Methods for efficiently synthesizing ω-amino fatty acid and α,ω-diamine

Abstract

Provided is a method for efficiently synthesizing an α,ω-diamine, comprising the following steps: (1) using an α,ω-dicarboxylic acid as a substrate, and synthesizing an ω-amino fatty acid under catalysis of a catalyst containing a first carboxylic acid reductase mutant; and (2) using an α,ω-dicarboxylic acid and the ω-amino fatty acid as substrates, synthesizing an α,ω-diamine under catalysis of a catalyst containing a second carboxylic acid reductase mutant, the first carboxylic acid reductase mutant having enhanced catalytic activity on the substrate α,ω-dicarboxylic acid and lowered catalytic activity on the ω-amino fatty acid, and / or, the second carboxylic acid reductase mutant having enhanced catalytic activity on both the substrates α,ω-dicarboxylic acid and ω-amino fatty acid. A method for efficiently synthesizing an ω-amino fatty acid is provided. Further disclosed are a series of carboxylic acid reductase mutants and the use thereof for the synthesis of an ω-amino fatty acid and an α,ω-diamine.

Description

Method for efficiently synthesizing ω-amino fatty acids and α,ω-diamines

[0001] This application claims priority to a patent application filed on September 15, 2023, entitled “Method for Efficient Synthesis of Ω-Amino Fatty Acids and α, Ω-Diamines” and application number CN 202311197881.6. Technical Field

[0002] The present invention belongs to the field of bioengineering technology, and more particularly, relates to a method for efficiently synthesizing ω-amino fatty acids and α,ω-diamines. Background Art

[0003] According to a Grand View Research report, the global nylon market reached $32.6 billion in 2022, with a projected compound annual growth rate of 6.4% from 2023 to 2030. ω-amino fatty acids are key monomers in nylon, with nylon 6 and nylon 66 accounting for over 90% of the nylon market. 6-Aminocaproic acid (6-ACA) is a monomer of nylon 6. Currently, the industrial production of 6-ACA primarily relies on chemical catalysis using benzene (via cyclohexane) and caprolactam as raw materials. Similarly, α,ω-diamines are widely used as nylon monomers, such as hexamethylenediamine, one of the two monomers of nylon 66. Currently, industrial production of hexamethylenediamine primarily relies on an energy-intensive, multi-stage chemical reaction developed by DuPont using butadiene as a starting material. While scalable, this process utilizes highly toxic hydrogen cyanide. In response to the call for "green biomanufacturing," in recent years, a growing number of researchers have attempted to synthesize ω-amino fatty acids and α,ω-diamines using renewable dibasic acids as substrates.

[0004] In 2020, the Yakunin team in Canada first reported the use of carboxylic acid reductase and transaminase to catalyze the reductive amination of carboxylic acid from adipic acid to 6-aminohexanoic acid, and then continued to catalyze the reductive amination of carboxylic acid of 6-aminohexanoic acid to synthesize hexamethylenediamine. The main catalyst form was pure enzyme, but the titer of the final product 6-aminohexanoic acid only reached 9.5mM, and the titer of hexamethylenediamine was only 3mM (J.Am.Chem.Soc., 2020, 142, 1038-1048). In 2023, the Deng Yu team of Jiangnan University constructed a redirected carboxylic acid reductase cascade for biocatalytic synthesis of hexamethylenediamine from adipic acid in Escherichia coli. Although the titer of hexamethylenediamine increased 567 times compared to the initial strain, it only reached 2.1mM (ACS Sustainable Chem.Eng., 2023, 11(15): 6011-6020). At the same time, our laboratory team successfully screened multiple carboxylic acid reductases (CARs) with high substrate specificity and catalytic efficiency through virtual screening. They then catalyzed the synthesis of 6-aminocaproic acid from adipic acid in a cascade with transaminases, with a conversion rate of nearly 90%. However, the substrate loading was only 5 mM, and the yield of hexamethylenediamine was only 14% (Biotechnol Bioeng, 2023, 120, 1773-1783). In order to further improve the substrate loading and yield, it is urgent to modify the CAR to further improve its catalytic efficiency for adipic acid, while reducing its enzyme activity for the product 6-aminocaproic acid to ensure a high yield of 6-aminocaproic acid. Subsequently, the substrate spectrum was expanded to other chain lengths of α, ω-dicarboxylic acids (C3-C12) to synthesize ω-amino fatty acids. In 2020, Levi Kramer et al. from the University of Nebraska in the United States carried out some modifications to improve the catalytic efficiency of CAR for AA. The catalytic efficiency of the optimal mutant increased by 17 times, but the final product was hexanediol, and the catalytic efficiency and substrate specificity for 6-aminocaproic acid were not considered (ACS Synth. Biol., 2020, 9, 1632-1637). At the same time, in order to efficiently synthesize α, ω-diamines, further modifications of CAR are needed to improve its catalytic activity for ω-amino fatty acids. Currently, only Yakunin et al. have improved the catalytic efficiency of CAR for 6-ACA by 9.1 times by analyzing the crystal structure of CAR from Mycobacterium abscessus, but the final substrate loading is only 10 mM (J. Am. Chem. Soc., 2020, 142, 1038-1048).

[0005] There are also many patent reports at home and abroad that use bio-enzymatic methods to catalyze the synthesis of various different ω-amino fatty acids, such as 5-aminovaleric acid (CN106011191A), 6-aminohexanoic acid, etc. (CN113430119A, US20070254341A1, TW1627153B, US20190271014A1). However, the substrates of these routes (lysine, 6-amino-2-hydroxyhexanoic acid, α-ketopimelic acid, 6-hydroxyhexanoic acid) are all non-biobased, and the products are single, and the broad spectrum of the component enzymes is poor. There are also reports on the biological synthesis of α,ω-diamines of different chain lengths, such as 1,4-butanediamine (CN116355824A), pentamethylenediamine (CN116376789A), hexamethylenediamine (US20190271014A1, US9,580,732B2), 1,8-octanediamine and 1,10-decanediamine (US9,580,732B2). The substrates of these routes are mainly L-glutamic acid, lysine, 6-hydroxyhexanoic acid and α,ω-diol. In 2021, Wang Dan and others from Chongqing University transferred the recombinant plasmid composition into Escherichia coli BL21 to obtain two genetically engineered bacteria. Lysine (5g / L) was used as a fermentation substrate and cultured with the genetically engineered bacteria LRAD to obtain 2.18-6.65g / L hexamethylenediamine (CN113755419A).

[0006] Patents also report the simultaneous synthesis of nylon monomers such as 6-aminocaproic acid and hexamethylenediamine through a designed cascade route. In 2015, Botes et al. from Invista Technologies GmbH in Switzerland described a biochemical pathway for producing 2-aminopimelane from 2,6-diaminopimelane, and for converting 2-aminopimelane into 6-aminocaproic acid, hexamethylenediamine, and the like, but the specific product titers were not detailed (CN106574283A). Schaffer et al. from Evonik Degussa GmbH in Germany proposed the production of amines and diamines from carboxylic acids or dicarboxylic acids or their monoesters, but only aminoundecanoic acid methyl ester was used in the specific examples (CN104937104A). Subsequently, Yakunin's team at the University of Toronto synthesized 6-aminocaproic acid and hexamethylenediamine using 20 mM adipic acid as a substrate, but the maximum conversion efficiency of 6-aminocaproic acid reached only 21.3 ± 0.8%, and the hexamethylenediamine yield was not clearly stated in the patent (CN116064690A).

[0007] Therefore, there is an urgent need in this field to solve the problems of low catalytic activity and poor substrate specificity of carboxylic acid reductase (CAR), the rate-limiting enzyme in the multi-enzyme cascade catalysis of α,ω-dicarboxylic acids (C3-C12) to synthesize ω-amino fatty acids and α,ω-diamines.

[0008] Summary of the Invention

[0009] The object of the present invention is to provide a method for efficiently synthesizing α,ω-diamine, which comprises the following steps: (1) using α,ω-dicarboxylic acid as a substrate and using a catalyst containing a first carboxylic acid reductase mutant to catalyze the synthesis of ω-amino fatty acid; (2) using α,ω-dicarboxylic acid and ω-amino fatty acid as substrates and using a catalyst containing a second carboxylic acid reductase mutant to catalyze the synthesis of α,ω-diamine; wherein the first carboxylic acid reductase mutant has enhanced catalytic activity for the substrate α,ω-dicarboxylic acid and reduced catalytic activity for ω-amino fatty acid; and / or wherein the second carboxylic acid reductase mutant has enhanced catalytic activity for both the substrate α,ω-dicarboxylic acid and ω-amino fatty acid. The present invention also provides a method for efficiently synthesizing ω-amino fatty acid. At the same time, the present invention also discloses a series of carboxylic acid reductase mutants and their use in synthesizing ω-amino fatty acid and α,ω-diamine.

