Ala Val Leu Met Ala

180 185 190

Pro Ser Pro Asn Met Phe Gln Asp Ser Asp Gly Asn Leu Leu Lys Asp

195 200 205

Glu Asn Gly Glu Leu Leu Ser Val Lys Tyr Thr Arg Arg Met Ile Glu

210 215 220

Asn Tyr Gly Pro Glu Gln Val Ala Ala Val Ile Thr Glu Val Ser Gln

225 230 235 240

Gly Ala Gly Ser Ala Met Pro Pro Tyr Glu Tyr Ile Pro Gln Ile Arg

245 250 255

Lys Met Thr Lys Glu Leu Gly Val Leu Trp Ile Asn Asp Glu Val Leu

260 265 270

Thr Gly Phe Gly Arg Thr Gly Lys Trp Phe Gly Tyr Gln His Tyr Gly

275 280 285

Val Gln Pro Asp Ile Ile Thr Met Gly Lys Gly Leu Ser Ser Ser Ser

290 295 300

Leu Pro Ala Gly Ala Val Leu Val Ser Lys Glu Ile Ala Ala Phe Met

305 310 315 320

Asp Lys His Arg Trp Glu Ser Val Ser Thr Tyr Ala Gly His Pro Val

325 330 335

Ala Met Ala Ala Val Cys Ala Asn Leu Glu Val Met Met Glu Glu Asn

340 345 350

Phe Val Glu Gln Ala Lys Asp Ser Gly Glu Tyr Ile Arg Ser Lys Leu

355 360 365

Glu Leu Leu Gln Glu Lys His Lys Ser Ile Gly Asn Phe Asp Gly Tyr

370 375 380

Gly Leu Leu Trp Ile Val Asp Ile Val Asn Ala Lys Thr Lys Thr Pro

385 390 395 400

Tyr Val Lys Leu Asp Arg Asn Phe Thr His Gly Met Asn Pro Asn Gln

405 410 415

Ile Pro Thr Gln Ile Ile Met Lys Lys Ala Leu Glu Lys Gly Val Leu

420 425 430

Ile Gly Gly Val Met Pro Asn Thr Met Arg Ile Gly Ala Ser Leu Asn

435 440 445

Val Ser Arg Gly Asp Ile Asp Lys Ala Met Asp Ala Leu Asp Tyr Ala

450 455 460

Leu Asp Tyr Leu Glu Ser Gly Glu Trp Gln Gln Ser

465 470 475

<210> 33

<211> 1344

<212> DNA

<213> Artificial sequences

<220>

<223>

<400> 33

atgaaccagc cgctgaatgt ggccccgccg gttagcagcg aactgaatct gcgtgcccat 60

tggatgccgt ttagcgcaaa tcgtaatttt cagaaagatc cgcgtattat tgttgccgca 120

gaaggtagtt ggctgaccga tgataaaggc cgcaaagtgt atgatagtct gagtggcctg 180

tggacctgcg gtgcaggcca tagccgtaaa gaaattcagg aagcagtggc acgccagctg 240

ggcaccctgg attatagccc gggttttcag tatggccatc cgctgagttt tcagctggca 300

gaaaaaattg ccggtctgct gccgggtgaa ctgaatcatg ttttctttac cggtagtggc 360

agcgaatgcg ccgataccag cattaagatg gcccgtgcat attggcgcct gaaaggtcag 420

ccgcagaaaa ccaaactgat tggccgtgca cgcggttatc atggcgtgaa tgttgccggc 480

accagcctgg gcggcattgg tggtaatcgc aaaatgtttg gtcagctgat ggatgtggat 540

catctgccgc ataccctgca gccgggcatg gcattcactc gtggtatggc acagaccggc 600

ggcgttgaac tggcaaatga actgctgaaa ctgattgaac tgcatgatgc cagtaatatt 660

gccgcagtga ttgtggaacc gatgagtggc agtgcaggtg ttctggtgcc gccggtgggt 720

tatctgcagc gtctgcgtga aatttgtgat cagcataata ttctgctgat ttttgatgaa 780

gtgatcaccg catttggccg tctgggtaca tatagcggtg ccgaatattt tggtgtgacc 840

ccggatctga tgaatgtggc aaaacaggtg accaatggtg ccgtgccgat gggcgcagtt 900

attgcaagca gcgaaatcta tgataccttt atgaatcagg ccctgccgga acatgccgtg 960

gaattttctc atggttatac ctatagtgca catccggttg cctgtgccgc cggcctggca 1020

gcactggata ttctggcccg tgataatctg gtgcagcaga gtgcagaact ggcaccgcat 1080

tttgaaaaag gtctgcatgg tctgcagggc gccaaaaatg ttattgatat tcgtaattgc 1140

ggcctggccg gcgccattca gattgcaccg cgtgatggtg acccgaccgt tcgcccgttt 1200

gaagccggca tgaaactgtg gcagcagggt ttttatgtgc gctttggcgg cgataccctg 1260

cagtttggtc cgacctttaa tgcacgcccg gaagaactgg atcgcctgtt tgatgcagtg 1320

ggtgaagcac tgaatggtat tgcc 1344

<210> 34

<211> 448

<212> PRT

<213> Pseudomonas aeruginosa (P. aeruginosa)