[0010] In a first aspect of the present invention, a method for efficiently synthesizing α,ω-diamine is provided, comprising the following steps:

[0011] (1) Using α,ω-dicarboxylic acid as a substrate and a catalyst containing a first carboxylic acid reductase mutant, catalyzes the synthesis of ω-amino fatty acids;

[0012] (2) using α,ω-dicarboxylic acid and ω-amino fatty acid as substrates and using a catalyst containing a second carboxylic acid reductase mutant to catalyze the synthesis of α,ω-diamine;

[0013] wherein the first carboxylic acid reductase mutant has enhanced catalytic activity towards substrate α,ω-dicarboxylic acid and reduced catalytic activity towards ω-amino fatty acid; and / or,

[0014] The second carboxylic acid reductase mutant has enhanced catalytic activity towards substrates α, ω-dicarboxylic acid and ω-amino fatty acid.

[0015] In one or more embodiments, the first carboxylic acid reductase mutant is:

[0016] (a1) a protein having an amino acid sequence corresponding to SEQ ID No. 2, with one or more amino acids at positions 303, 306, 342, 344, 395, 418, and 426 mutated;

[0017] (b1) a protein derived from (a1) which is formed by substituting, deleting or adding one or more amino acid residues in the amino acid sequence of protein (a1) and has the functions of protein (a1), except that one or more amino acids at positions 303, 306, 342, 344, 395, 418 and 426 of SEQ ID No. 2 are identical to the amino acids at the corresponding positions in protein (a1) after mutation;

[0018] (c1) a protein derived from (a1) that has an amino acid sequence homology of 80% or more (e.g., 85% or more, 88% or more, 90% or more, 92% or more, 95% or more, 98% or more, or 99% or more) with the protein (a1) and has the functions of the protein (a1), except that one or more of the amino acids at positions 303, 306, 342, 344, 395, 418, and 426 of SEQ ID No. 2 are identical to the amino acids after mutation of the corresponding positions in the protein (a);

[0019] (d1) An active fragment of the (a1) protein, which comprises a structure that interacts with a carboxylic acid substrate in the spatial structure of a carboxylic acid reductase, and one or more amino acids at positions 303, 306, 342, 344, 395, 418, and 426 corresponding to SEQ ID No. 2 are identical to the amino acids after mutation at the corresponding positions of the (a1) protein.

[0020] In one or more embodiments, the first carboxylic acid reductase mutant is selected from the following group:

[0021] (1) Alanine (Ala) at position 303 was mutated to lysine (Lys);

[0022] (2) leucine (Leu) at position 306 is mutated to lysine (Lys) or arginine (Arg);

[0023] (3) Leucine (Leu) at position 342 is mutated to lysine (Lys) or arginine (Arg);

[0024] (4) Valine (Val) at position 344 was mutated to lysine (Lys);

[0025] (5) Glycine (Gly) at position 395 was mutated to lysine (Lys);

[0026] (6) glycine (Gly) at position 418 is mutated to histidine (His), lysine (Lys), asparagine (Asn), or serine (Ser);

[0027] (7) Glycine (Gly) at position 426 was mutated to histidine (His).

[0028] In one or more embodiments, the second carboxylic acid reductase mutant is:

[0029] (a2) a protein having an amino acid sequence corresponding to SEQ ID No. 2, with one or more amino acids at positions 284, 303, 306, 342, 393, 395, 418, or 426 mutated;

[0030] (b2) a protein derived from (a2) which is formed by substituting, deleting or adding one or more amino acid residues in the amino acid sequence of protein (a2) and has the functions of protein (a2), except that one or more amino acids at positions 284, 303, 306, 342, 393, 395, 418 and 426 corresponding to SEQ ID No. 2 are identical to the amino acids at the corresponding positions in protein (a2) after mutation;

[0031] (c2) a protein derived from (a2) that has an amino acid sequence homology of 80% or more (e.g., 85% or more, 88% or more, 90% or more, 92% or more, 95% or more, 98% or more, or 99% or more) to the protein (a2) and has the function of the protein (a2), except that one or more of the amino acids at positions 284, 303, 306, 342, 393, 395, 418, and 426 corresponding to SEQ ID No. 2 are identical to the amino acids after mutation of the corresponding positions in the protein (a);

[0032] (d2) An active fragment of the (a2) protein, which comprises a structure that interacts with a carboxylic acid substrate in the spatial structure of a carboxylic acid reductase, and one or more amino acids at positions 284, 303, 306, 342, 393, 395, 418, and 426 corresponding to SEQ ID No. 2 are identical to the amino acids after mutation at the corresponding positions of the (a2) protein.

[0033] In one or more embodiments, the second carboxylic acid reductase mutant is selected from the following group, or a combination of two or more of the following groups:

[0034] (8) Leucine (Leu) at position 284 is mutated to aspartic acid (Asp), glutamic acid (Glu), glycine (Gly), asparagine (Asn), threonine (Thr), valine (Val), or tryptophan (Trp);

[0035] (9) Alanine (Ala) at position 303 is mutated to glutamic acid (Glu), methionine (Met), or valine (Val);

[0036] (10) Leucine (Leu) at position 306 is mutated to methionine (Met) or valine (Val);

[0037] (11) Leucine (Leu) at position 342 is mutated to aspartic acid (Asp), glutamic acid (Glu), lysine (Lys), methionine (Met), asparagine (Asn), glutamine (Gln), valine (Val), or tryptophan (Trp);

[0038] (12) Glycine (Gly) at position 393 is mutated to aspartic acid (Asp) or glutamic acid (Glu);

[0039] (13) Glycine (Gly) at position 395 was mutated to glutamic acid (Glu);

[0040] (14) Glycine (Gly) at position 418 is mutated to aspartic acid (Asp) or glutamic acid (Glu);

[0041] (15) Glycine (Gly) at position 426 was mutated to serine (Ser).

[0042] In one or more embodiments, the second carboxylic acid reductase mutant is selected from the following group:

[0043] (16) Leucine (Leu) at position 284 mutated to aspartic acid (Asp), alanine (Ala) at position 303 mutated to methionine (Met), leucine (Leu) at position 306 mutated to isoleucine (Ile), leucine (Leu) at position 342 mutated to glutamic acid (Glu), and glycine (Gly) at position 393 mutated to alanine (Ala);

[0044] (17) Leucine (Leu) at position 284 mutated to aspartic acid (Asp), Leucine (Leu) at position 306 mutated to methionine (Met), and Leucine (Leu) at position 342 mutated to glutamic acid (Glu);

[0045] (18) Leucine (Leu) at position 284 mutated to aspartic acid (Asp), alanine (Ala) at position 303 mutated to isoleucine (Ile), leucine (Leu) at position 306 mutated to isoleucine (Ile), leucine (Leu) at position 342 mutated to glutamic acid (Glu), and glycine (Gly) at position 418 mutated to aspartic acid (Asp);

[0046] (19) Leucine (Leu) at position 284 mutated to isoleucine (Ile), Leucine (Leu) at position 306 mutated to methionine (Met), Leucine (Leu) at position 342 mutated to glutamic acid (Glu), and glycine (Gly) at position 393 mutated to glutamic acid (Glu);

[0047] (20) Leucine (Leu) at position 284 mutated to aspartic acid (Asp), Leucine (Leu) at position 306 mutated to methionine (Met), Leucine (Leu) at position 342 mutated to glutamic acid (Glu), glycine (Gly) at position 393 mutated to alanine (Ala), and glycine (Gly) at position 418 mutated to glutamic acid (Glu);

[0048] (21) Leucine (Leu) at position 284 mutated to aspartic acid (Asp), Leucine (Leu) at position 306 mutated to methionine (Met), Leucine (Leu) at position 342 mutated to glutamic acid (Glu), and glycine (Gly) at position 393 mutated to glutamic acid (Glu);

[0049] (22) Leucine (Leu) at position 284 mutated to glutamic acid (Glu), Leucine (Leu) at position 306 mutated to methionine (Met), Leucine (Leu) at position 342 mutated to glutamic acid (Glu), glycine (Gly) at position 393 mutated to alanine (Ala), and glycine (Gly) at position 426 mutated to serine (Ser);