<400> 34

Met Asn Gln Pro Leu Asn Val Ala Pro Pro Val Ser Ser Glu Leu Asn

1 5 10 15

Leu Arg Ala His Trp Met Pro Phe Ser Ala Asn Arg Asn Phe Gln Lys

20 25 30

Asp Pro Arg Ile Ile Val Ala Ala Glu Gly Ser Trp Leu Thr Asp Asp

35 40 45

Lys Gly Arg Lys Val Tyr Asp Ser Leu Ser Gly Leu Trp Thr Cys Gly

50 55 60

Ala Gly His Ser Arg Lys Glu Ile Gln Glu Ala Val Ala Arg Gln Leu

65 70 75 80

Gly Thr Leu Asp Tyr Ser Pro Gly Phe Gln Tyr Gly His Pro Leu Ser

85 90 95

Phe Gln Leu Ala Glu Lys Ile Ala Gly Leu Leu Pro Gly Glu Leu Asn

100 105 110

His Val Phe Phe Thr Gly Ser Gly Ser Glu Cys Ala Asp Thr Ser Ile

115 120 125

Lys Met Ala Arg Ala Tyr Trp Arg Leu Lys Gly Gln Pro Gln Lys Thr

130 135 140

Lys Leu Ile Gly Arg Ala Arg Gly Tyr His Gly Val Asn Val Ala Gly

145 150 155 160

Thr Ser Leu Gly Gly Ile Gly Gly Asn Arg Lys Met Phe Gly Gln Leu

165 170 175

Met Asp Val Asp His Leu Pro His Thr Leu Gln Pro Gly Met Ala Phe

180 185 190

Thr Arg Gly Met Ala Gln Thr Gly Gly Val Glu Leu Ala Asn Glu Leu

195 200 205

Leu Lys Leu Ile Glu Leu His Asp Ala Ser Asn Ile Ala Ala Val Ile

210 215 220

Val Glu Pro Met Ser Gly Ser Ala Gly Val Leu Val Pro Pro Val Gly

225 230 235 240

Tyr Leu Gln Arg Leu Arg Glu Ile Cys Asp Gln His Asn Ile Leu Leu

245 250 255

Ile Phe Asp Glu Val Ile Thr Ala Phe Gly Arg Leu Gly Thr Tyr Ser

260 265 270

Gly Ala Glu Tyr Phe Gly Val Thr Pro Asp Leu Met Asn Val Ala Lys

275 280 285

Gln Val Thr Asn Gly Ala Val Pro Met Gly Ala Val Ile Ala Ser Ser

290 295 300

Glu Ile Tyr Asp Thr Phe Met Asn Gln Ala Leu Pro Glu His Ala Val

305 310 315 320

Glu Phe Ser His Gly Tyr Thr Tyr Ser Ala His Pro Val Ala Cys Ala

325 330 335

Ala Gly Leu Ala Ala Leu Asp Ile Leu Ala Arg Asp Asn Leu Val Gln

340 345 350

Gln Ser Ala Glu Leu Ala Pro His Phe Glu Lys Gly Leu His Gly Leu

355 360 365

Gln Gly Ala Lys Asn Val Ile Asp Ile Arg Asn Cys Gly Leu Ala Gly

370 375 380

Ala Ile Gln Ile Ala Pro Arg Asp Gly Asp Pro Thr Val Arg Pro Phe

385 390 395 400

Glu Ala Gly Met Lys Leu Trp Gln Gln Gly Phe Tyr Val Arg Phe Gly

405 410 415

Gly Asp Thr Leu Gln Phe Gly Pro Thr Phe Asn Ala Arg Pro Glu Glu

420 425 430

Leu Asp Arg Leu Phe Asp Ala Val Gly Glu Ala Leu Asn Gly Ile Ala

435 440 445

Claims (16)

1. a synthetic method of chiral 2-amino-1-butanol, comprises the steps: (A) Using 1,2-butanediol as a substrate, 2-keto-1-butanol is generated by the catalytic reaction of enzyme A and its coenzyme; (B) Using the 2-keto-1-butanol generated in step (A) as a substrate, the chiral 2-amino-1-butanol is generated by the catalytic reaction of enzyme B and its coenzyme; The enzyme A and the enzyme B are any of the following three combinations: The first combination: The enzyme A is selected from any of the following: (a1) alcohol dehydrogenase derived from Lactobacillus brevis , the amino acid sequence is SEQ ID No. 2; (a2) alcohol dehydrogenase derived from Bacillus stearothermophilus ( Geobacillus stearothermophilus ), the amino acid sequence is SEQ ID No.4; (a3) Alcohol dehydrogenase derived from Thermoanaerobacter brockii , the amino acid sequence is SEQ ID No.6; (a4) an alcohol dehydrogenase derived from Lactobacillus kefiri , the amino acid sequence of which is SEQ ID No.8; (a5) alcohol dehydrogenase derived from Bacillaceae , the amino acid sequence is SEQ ID No.10; (a6) an alcohol dehydrogenase derived from Leifsonia sp., the amino acid sequence of which is SEQ ID No. 12; (a7) Alcohol dehydrogenase derived from Thermoanaerobacter wiegelii , the amino acid sequence is SEQ ID No. 14; (a8) Compared with the alcohol dehydrogenase derived from Lactobacillus pumilus shown in SEQ ID No. 2, only the following mutation I11V exists; (a9) Compared with the alcohol dehydrogenase derived from Lactobacillus pumilus shown in SEQ ID No. 2, only the following mutation exists: G37D; (a10) A carbonyl reductase derived from Spora sp. ochra, the amino acid sequence is SEQ ID No. 16; (a11) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of any of the proteins defined in (a1)-(a10); The enzyme B is selected from any of the following: (b1) Leucine dehydrogenase derived from Bacillus stearothermophilus, the amino acid sequence is SEQ ID No. 18; (b2) Compared with the leucine dehydrogenase derived from Bacillus stearothermophilus shown in SEQ ID No. 18, there are only the following mutations: K68S/N261L; (b3) Compared with the leucine dehydrogenase derived from Bacillus stearothermophilus shown in SEQ ID No. 18, only the following mutation exists: K68Y/N261C; (b4) ATA-117 transaminase from Codexis, the amino acid sequence is SEQ ID No.20; (b5) A transaminase derived from Aspergillus terreus , the amino acid sequence is SEQ ID No.22; (b6) A transaminase derived from Aspergillus fumigatus , the amino acid sequence is SEQ ID No. 24; (b7) A transaminase derived from Neosartorya fischeri , the amino acid sequence is SEQ ID No. 26; (b8) A transaminase derived from Gibberella zeae , the amino acid sequence is SEQ ID No.28; (b9) a transaminase derived from Mycobacterium vanbaalenii , the amino acid sequence is SEQ ID No. 30; (b10) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of any of the proteins defined in (b1)-(b9); The second combination: The enzyme A is selected from any of the following: (a1) alcohol dehydrogenase derived from Lactobacillus brevis , the amino acid sequence is SEQ ID No. 2; (a2) alcohol dehydrogenase derived from Bacillus stearothermophilus ( Geobacillus stearothermophilus ), the amino acid sequence is SEQ ID No.4; (a3) Alcohol dehydrogenase derived from Thermoanaerobacter brockii , the amino acid sequence is SEQ ID No.6; (a4) an alcohol dehydrogenase derived from Lactobacillus kefiri , the amino acid sequence of which is SEQ ID No.8; (a5) alcohol dehydrogenase derived from Bacillaceae , the amino acid sequence is SEQ ID No.10; (a6) an alcohol dehydrogenase derived from Leifsonia sp., the amino acid sequence of which is SEQ ID No. 12; (a8) Compared with the alcohol dehydrogenase derived from Lactobacillus pumilus shown in SEQ ID No. 2, only the following mutation I11V exists; (a9) Compared with the alcohol dehydrogenase derived from Lactobacillus pumilus shown in SEQ ID No. 2, only the following mutation