[0050] (23) Leucine (Leu) at position 284 mutated to phenylalanine (Phe), Leucine (Leu) at position 306 mutated to isoleucine (Ile), Leucine (Leu) at position 342 mutated to aspartic acid (Asp), and glycine (Gly) at position 393 mutated to glutamic acid (Glu);

[0051] (24) Leucine (Leu) at position 284 mutated to phenylalanine (Phe), Leucine (Leu) at position 306 mutated to methionine (Met), Leucine (Leu) at position 342 mutated to aspartic acid (Asp), and glycine (Gly) at position 393 mutated to glutamic acid (Glu);

[0052] (25) Alanine (Ala) at position 303 mutated to methionine (Met), leucine (Leu) at position 306 mutated to isoleucine (Ile), leucine (Leu) at position 342 mutated to aspartic acid (Asp), and glycine (Gly) at position 393 mutated to glutamic acid (Glu);

[0053] (26) Alanine (Ala) at position 303 mutated to methionine (Met), leucine (Leu) at position 306 mutated to methionine (Met), leucine (Leu) at position 342 mutated to aspartic acid (Asp), and glycine (Gly) at position 393 mutated to glutamine (Gln);

[0054] (27) Leucine (Leu) at position 284 mutated to aspartic acid (Asp), Leucine (Leu) at position 306 mutated to methionine (Met), Leucine (Leu) at position 342 mutated to aspartic acid (Asp), and glycine (Gly) at position 393 mutated to glutamic acid (Glu);

[0055] (28) Leucine (Leu) at position 306 mutated to isoleucine (Ile), Leucine (Leu) at position 342 mutated to aspartic acid (Asp), and glycine (Gly) at position 418 mutated to aspartic acid (Asp);

[0056] (29) Leucine (Leu) at position 284 mutated to glutamic acid (Glu), alanine (Ala) at position 303 mutated to isoleucine (Ile), leucine (Leu) at position 306 mutated to isoleucine (Ile), leucine (Leu) at position 342 mutated to glutamic acid (Glu), and glycine (Gly) at position 393 mutated to glutamic acid (Glu);

[0057] (30) Leucine (Leu) at position 284 mutated to aspartic acid (Asp), alanine (Ala) at position 303 mutated to methionine (Met), leucine (Leu) at position 306 mutated to isoleucine (Ile), leucine (Leu) at position 342 mutated to glutamic acid (Glu), glycine (Gly) at position 393 mutated to alanine (Ala), and glycine (Gly) at position 418 mutated to aspartic acid (Asp);

[0058] (31) Leucine (Leu) at position 284 mutated to glutamine (Gln), Leucine (Leu) at position 306 mutated to methionine (Met), Leucine (Leu) at position 342 mutated to glutamic acid (Glu), glycine (Gly) at position 418 mutated to glutamic acid (Glu), and glycine (Gly) at position 426 mutated to serine (Ser);

[0059] (32) Leucine (Leu) at position 284 mutated to aspartic acid (Asp), alanine (Ala) at position 303 mutated to isoleucine (Ile), leucine (Leu) at position 306 mutated to isoleucine (Ile), leucine (Leu) at position 342 mutated to aspartic acid (Asp), glycine (Gly) at position 393 mutated to glutamic acid (Glu), and glycine (Gly) at position 418 mutated to glutamic acid (Glu);

[0060] (33) Leucine (Leu) at position 284 mutated to phenylalanine (Phe), Leucine (Leu) at position 306 mutated to isoleucine (Ile), Leucine (Leu) at position 342 mutated to glutamic acid (Glu), and glycine (Gly) at position 393 mutated to glutamine (Gln).

[0061] In one or more embodiments, the α,ω-dicarboxylic acids include malonic acid (C3), succinic acid (C4), glutaric acid (C5), adipic acid (C6), pimelic acid (C7), suberic acid (C8), azelaic acid (C9), sebacic acid (C10), undecanedioic acid (C11), and dodecanedioic acid (C12).

[0062] In one or more embodiments, the ω-amino fatty acid is 3-aminopropionic acid (C3), 4-aminobutyric acid (C4), 5-aminopentanoic acid (C5), 6-aminohexanoic acid (C6), 7-aminoheptanoic acid (C7), 8-aminooctanoic acid (C8), 9-aminononanoic acid (C9), 10-aminodecanoic acid (C10), 11-aminoundecanoic acid (C11), 12-aminododecanoic acid (C12) or a derivative thereof.

[0063] In one or more embodiments, the α,ω-diamine is a short-chain α,ω-diamine, a medium-chain α,ω-diamine, a medium-long chain α,ω-diamine, or a long-chain α,ω-diamine.

[0064] In one or more embodiments, the short chain refers to a carbon chain with a length of 5C or less (e.g., 3C-5C), the medium chain refers to a carbon chain with a length of 6C-9C (6C, as a nylon monomer, accounts for the largest proportion), the medium-long chain refers to a carbon chain with a length of 10C-12C, and the long chain refers to a carbon chain with a length of 13C or more.

[0065] In one or more embodiments, the “above” or “below” means to include the number.

[0066] In one or more embodiments, the 3C-5C, 6C-9C, and 10C-12C mentioned above all refer to inclusive endpoint values.

[0067] In one or more embodiments, the α,ω-diamine includes: 1,3-propylenediamine (C3), 1,4-butanediamine (C4), 1,5-pentanediamine (C5), 1,6-hexanediamine (C6), 1,7-heptanediamine (C7), 1,8-octanediamine (C8), 1,9-nonanediamine (C9), 1,10-decanediamine (C10), 1,11-undecanediamine (C11), 1,12-dodecanediamine (C12) or derivatives thereof.

[0068] In one or more embodiments, the catalyst further comprises one or more of: transaminase, glucose dehydrogenase, and polyphosphate kinase.

[0069] In one or more embodiments, the transaminase uses an organic amine as an amino group donor.

[0070] In one or more embodiments, the amino donor includes: an aliphatic amine donor (such as isopropylamine, alanine, glutamic acid, etc.), an aromatic amino donor (such as benzylamine, etc.).

[0071] In one or more embodiments, the glucose dehydrogenase uses glucose and NADP + As substrate, it catalyzes the oxidation of glucose and NADP + Reduced to NADPH.

[0072] In one or more embodiments, the glucose dehydrogenase is glucose dehydrogenase BmGDH.

[0073] In one or more embodiments, the polyphosphate kinase uses sodium hexametaphosphate as a substrate and catalyzes AMP to generate ATP.

[0074] In one or more embodiments, the polyphosphate kinase is polyphosphate kinase PPK12.

[0075] In a second aspect of the present invention, a method for efficiently synthesizing ω-amino fatty acids is provided, comprising: using α,ω-dicarboxylic acid as a substrate and a first carboxylic acid reductase mutant as a catalyst to catalyze the synthesis of ω-amino fatty acids; wherein the first carboxylic acid reductase mutant has enhanced catalytic activity for the substrate α,ω-dicarboxylic acid and reduced catalytic activity for ω-amino fatty acids.

[0076] In one or more embodiments, the first carboxylic acid reductase mutant is:

[0077] (a1) a protein having an amino acid sequence corresponding to SEQ ID No. 2, with one or more amino acids at positions 303, 306, 342, 344, 395, 418, and 426 mutated;

[0078] (b1) a protein derived from (a1) which is formed by substituting, deleting or adding one or more amino acid residues in the amino acid sequence of protein (a1) and has the functions of protein (a1), except that one or more amino acids at positions 303, 306, 342, 344, 395, 418 and 426 of SEQ ID No. 2 are identical to the amino acids at the corresponding positions in protein (a1) after mutation;

[0079] (c1) a protein derived from (a1) that has an amino acid sequence homology of 80% or more (e.g., 85% or more, 88% or more, 90% or more, 92% or more, 95% or more, 98% or more, or 99% or more) with the protein (a1) and has the functions of the protein (a1), except that one or more of the amino acids at positions 303, 306, 342, 344, 395, 418, and 426 of SEQ ID No. 2 are identical to the amino acids after mutation of the corresponding positions in the protein (a);

[0080] (d1) An active fragment of the (a1) protein, which comprises a structure that interacts with a carboxylic acid substrate in the spatial structure of a carboxylic acid reductase, and one or more amino acids at positions 303, 306, 342, 344, 395, 418, and 426 corresponding to SEQ ID No. 2 are identical to the amino acids after mutation at the corresponding positions of the (a1) protein.

[0081] In one or more embodiments, the first carboxylic acid reductase mutant is selected from the following group:

[0082] (1) Alanine (Ala) at position 303 was mutated to lysine (Lys);

[0083] (2) leucine (Leu) at position 306 is mutated to lysine (Lys) or arginine (Arg);

[0084] (3) Leucine (Leu) at position 342 is mutated to lysine (Lys) or arginine (Arg);

[0085] (4) Valine (Val) at position 344 was mutated to lysine (Lys);

[0086] (5) Glycine (Gly) at position 395 was mutated to lysine (Lys);

[0087] (6) glycine (Gly) at position 418 is mutated to histidine (His), lysine (Lys), asparagine (Asn), or serine (Ser);

[0088] (7) Glycine (Gly) at position 426 was mutated to histidine (His).

[0089] In a third aspect of the present invention, a carboxylic acid reductase mutant is provided, wherein the catalytic activity of the mutant towards a substrate is altered compared to that of a wild-type carboxylic acid reductase.

[0090] In one or more embodiments, when the substrate is an α, ω-dicarboxylic acid, the carboxylic acid reductase mutant has enhanced catalytic activity for the substrate α, ω-dicarboxylic acid and reduced catalytic activity for ω-amino fatty acid; preferably, the carboxylic acid reductase mutant is the first carboxylic acid reductase mutant defined in any embodiment of the present invention.

[0091] In one or more embodiments, when the substrate is an α, ω-dicarboxylic acid and an ω-amino fatty acid, the catalytic activity of the carboxylic acid reductase mutant on the substrates α, ω-dicarboxylic acid and ω-amino fatty acid is enhanced; preferably, the carboxylic acid reductase mutant is the second carboxylic acid reductase mutant defined in any embodiment of the present invention.

[0092] In the fourth aspect of the present invention, an isolated polynucleotide is provided, wherein the nucleic acid encodes the carboxylic acid reductase mutant of the present invention, or the nucleic acid encodes the first carboxylic acid reductase mutant or the second carboxylic acid reductase mutant defined in any embodiment of the present invention.

[0093] In the fifth aspect of the present invention, a vector is provided, which contains the polynucleotide of the present invention; preferably, the vector is an expression vector or an expression transformant; more preferably, the expression transformant comprises an expression vector.

[0094] In a sixth aspect of the present invention, a genetically engineered host cell is provided, which contains the vector of the present invention, or has the polynucleotide of the present invention integrated into its genome.

[0095] In a seventh aspect of the present invention, an application selected from the following is provided:

[0096] (a) Use of the carboxylic acid reductase mutant of the present invention, or the first carboxylic acid reductase mutant or the second carboxylic acid reductase mutant defined in any embodiment of the present invention, in catalyzing the synthesis of α,ω-diamines;

[0097] (b) Use of the first carboxylic acid reductase mutant of the present invention, or the first carboxylic acid reductase mutant defined in any embodiment of the present invention, in catalyzing the synthesis of ω-amino fatty acids;

[0098] (c) Use of the carboxylic acid reductase mutant of the present invention, or the first carboxylic acid reductase mutant or the second carboxylic acid reductase mutant as defined in any embodiment of the present invention, in the preparation of a catalyst for catalytic synthesis of ω-amino fatty acids and / or α,ω-diamines.

[0099] In one or more embodiments, the first carboxylic acid reductase mutant of the present invention, or the first carboxylic acid reductase mutant defined in any embodiment of the present invention can be used to prepare a catalyst for catalytic synthesis of ω-amino fatty acids, and can also be used to prepare a catalyst for catalytic synthesis of α,ω-diamines.

[0100] In one or more embodiments, the second carboxylic acid reductase mutant of the present invention, or the second carboxylic acid reductase mutant defined in any embodiment of the present invention, can be used to prepare a catalyst for catalyzing the synthesis of α,ω-diamines.

[0101] Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0102] Figure 1. Reaction scheme for the synthesis of BOC-protected ω-amino fatty acids.

[0103] Figure 2. Schematic diagram of the multi-enzyme cascade catalysis of the synthesis of ω-amino fatty acids and α,ω-diamines of different chain lengths from α,ω-dicarboxylic acids. DETAILED DESCRIPTION

[0104] In response to the problems of low catalytic activity and poor substrate specificity of carboxylic acid reductase (CAR), the rate-limiting enzyme in the multi-enzyme cascade catalysis system for the synthesis of ω-amino fatty acids and α,ω-diamines from α,ω-dicarboxylic acids (C3-C12), towards the model substrates adipic acid (AA) and 6-aminohexanoic acid (6-ACA), the inventors have conducted in-depth research and provided a method for efficiently synthesizing ω-amino fatty acids and α,ω-diamines. By site-directed mutagenesis of carboxylic acid reductase, the catalytic efficiency of carboxylic acid reductase towards AA and 6-ACA can be precisely regulated to efficiently synthesize ω-amino fatty acids and α,ω-diamines.

[0105] When efficiently synthesizing ω-amino fatty acids, the carboxylic acid reductase mutant provided by the present invention has a higher catalytic efficiency for α, ω-dicarboxylic acids compared to the wild-type carboxylic acid reductase, while the catalytic efficiency for the product ω-amino fatty acids is reduced, thereby further increasing the substrate loading and increasing the yield of ω-amino fatty acids. When efficiently synthesizing α, ω-diamines, the carboxylic acid reductase mutant provided by the present invention has improved catalytic efficiency for both α, ω-dicarboxylic acids and ω-amino fatty acids compared to the wild-type carboxylic acid reductase, especially for ω-amino fatty acids. cat / K mThe efficiency is greatly improved (101 times), thereby efficiently synthesizing α, ω-diamines. The present invention also expands the substrate spectrum to other α, ω-dicarboxylic acids (C3-C12) to synthesize ω-amino fatty acids and α, ω-diamines. The present invention also provides a protein and gene of a carboxylic acid reductase mutant, a recombinant expression vector and a recombinant expression transformant containing the gene, a method for preparing a recombinant transformant culture that can express the carboxylic acid reductase mutant, and the use of the carboxylic acid reductase mutant or recombinant transformant culture and a transaminase ω-TA, a glucose dehydrogenase GDH / formate dehydrogenase FDH and a polyphosphate kinase PPK12 cascade catalyzed synthesis of ω-amino fatty acids and α, ω-diamines from α, ω-dicarboxylic acids.

[0106] As used herein, unless otherwise specified, the "carboxylic acid reductase mutant" and "recombinant carboxylic acid reductase mutant" are used interchangeably and refer to a polypeptide corresponding to a wild-type carboxylic acid reductase after a mutation occurs near its substrate binding pocket or a polypeptide with altered catalytic activity, preferably a polypeptide corresponding to a mutation at one or more positions 284, 303, 306, 342, 344, 393, 395, 418, or 426 of its sequence.

[0107] As used herein, "first" and "second" are merely used to distinguish the carboxylic acid reductase mutants used in different steps, and do not represent the order or precedence.

[0108] If a wild-type carboxylic acid reductase is desired, it can be a protein with an amino acid sequence such as SEQ ID No. 2, or a functionally equivalent variant or active fragment of the protein. Preferably, the wild-type carboxylic acid reductase is derived from Mycobacteroides abscessus; however, it should be understood that homologs of carboxylic acid reductases derived from other species that share homology and functionally equivalent functions are also encompassed by the present invention. If a polynucleotide representing a wild-type carboxylic acid reductase is desired, it can be a polynucleotide encoding the protein, such as the polynucleotide with a nucleotide sequence such as SEQ ID No. 1, or a polynucleotide including additional coding and / or non-coding sequences.

[0109] As used herein, "isolated carboxylic acid reductase mutant" means that the carboxylic acid reductase mutant is substantially free of other proteins, lipids, carbohydrates, or other substances with which it is naturally associated. Those skilled in the art can purify the carboxylic acid reductase mutant using standard protein purification techniques. Substantially pure protein can produce a single major band on a non-reducing polyacrylamide gel.

[0110] As used herein, "substrate binding pocket" refers to a position in the spatial structure of a carboxylic acid reductase mutant that interacts with (binds to) a substrate.

[0111] The protein of the present invention can be a recombinant protein, a natural protein, or a synthetic protein, preferably a recombinant protein. The protein of the present invention can be a naturally purified product, or a chemically synthesized product, or produced using recombinant technology from a prokaryotic or eukaryotic host (e.g., bacteria, yeast, higher plants, insects, and mammalian cells).