exists: G37D; (a12) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the proteins as defined in any of (a1)-(a6) and (a8)-(a9); The enzyme B is selected from any of the following: (b9) a transaminase derived from Mycobacterium vanbaalenii , the amino acid sequence is SEQ ID No. 30; (b11) A transaminase derived from Bacillus megaterium , the amino acid sequence is SEQ ID No. 32; (b12) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein as defined in (b9) or (b11); The third combination: The enzyme A is selected from any of the following: (a7) Alcohol dehydrogenase derived from Thermoanaerobacter wiegelii , the amino acid sequence is SEQ ID No. 14; (a10) A carbonyl reductase derived from Spora sp. ochra, the amino acid sequence is SEQ ID No. 16; (a13) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein as defined in (a7) or (a10); The enzyme B is selected from any of the following: (b11) A transaminase derived from Bacillus megaterium , the amino acid sequence is SEQ ID No. 32; (b13) A transaminase derived from Pseudomonas aeruginosa ( P. aeruginosa ), the amino acid sequence is SEQ ID No. 34; (b14) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein as defined in (b11) or (b13); The chiral 2-amino-1-butanol that the enzyme in the first combination is used to synthesize is ( S )-2-amino-1-butanol; the second combination and the third The chiral 2-amino-1-butanol used for synthesis by the enzymes in this combination is ( R )-2-amino-1-butanol. 2. method according to claim 1 is characterized in that: in described method, described enzyme A and described enzyme B are crude enzyme liquid, crude enzyme liquid freeze-dried powder, pure enzyme or whole cell form of catalysis. 3. method according to claim 2 is characterized in that: described crude enzyme liquid, described crude enzyme liquid freeze-dried powder and described pure enzyme are all prepared according to the method comprising the following steps: in host cell, express all The enzyme A and/or the enzyme B are obtained to obtain recombinant cells; the recombinant cells are lysed to obtain the crude enzyme solution, the lyophilized powder of the crude enzyme solution or the pure enzyme. 4. The method according to claim 2, wherein the whole cell is prepared according to a method comprising the following steps: expressing the enzyme A and the enzyme B in the host cell, and the obtained recombinant cell is the whole cell. 5. method according to claim 4 is characterized in that: described recombinant cell is prepared according to the method comprising the following steps: import nucleic acid capable of expressing described enzyme A and described enzyme B into described host cell molecule, the recombinant cells expressing the enzyme A and the enzyme B are obtained after induction and culture. 6. The method according to claim 5, wherein: the nucleic acid molecules capable of expressing the enzyme A and the enzyme B are introduced into the host cell in the form of a recombinant vector; the recombinant vector is a carrier A bacterial plasmid, bacteriophage, yeast plasmid or retroviral packaging plasmid having the genes encoding said enzyme A and said enzyme B. 7. The method according to claim 5, wherein the host cell is a prokaryotic cell or a lower eukaryotic cell. 8. The method according to claim 7, wherein the prokaryotic cells are bacteria; the lower eukaryotic cells are yeast cells. 