[0112] The present invention also includes fragments, derivatives and analogs of the carboxylic acid reductase mutants. As used herein, the terms "fragment", "derivative" and "analog" refer to proteins that substantially retain the same biological function or activity as the natural carboxylic acid reductase mutants of the present invention. The protein fragments, derivatives or analogs of the present invention can be (i) proteins in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) proteins with a substituent group in one or more amino acid residues, or (iii) proteins formed by the fusion of an additional amino acid sequence to this protein sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the protein, or a fusion protein). According to the definitions herein, these fragments, derivatives and analogs are within the scope known to those skilled in the art. However, the amino acid sequences of the carboxylic acid reductase mutants and their fragments, derivatives and analogs must contain the mutations described above in the present invention; preferably, the mutations are mutations corresponding to one or more amino acids at positions 284, 303, 306, 342, 344, 393, 395, 418 and 426 of SEQ ID NO: 2.

[0113] In the present invention, the term "carboxylic acid reductase mutant" also includes (but is not limited to): deletion, insertion, replacement and / or substitution of several (usually 1-20, more preferably 1-10, and even more preferably 1-8, 1-5, 1-3 or 1-2) amino acids, and addition or deletion of one or several (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and / or N-terminus. For example, in the art, when amino acids with similar or similar properties are substituted, the function of the protein is generally not changed. For another example, the addition or deletion of one or several amino acids at the C-terminus and / or N-terminus generally does not change the function of the protein. The term also includes active fragments and active derivatives of carboxylic acid reductase mutants. However, among these variant forms, the mutations described above in the present invention must exist; preferably, the mutations are mutations corresponding to one or more amino acids at positions 284, 303, 306, 342, 344, 393, 395, 418, and 426 of SEQ ID NO: 2.

[0114] In the present invention, the term "carboxylic acid reductase mutant" also includes (but is not limited to): derivative proteins that retain their protein activity and have an amino acid sequence identity of 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more, such as 98% or more, or 99% or more, with the amino acid sequence of the carboxylic acid reductase mutant. Similarly, these derivative proteins must contain the mutations described in the present invention; preferably, the mutations are mutations corresponding to one or more amino acids at positions 284, 303, 306, 342, 344, 393, 395, 418, or 426 of SEQ ID NO: 2.

[0115] As used herein, “adipic acid” and “AA” are used interchangeably, and “α,ω-dicarboxylic acid” includes “adipic acid.” Generally, “α,ω-dicarboxylic acid” refers to “adipic acid” herein.

[0116] As used herein, "ω-amino fatty acid" and "6-amino fatty acid" are used interchangeably, and "ω-amino fatty acid" includes "6-aminocaproic acid". Generally, "ω-amino fatty acid" refers to "6-aminocaproic acid". The "6-aminocaproic acid", "6-ACA" and "ACA" are used interchangeably.

[0117] As used herein, “1,6-hexanediamine” and “hexamethylenediamine” are used interchangeably, and “α,ω-diamine” includes “1,6-hexamethylenediamine.” Generally, “α,ω-diamine” refers to “1,6-hexamethylenediamine.”

[0118] As used herein, "catalytic reaction", "enzyme-linked reaction" and "enzymatic reaction" are used interchangeably and all refer to reactions carried out under the catalysis of an enzyme. In this article, the "multi-enzyme linked reaction", "multi-enzyme cascade reaction" and "multi-stage enzyme cascade reaction" are used interchangeably and include at least two enzyme cascade reactions. In a specific embodiment, the multi-enzyme cascade reaction for synthesizing α, ω-diamines includes: using a first carboxylic acid reductase mutant as a catalyst to catalyze the multi-enzyme cascade reaction for synthesizing ω-amino fatty acids (i.e., module I), and using a second carboxylic acid reductase mutant as a catalyst to catalyze the multi-enzyme cascade reaction for synthesizing α, ω-diamines (i.e., module II). The conditions for the multi-enzyme cascade reaction are known to those skilled in the art, and can be carried out, for example, at room temperature (e.g., 20-40° C.), normal pressure and pH 6.0-7.5.

[0119] As used herein, "catalytic efficiency" and "catalytic activity" are used interchangeably.

[0120] Method for efficiently synthesizing ω-amino fatty acids

[0121] The present invention provides a method for efficiently synthesizing ω-amino fatty acids, comprising using a first carboxylic acid reductase mutant as a catalyst. Because the first carboxylic acid reductase mutant has high catalytic efficiency for α,ω-dicarboxylic acids and low catalytic efficiency for ω-amino fatty acids, substrate loading is further increased, resulting in a higher yield of ω-amino fatty acids.

[0122] As used herein, the term "catalytic efficiency" generally refers to the catalytic activity of an enzyme on a substrate. In some specific embodiments, the catalytic efficiency of an enzyme is represented by a specific activity value.

[0123] In one or more embodiments, the improved "catalytic efficiency" refers to an increase in catalytic efficiency of 5%, 10%, 15%, 20%, 30% or more compared to the "wild type" or "control".

[0124] In one or more embodiments, the reduction in "catalytic efficiency" refers to a reduction in catalytic efficiency of 5%, 10%, 15%, 20%, 30% or less compared to the "wild type" or "control". As used in the present invention, the "first carboxylic acid reductase mutant" corresponds to the wild-type carboxylic acid reductase, and the catalytic efficiency for α, ω-dicarboxylic acid and ω-amino fatty acid is changed. For example, the catalytic efficiency for α, ω-dicarboxylic acid is improved, while the catalytic efficiency for ω-amino fatty acid is reduced, corresponding to the wild-type carboxylic acid reductase. Since α, ω-dicarboxylic acid is the substrate and ω-amino fatty acid is the product, it can also be said that the substrate specificity and / or substrate specificity of the "first carboxylic acid reductase mutant" has changed, that is, the specificity and / or specificity for the substrate α, ω-dicarboxylic acid is enhanced. In one or more embodiments, corresponding to the wild-type carboxylic acid reductase, the first carboxylic acid reductase mutant is mutated at one or more amino acids at positions 303, 306, 342, 344, 395, 418, and 426 of the amino acid sequence shown in SEQ ID No. 2.

[0125] In one or more embodiments, the first carboxylic acid reductase mutant is mutated at one or more amino acids corresponding to alanine (Ala) at position 303, leucine (Leu) at position 306, leucine (Leu) at position 342, valine (Val) at position 344, glycine (Gly) at position 395, glycine (Gly) at position 418, and glycine (Gly) at position 426 of the amino acid sequence shown in SEQ ID No. 2. In one or more preferred embodiments, the first carboxylic acid reductase mutant is mutated at one or more of the following amino acid positions corresponding to the amino acid sequence shown in SEQ ID No. 2:

[0126] (1) Alanine (Ala) at position 303 was mutated to lysine (Lys);

[0127] (2) leucine (Leu) at position 306 is mutated to lysine (Lys) or arginine (Arg);

[0128] (3) Leucine (Leu) at position 342 is mutated to lysine (Lys) or arginine (Arg);

[0129] (4) Valine (Val) at position 344 was mutated to lysine (Lys);

[0130] (5) Glycine (Gly) at position 395 was mutated to lysine (Lys);

[0131] (6) glycine (Gly) at position 418 is mutated to histidine (His), lysine (Lys), asparagine (Asn), or serine (Ser);

[0132] (7) Glycine (Gly) at position 426 was mutated to histidine (His).

[0133] In one or more embodiments, the method for efficiently synthesizing ω-amino fatty acids comprises: utilizing the first carboxylic acid reductase mutant described in the present invention, using α,ω-dicarboxylic acid as a substrate, and catalyzing the synthesis of ω-amino fatty acids through a multi-enzyme cascade reaction under the condition of providing an amino donor.

[0134] Method for efficiently synthesizing α, ω-diamine

[0135] The present invention provides a method for efficiently synthesizing α,ω-diamines, comprising using a second carboxylic acid reductase mutant as a catalyst to catalyze the synthesis of the α,ω-diamines. Because the second carboxylic acid reductase mutant has high catalytic efficiency for both α,ω-dicarboxylic acids and ω-amino fatty acids, the α,ω-diamines can be efficiently synthesized.

[0136] In one or more embodiments, the method for efficiently synthesizing α,ω-diamines, in addition to the step of using the second carboxylic acid reductase mutant as a catalyst to catalyze the synthesis of α,ω-diamines (module II), also includes a step of using the first carboxylic acid reductase mutant as a catalyst to efficiently synthesize ω-amino fatty acids (module I).

[0137] As used herein, the term "catalytic efficiency" generally refers to the catalytic activity of an enzyme on a substrate. In some specific embodiments, the catalytic efficiency of an enzyme is represented by a specific activity value.

[0138] In one or more embodiments, the improved "catalytic efficiency" refers to an increase in catalytic efficiency of 5%, 10%, 15%, 20%, 30% or more compared to the "wild type" or "control." As used herein, the "second carboxylic acid reductase mutant" corresponds to the wild-type carboxylic acid reductase, and has altered catalytic efficiency for α,ω-dicarboxylic acids and ω-amino fatty acids. For example, the catalytic efficiency for α,ω-dicarboxylic acids is improved compared to the wild-type carboxylic acid reductase, and the catalytic efficiency for ω-amino fatty acids is also improved. Since, in the process of synthesizing α,ω-diamines, α,ω-dicarboxylic acids are substrates and ω-amino fatty acids are intermediates, it can also be said that the specificity and / or specificity of the "second carboxylic acid reductase mutant" for the substrate and intermediate products has changed, i.e., the specificity and / or specificity for the substrate α,ω-dicarboxylic acid and the intermediate product ω-amino fatty acid has been enhanced. In one or more embodiments, corresponding to the wild-type carboxylic acid reductase, the second carboxylic acid reductase mutant is mutated at one or more amino acids at positions 284, 303, 306, 342, 393, 395, 418, and 426 of the amino acid sequence shown in SEQ ID No. 2.

[0139] In one or more embodiments, the second carboxylic acid reductase mutant is mutated at one or more amino acids corresponding to leucine (Leu) at position 284, alanine (Ala) at position 303, leucine (Leu) at position 306, leucine (Leu) at position 342, glycine (Gly) at position 393, glycine (Gly) at position 395, glycine (Gly) at position 418, and glycine (Gly) at position 426 of the amino acid sequence shown in SEQ ID No. 2.

[0140] In one or more preferred embodiments, corresponding to the amino acid sequence shown in SEQ ID No. 2, the second carboxylic acid reductase mutant is mutated at one or more of the following amino acid positions:

[0141] (8) Leucine (Leu) at position 284 is mutated to aspartic acid (Asp), glutamic acid (Glu), glycine (Gly), asparagine (Asn), threonine (Thr), valine (Val), or tryptophan (Trp);

[0142] (9) Alanine (Ala) at position 303 is mutated to glutamic acid (Glu), methionine (Met), or valine (Val);

[0143] (10) Leucine (Leu) at position 306 is mutated to methionine (Met) or valine (Val);

[0144] (11) Leucine (Leu) at position 342 is mutated to aspartic acid (Asp), glutamic acid (Glu), lysine (Lys), methionine (Met), asparagine (Asn), glutamine (Gln), valine (Val), or tryptophan (Trp);

[0145] (12) Glycine (Gly) at position 393 is mutated to aspartic acid (Asp) or glutamic acid (Glu);

[0146] (13) Glycine (Gly) at position 395 was mutated to glutamic acid (Glu);

[0147] (14) Glycine (Gly) at position 418 mutated to aspartic acid (Asp) or glutamic acid (Glu); (15) Glycine (Gly) at position 426 mutated to serine (Ser).

[0148] In one or more embodiments, the second carboxylic acid reductase mutant has multiple amino acid mutations in the amino acid sequence corresponding to SEQ ID No. 2, and the mutated amino acids are selected from any one of the following groups:

[0149] Nos. 284, 303, 306, 342 and 393; or

[0150] Nos. 284, 306 and 342; or

[0151] Nos. 284, 303, 306, 342 and 418; or

[0152] Nos. 284, 306, 342 and 393; or

[0153] 284th, 306th, 342nd, 393rd and 418th; or

[0154] 284th, 306th; 342nd and 393rd; or

[0155] 284th, 306th, 342nd, 393rd and 426th; or

[0156] Nos. 284, 306, 342 and 393; or

[0157] Nos. 284, 306, 342 and 393; or

[0158] 303rd, 306th, 342nd and 393rd; or

[0159] 303rd, 306th, 342nd and 393rd; or

[0160] Nos. 284, 306, 342 and 393; or

[0161] Nos. 306, 342 and 418; or

[0162] Nos. 284, 303, 306, 342 and 393; or

[0163] 284th, 303rd, 306th, 342nd, 393rd and 418th; or

[0164] Nos. 284, 306, 342, 418 and 426; or

[0165] No. 284, No. 303, No. 306, No. 342, No. 393 and No. 418; or No. 284, No. 306, No. 342 and No. 393.

[0166] In one or more embodiments, the second carboxylic acid reductase mutant has multiple amino acid mutations in the amino acid sequence corresponding to SEQ ID No. 2, and the mutated amino acids are selected from any one of the following groups:

[0167] Leucine (Leu) at position 284, alanine (Ala) at position 303, leucine (Leu) at position 306, leucine (Leu) at position 342, and glycine (Gly) at position 393; or

[0168] Leucine (Leu) at position 284, Leucine (Leu) at position 306, and Leucine (Leu) at position 342; or

[0169] Leucine (Leu) at position 284, alanine (Ala) at position 303, leucine (Leu) at position 306, leucine (Leu) at position 342, and glycine (Gly) at position 418; or

[0170] Leucine (Leu) at position 284, Leucine (Leu) at position 306, Leucine (Leu) at position 342, and Glycine (Gly) at position 393; or

[0171] Leucine (Leu) at position 284, Leucine (Leu) at position 306, Leucine (Leu) at position 342, Glycine (Gly) at position 393, and Glycine (Gly) at position 418; or

[0172] Leucine (Leu) at position 284, Leucine (Leu) at position 306, Leucine (Leu) at position 342, and Glycine (Gly) at position 393; or

[0173] Leucine (Leu) at position 284, Leucine (Leu) at position 306, Leucine (Leu) at position 342, Glycine (Gly) at position 393, and Glycine (Gly) at position 426; or

[0174] Leucine (Leu) at position 284, Leucine (Leu) at position 306, Leucine (Leu) at position 342, and Glycine (Gly) at position 393; or

[0175] Leucine (Leu) at position 284, Leucine (Leu) at position 306, Leucine (Leu) at position 342, and Glycine (Gly) at position 393; or

[0176] Alanine (Ala) at position 303, Leucine (Leu) at position 306, Leucine (Leu) at position 342, and Glycine (Gly) at position 393; or

[0177] Alanine (Ala) at position 303, Leucine (Leu) at position 306, Leucine (Leu) at position 342, and Glycine (Gly) at position 393; or

[0178] Leucine (Leu) at position 284, Leucine (Leu) at position 306, Leucine (Leu) at position 342, and Glycine (Gly) at position 393; or

[0179] Leucine (Leu) at position 306, Leucine (Leu) at position 342, and Glycine (Gly) at position 418; or

[0180] Leucine (Leu) at position 284, alanine (Ala) at position 303, leucine (Leu) at position 306, leucine (Leu) at position 342, and glycine (Gly) at position 393; or

[0181] Leucine (Leu) at position 284, alanine (Ala) at position 303, leucine (Leu) at position 306, leucine (Leu) at position 342, glycine (Gly) at position 393, and glycine (Gly) at position 418; or

[0182] Leucine (Leu) at position 284, Leucine (Leu) at position 306, Leucine (Leu) at position 342, Glycine (Gly) at position 418, and Glycine (Gly) at position 426; or

[0183] Leucine (Leu) at position 284, alanine (Ala) at position 303, leucine (Leu) at position 306, leucine (Leu) at position 342, glycine (Gly) at position 393, and glycine (Gly) at position 418; or

[0184] Leucine (Leu) at position 284, Leucine (Leu) at position 306, Leucine (Leu) at position 342, and Glycine (Gly) at position 393.