9. The method according to claim 8, wherein the bacteria are Escherichia coli. 10. method according to claim 1 is characterized in that: the sequence of the coding gene of the alcohol dehydrogenase derived from Lactobacillus pumilus is SEQ ID No.1 or at its 5' end and/or 3' end The fusion sequence obtained after connecting the tag coding sequence; The sequence of the coding gene of the alcohol dehydrogenase derived from Bacillus stearothermophilus is SEQ ID No.3 or the fusion sequence obtained after connecting the tag coding sequence at its 5' end and/or 3' end; The sequence of the encoding gene of the alcohol dehydrogenase derived from thermoanaerobic bacillus is SEQ ID No.5 or the fusion sequence obtained after connecting the tag encoding sequence at its 5' end and/or 3' end; The sequence of the coding gene of the alcohol dehydrogenase derived from Lactobacillus Caucasus yogurt is SEQ ID No.7 or the fusion sequence obtained after connecting the tag coding sequence at its 5' end and/or 3' end; The sequence of the coding gene of the alcohol dehydrogenase derived from Bacillus family is SEQ ID No.9 or the fusion sequence obtained after connecting the tag coding sequence at its 5' end and/or 3' end; The sequence of the coding gene of the alcohol dehydrogenase derived from the genus Lysella is SEQ ID No.11 or the fusion sequence obtained after connecting the tag coding sequence at its 5' end and/or 3' end; The sequence of the coding gene of the alcohol dehydrogenase derived from Thermoanaerobic virgiii is SEQ ID No.13 or the fusion sequence obtained after connecting the tag coding sequence at its 5' end and/or 3' end; The sequence of the encoding gene of the carbonyl reductase derived from Sporax ochra is SEQ ID No. 15 or the fusion sequence obtained after connecting the tag encoding sequence at its 5' end and/or 3' end; The sequence of the coding gene of the leucine dehydrogenase derived from Bacillus stearothermophilus is SEQ ID No.17 or the fusion sequence obtained after connecting the tag coding sequence at its 5' end and/or 3' end; The sequence of the coding gene of the ATA-117 transaminase of the Codexis company is SEQ ID No.19 or the fusion sequence obtained after connecting the tag coding sequence at its 5' end and/or 3' end; The sequence of the encoding gene of the transaminase derived from Aspergillus terreus is SEQ ID No.21 or the fusion sequence obtained after connecting the tag encoding sequence at its 5' end and/or 3' end; The sequence of the encoding gene of the transaminase derived from Aspergillus fumigatus is SEQ ID No.23 or the fusion sequence obtained after connecting the tag encoding sequence at its 5' end and/or 3' end; The sequence of the encoding gene of the transaminase derived from Neosartorius fischeri is SEQ ID No.25 or the fusion sequence obtained after connecting the tag encoding sequence at its 5' end and/or 3' end; The sequence of the coding gene of the transaminase derived from Gibberella zeae is SEQ ID No.27 or the fusion sequence obtained after connecting the tag coding sequence at its 5' end and/or 3' end; The sequence of the encoding gene of the transaminase derived from Mycobacterium is SEQ ID No.29 or the fusion sequence obtained after connecting the tag encoding sequence at its 5' end and/or 3' end; The sequence of the encoding gene of the transaminase derived from Bacillus megaterium is SEQ ID No.31 or the fusion sequence obtained after connecting the tag encoding sequence at its 5' end and/or 3' end; The sequence of the encoding gene of the transaminase derived from Pseudomonas aeruginosa is SEQ ID No.33 or the fusion sequence obtained after connecting the tag encoding sequence at its 5' end and/or 3' end; The sequence of the coding gene of the Lactobacillus brevis-derived alcohol dehydrogenase mutant is any one of the following (g1)-(g3): (g1) Compared with SEQ ID No.1, only the following mutations exist: A31G/T33G; (g2) Compared to SEQ ID No. 1, there are only the following mutations: G110A/C111T; (g3) ligated at the 5' and/or 3' ends of the sequences defined by (g1) or (g2) The fusion sequence obtained after the tag coding sequence; The sequence of the coding gene of the mutant of leucine dehydrogenase derived from Bacillus stearothermophilus is any one of the following (h1)-(h3): (h1) compared with SEQ ID No. 17, only The following mutations exist: A203G/A204C/A781C/A782T/C783G; (h2) Compared with SEQ ID No. 17, only the following mutations exist: A202T/A204T/A781T/A782G; (h3) in (h1) or (h2) The fusion sequence obtained by linking the 5' end and/or 3' end of the defined sequence with the tag coding sequence. 