[0185] In one or more preferred embodiments, corresponding to the amino acid sequence shown in SEQ ID No. 2, the second carboxylic acid reductase mutant is selected from the following group:

[0186] (16) Leucine (Leu) at position 284 mutated to aspartic acid (Asp), alanine (Ala) at position 303 mutated to methionine (Met), leucine (Leu) at position 306 mutated to isoleucine (Ile), leucine (Leu) at position 342 mutated to glutamic acid (Glu), and glycine (Gly) at position 393 mutated to alanine (Ala);

[0187] (17) Leucine (Leu) at position 284 mutated to aspartic acid (Asp), Leucine (Leu) at position 306 mutated to methionine (Met), and Leucine (Leu) at position 342 mutated to glutamic acid (Glu);

[0188] (18) Leucine (Leu) at position 284 mutated to aspartic acid (Asp), alanine (Ala) at position 303 mutated to isoleucine (Ile), leucine (Leu) at position 306 mutated to isoleucine (Ile), leucine (Leu) at position 342 mutated to glutamic acid (Glu), and glycine (Gly) at position 418 mutated to aspartic acid (Asp);

[0189] (19) Leucine (Leu) at position 284 mutated to isoleucine (Ile), Leucine (Leu) at position 306 mutated to methionine (Met), Leucine (Leu) at position 342 mutated to glutamic acid (Glu), and glycine (Gly) at position 393 mutated to glutamic acid (Glu);

[0190] (20) Leucine (Leu) at position 284 mutated to aspartic acid (Asp), Leucine (Leu) at position 306 mutated to methionine (Met), Leucine (Leu) at position 342 mutated to glutamic acid (Glu), glycine (Gly) at position 393 mutated to alanine (Ala), and glycine (Gly) at position 418 mutated to glutamic acid (Glu);

[0191] (21) Leucine (Leu) at position 284 mutated to aspartic acid (Asp), Leucine (Leu) at position 306 mutated to methionine (Met), Leucine (Leu) at position 342 mutated to glutamic acid (Glu), and glycine (Gly) at position 393 mutated to glutamic acid (Glu);

[0192] (22) Leucine (Leu) at position 284 mutated to glutamic acid (Glu), Leucine (Leu) at position 306 mutated to methionine (Met), Leucine (Leu) at position 342 mutated to glutamic acid (Glu), glycine (Gly) at position 393 mutated to alanine (Ala), and glycine (Gly) at position 426 mutated to serine (Ser);

[0193] (23) Leucine (Leu) at position 284 mutated to phenylalanine (Phe), Leucine (Leu) at position 306 mutated to isoleucine (Ile), Leucine (Leu) at position 342 mutated to aspartic acid (Asp), and glycine (Gly) at position 393 mutated to glutamic acid (Glu);

[0194] (24) Leucine (Leu) at position 284 mutated to phenylalanine (Phe), Leucine (Leu) at position 306 mutated to methionine (Met), Leucine (Leu) at position 342 mutated to aspartic acid (Asp), and glycine (Gly) at position 393 mutated to glutamic acid (Glu);

[0195] (25) Alanine (Ala) at position 303 mutated to methionine (Met), leucine (Leu) at position 306 mutated to isoleucine (Ile), leucine (Leu) at position 342 mutated to aspartic acid (Asp), and glycine (Gly) at position 393 mutated to glutamic acid (Glu);

[0196] (26) Alanine (Ala) at position 303 mutated to methionine (Met), leucine (Leu) at position 306 mutated to methionine (Met), leucine (Leu) at position 342 mutated to aspartic acid (Asp), and glycine (Gly) at position 393 mutated to glutamine (Gln);

[0197] (27) Leucine (Leu) at position 284 mutated to aspartic acid (Asp), Leucine (Leu) at position 306 mutated to methionine (Met), Leucine (Leu) at position 342 mutated to aspartic acid (Asp), and glycine (Gly) at position 393 mutated to glutamic acid (Glu);

[0198] (28) Leucine (Leu) at position 306 mutated to isoleucine (Ile), Leucine (Leu) at position 342 mutated to aspartic acid (Asp), and glycine (Gly) at position 418 mutated to aspartic acid (Asp);

[0199] (29) Leucine (Leu) at position 284 mutated to glutamic acid (Glu), alanine (Ala) at position 303 mutated to isoleucine (Ile), leucine (Leu) at position 306 mutated to isoleucine (Ile), leucine (Leu) at position 342 mutated to glutamic acid (Glu), and glycine (Gly) at position 393 mutated to glutamic acid (Glu);

[0200] (30) Leucine (Leu) at position 284 mutated to aspartic acid (Asp), alanine (Ala) at position 303 mutated to methionine (Met), leucine (Leu) at position 306 mutated to isoleucine (Ile), leucine (Leu) at position 342 mutated to glutamic acid (Glu), glycine (Gly) at position 393 mutated to alanine (Ala), and glycine (Gly) at position 418 mutated to aspartic acid (Asp);

[0201] (31) Leucine (Leu) at position 284 mutated to glutamine (Gln), Leucine (Leu) at position 306 mutated to methionine (Met), Leucine (Leu) at position 342 mutated to glutamic acid (Glu), glycine (Gly) at position 418 mutated to glutamic acid (Glu), and glycine (Gly) at position 426 mutated to serine (Ser);

[0202] (32) Leucine (Leu) at position 284 mutated to aspartic acid (Asp), alanine (Ala) at position 303 mutated to isoleucine (Ile), leucine (Leu) at position 306 mutated to isoleucine (Ile), leucine (Leu) at position 342 mutated to aspartic acid (Asp), glycine (Gly) at position 393 mutated to glutamic acid (Glu), and glycine (Gly) at position 418 mutated to glutamic acid (Glu);

[0203] (33) Leucine (Leu) at position 284 mutated to phenylalanine (Phe), Leucine (Leu) at position 306 mutated to isoleucine (Ile), Leucine (Leu) at position 342 mutated to glutamic acid (Glu), and glycine (Gly) at position 393 mutated to glutamine (Gln).

[0204] In one or more embodiments, the method for efficiently synthesizing α,ω-diamines uses α,ω-dicarboxylic acids as substrates, and the α,ω-dicarboxylic acids include but are not limited to: malonic acid (C3), succinic acid (C4), glutaric acid (C5), adipic acid (C6), pimelic acid (C7), suberic acid (C8), azelaic acid (C9), sebacic acid (C10), undecanedioic acid (C11) and dodecanedioic acid (C12).

[0205] In one or more embodiments, the ω-amino fatty acid is 3-aminopropionic acid (C3), 4-aminobutyric acid (C4), 5-aminopentanoic acid (C5), 6-aminohexanoic acid (C6), 7-aminoheptanoic acid (C7), 8-aminooctanoic acid (C8), 9-aminononanoic acid (C9), 10-aminodecanoic acid (C10), 11-aminoundecanoic acid (C11), 12-aminododecanoic acid (C12) or a derivative thereof.

[0206] In one or more embodiments, the method for efficiently synthesizing α,ω-diamines uses an organic ammonia as an amino donor. The amino donor includes aliphatic amine donors such as isopropylamine, alanine, and glutamic acid, or aromatic amino donors such as benzylamine. Those skilled in the art will appreciate that any amino donor that can be used to synthesize ω-amino fatty acids and / or α,ω-diamines can be used in the present invention.

[0207] In one or more embodiments, the catalyst includes, but is not limited to, transaminase, glucose dehydrogenase, and polyphosphate kinase in addition to the carboxylic acid reductase mutant.

[0208] In one or more embodiments, the catalyst is in the form of whole cells, a crude cell extract (crude enzyme solution), or a pure enzyme. The crude cell extract may be a crude enzyme solution containing the carboxylic acid reductase mutant obtained by fragmenting and isolating host cells into which the transformant of the present invention has been introduced, or a lyophilized enzyme powder obtained by lyophilizing the crude enzyme solution. The pure enzyme may be a purified enzyme obtained by purifying the crude enzyme solution.

[0209] In some specific embodiments, the carboxylic acid reductase mutants of the present invention can also increase product concentration when used in combination with other catalysts. For example, when the carboxylic acid reductase mutants of the present invention are used with isopropylamine as an amino group donor, due to the high catalytic efficiency of the carboxylic acid reductase mutants of the present invention, they can further efficiently catalyze the intermediate ω-amino fatty acid, facilitating the equilibrium shift of the previous transaminase step, thereby obtaining a higher concentration of α,ω-diamine.

[0210] In one or more embodiments, the glucose dehydrogenase uses glucose and NADP + As substrate, it catalyzes the oxidation of glucose and NADP+ In one or more specific embodiments, the glucose dehydrogenase is glucose dehydrogenase BmGDH (NCBI Reference Sequence: 1GCO_A) derived from Bacillus megaterium IWG3, which can be used for NADPH regeneration.

[0211] In one or more embodiments, the polyphosphate kinase uses sodium hexametaphosphate as a substrate to catalyze AMP to generate ATP. In one or more specific embodiments, the polyphosphate kinase is polyphosphate kinase PPK12 (NCBI Reference Sequence: HCY06753.1) from Erysipelotrichaceae bacteria, which can be used for ATP regeneration.

[0212] In one or more embodiments, the α,ω-diamine can be a short-chain α,ω-diamine, a medium-chain α,ω-diamine, a medium-long chain α,ω-diamine, or a long-chain α,ω-diamine.

[0213] In one or more embodiments, the short chain refers to a carbon chain with a length of 5C or less, such as a carbon chain with a length of 3C-5C. The medium chain refers to a carbon chain with a length of 6C-9C. The medium-long chain refers to a carbon chain with a length of 10C-12C. The long chain refers to a carbon chain with a length of 13C or more.