11. The method according to any one of claims 1-10, wherein in step (A) and step (B), the temperature of the catalytic reaction is both 25-37°C; the time of the catalytic reaction is All were 4 to 48 hours. 12. The method according to any one of claims 1-10, characterized in that: when the enzyme A and the enzyme B are in the form of crude enzyme liquid, crude enzyme liquid freeze-dried powder or pure enzyme, catalysis occurs In the step (A) and step (B), the concentrations of the enzyme A and the enzyme B in the respective reaction systems are both 0.1 g/L to 10 g/L; when the enzyme A and the enzyme When B catalyzes in the form of whole cells co-expressing the enzyme A and the enzyme B, the wet weight of the whole cells per liter of the reaction system is 100 g. 13. The method according to any one of claims 1-10, characterized in that: when the enzyme A and the enzyme B are in the form of crude enzyme liquid, crude enzyme liquid freeze-dried powder or pure enzyme, catalysis occurs , in step (A), the catalytic reaction is carried out in the buffer shown in the following (k1); in step (B), the catalytic reaction is carried out in the buffer shown in any of the following (k2)-(k3). When the enzyme A and the enzyme B catalyze in the form of a whole cell co-expressing the enzyme A and the enzyme B, the catalytic reactions of both steps (A) and (B) are Performed in the buffer indicated below (k1); (k1) Phosphate buffer with a concentration of 50 to 100 mM and pH 6.5 to 8.0; (k2) NH ₃ ·H ₂ O or NH ₄ Cl buffer with a concentration of 100 mM to 2 M and a pH of 8.0 to 9.6 (k3) Phosphate buffer with a concentration of 50-100 mM, a concentration of isopropylamine of 250 mM-1 M, and a pH of 7.5-8.5. 14. The method according to any one of claims 1-10, characterized in that: when the enzyme A and the enzyme B are in the form of crude enzyme liquid, crude enzyme liquid freeze-dried powder or pure enzyme, catalysis occurs In step (A), in addition to 1,2-butanediol and the enzyme A and its coenzyme, the reaction system of the catalytic reaction also contains acetone. 15. The method according to any one of claims 1-10, characterized in that: when the enzyme A and the enzyme B are in the form of crude enzyme liquid, crude enzyme liquid freeze-dried powder or pure enzyme, catalysis occurs , in step (B), when the enzyme B is an amino acid dehydrogenase or a mutant of the amino acid dehydrogenase, the reaction system of the catalytic reaction contains 2-keto-1-butanol and the In addition to enzyme B and its coenzymes, it also contains glucose dehydrogenase and glucose. 16. Application of an enzyme system or its related products in the synthesis of chiral 2-amino-1-butanol; Described chiral 2-amino-1-butanol is ( S )-2-amino-1-butanol or ( R )-2-amino-1-butanol; When the chiral 2-amino-1-butanol is ( S )-2-amino-1-butanol, the enzyme system is recorded as enzyme system I, and the related product is recorded as related product I; the The enzyme system I includes the enzyme A and the enzyme B in the first combination in claim 1; the related product I is a nucleic acid molecule capable of expressing each enzyme in the enzyme system I, or contains the Expression cassettes, recombinant vectors, recombinant bacteria or transgenic cell lines of the nucleic acid molecules; When the chiral 2-amino-1-butanol is ( R )-2-amino-1-butanol, the enzyme system is recorded as enzyme system II, and the related product is recorded as related product II; the Enzyme system II includes the enzyme A and the enzyme B in the second combination or the third combination in claim 1; the related product II is capable of expressing each enzyme in the enzyme system II The nucleic acid molecule, or the expression cassette, recombinant vector, recombinant bacteria or transgenic cell line containing the nucleic acid molecule.

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