[0214] In one or more embodiments, the α,ω-diamine is 1,3-propylenediamine (C3), 1,4-butanediamine (C4), 1,5-pentanediamine (C5), 1,6-hexanediamine (C6), 1,7-heptanediamine (C7), 1,8-octanediamine (C8), 1,9-nonanediamine (C9), 1,10-decanediamine (C10), 1,11-undecanediamine (C11), 1,12-dodecanediamine (C12), or a derivative thereof.

[0215] Those skilled in the art know that when synthesizing α,ω-diamines of different lengths, it is necessary to select α,ω-dicarboxylic acids and / or ω-amino fatty acids of appropriate length as substrates based on the desired chain length of the α,ω-diamine product.

[0216] The method of synthesizing α,ω-diamine by multi-enzyme cascade reaction is known in the art. The conditions such as the enzymatic reaction system, reaction temperature, reaction time, etc. used in the method are all known to those skilled in the art or can be obtained by routine experiments, for example, see Biotechnol Bioeng, 2023, 120, 1773-1783. It should be understood that after knowing the carboxylic acid reductase mutant of the present invention, all methods that can use the carboxylic acid reductase mutant of the present invention to synthesize ω-amino fatty acids and / or α,ω-diamine are included in the scope of protection of the present invention.

[0217] Methods for determining ω-amino fatty acids and / or α,ω-diamines are also known in the art, including but not limited to high performance liquid chromatography, gas chromatography, liquid chromatography-mass spectrometry, and gas chromatography-mass spectrometry.

[0218] Nucleic acid, recombinant expression vector, recombinant expression transformant

[0219] The present invention also provides a polynucleotide sequence encoding the carboxylic acid reductase mutant or its conservative variant protein.

[0220] The polynucleotides of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand.

[0221] The polynucleotide encoding the mature protein of the mutant includes: a coding sequence encoding only the mature protein; a coding sequence of the mature protein and various additional coding sequences; a coding sequence of the mature protein (and optional additional coding sequences) and non-coding sequences.

[0222] A "polynucleotide encoding a protein" may include a polynucleotide encoding the protein, or may include additional coding and / or non-coding sequences.

[0223] The present invention also relates to a vector comprising the polynucleotide of the present invention, a host cell produced by genetic engineering using the vector or carboxylic acid reductase mutant coding sequence of the present invention, and a method for producing the protein of the present invention by recombinant technology.

[0224] The polynucleotide sequences of the present invention can be used to express or produce recombinant carboxylic acid reductase mutants by conventional recombinant DNA techniques. Generally, the following steps are involved:

[0225] (1) Transforming or transducing a suitable host cell with a polynucleotide (or variant) encoding a carboxylic acid reductase mutant of the present invention, or a recombinant expression vector containing the polynucleotide;

[0226] (2) Host cells cultured in a suitable culture medium;

[0227] (3) Isolate and purify proteins from culture medium or cells.

[0228] In the present invention, the carboxylic acid reductase mutant polynucleotide sequence can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses, or other vectors well known in the art. In short, any plasmid or vector can be used as long as it can replicate and be stable in the host. An important feature of an expression vector is that it usually contains an origin of replication, a promoter, a marker gene, and translation control elements.

[0229] In one or more embodiments, other polynucleotide sequences (e.g., polynucleotide sequences encoding other catalysts) other than the carboxylic acid reductase mutant can also be inserted into the recombinant expression vector. Other polynucleotide sequences can be inserted into the same recombinant expression vector as the carboxylic acid reductase mutant polynucleotide sequence, or can be inserted into different recombinant expression vectors.

[0230] Therefore, the present invention also provides a recombinant expression transformant, which includes the recombinant expression vector of the present invention into which the carboxylic acid reductase mutant polynucleotide sequence is inserted. The recombinant expression transformant can be prepared by simultaneously transforming and introducing two or more recombinant expression vectors of the present invention (e.g., a recombinant expression vector into which the carboxylic acid reductase mutant polynucleotide sequence is inserted and a recombinant expression vector into which another catalyst polynucleotide sequence is inserted) into a host cell using conventional methods in the art.

[0231] In one or more specific embodiments, the nucleic acid sequence encoding the carboxylic acid reductase mutant of the present invention can be linked to a pET30b plasmid, the nucleic acid sequence encoding the transaminase can be linked to a pETDuet-1 plasmid (e.g., the second multiple cloning site MCS II), the nucleic acid sequence encoding the glucose dehydrogenase can be linked to a pET-28a plasmid, and the nucleic acid sequence encoding the polyphosphate kinase can be linked to a pET-28a plasmid. In a preferred embodiment, in order to successfully express the carboxylic acid reductase mutant and exhibit catalytic activity, the nucleic acid sequence encoding the phosphopantetheine-modifying enzyme Bssfp (NCBI Reference Sequence: WP_003234549.1) from Bacilus subtilis can be linked to a pCDFDuet plasmid (e.g., the first multiple cloning site MCS I).

[0232] Methods well known to those skilled in the art can be used to construct expression vectors containing a carboxylic acid reductase mutant encoding DNA sequence and appropriate transcription / translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, etc. The DNA sequence can be effectively linked to an appropriate promoter in the expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. The expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selecting transformed host cells.

[0233] A vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell to enable it to express the protein.

[0234] In the present invention, the host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a plant cell. Representative examples of host cells include Escherichia coli (E. coli), Bacillus subtilis, Corynebacterium glutamicum, Saccharomyces cerevisiae, Streptomyces, and Agrobacterium. In a specific embodiment of the present invention, E. coli BL21 (DE3) is used as the host cell.

[0235] Those skilled in the art will appreciate how to select appropriate vectors, promoters, enhancers and host cells.

[0236] Compared with the prior art, the present invention has excellent technical effects:

[0237] 1. Compared with other enzyme catalysts for synthesizing ω-amino fatty acids and α,ω-diamines, the carboxylic acid reductase mutant provided by the present invention has the advantages of high catalytic activity, strong substrate specificity and a wide range of catalytic substrates. The substrate loading capacity and space-time yield both reach the highest level of currently reported biosynthesis, providing new reference and ideas for the industrial synthesis of nylon monomers by biotechnology.

[0238] 2. Because carboxylic acid reductase catalysis requires expensive cofactors NADPH and ATP, the cost of these cofactors increases with increasing substrate loading and expanding the reaction system. The present invention provides a glucose dehydrogenase (BmGDH) derived from Bacillus megaterium IWG3 (NCBI Reference Sequence: 1GCO_A) for NADPH regeneration, and a polyphosphate kinase (PPK12) derived from Erysipelotrichaceae bacteria (NCBI Reference Sequence: HCY06753.1) for ATP regeneration. These two enzymes can efficiently regenerate the cofactors NADPH and ATP, saving cofactor costs in the carboxylic acid reductase catalysis process and enabling industrial scale-up of production.

[0239] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally carried out under conventional conditions or under conditions recommended by the manufacturer.

[0240] Materials and methods

[0241] 1. Multi-enzyme cascade catalysis of α, ω-dicarboxylic acids to synthesize ω-amino fatty acids and α, ω-diamines

[0242] The schematic diagram of the multi-enzyme cascade catalyzing the synthesis of ω-amino fatty acids and α,ω-diamines with different chain lengths in α,ω-dicarboxylic acid is shown in Figure 2.

[0243] The enzymatic reaction was carried out in Hepes-Na buffer at pH 6.5 at 30°C. The reaction system for synthesizing ω-amino fatty acid module 1 included α,ω-dicarboxylic acid (C3-C12) with a final concentration of 100 mM, 1 mM NADP(H), 10 mM ATP, 120 mM MgCl2, 1 mM PLP, 120 mM glucose, 100 mM Poly P6 and 200 mM isopropylamine, as well as appropriate amounts of recombinant carboxylic acid reductase mutant, transaminase, glucose dehydrogenase and polyphosphate kinase.

[0244] The module 1 and module 2 reaction systems for synthesizing α,ω-diamines include α,ω-dicarboxylic acids (C3-C12) at a final concentration of 100 mM, 2 mM NADP(H), 10 mM ATP, 240 mM MgCl2, 1 mM PLP, 240 mM glucose, 200 mM Poly P6, and 400 mM isopropylamine, as well as appropriate amounts of recombinant carboxylic acid reductase mutants, transaminases, glucose dehydrogenases, and polyphosphate kinases.

[0245] Reactions were performed in an EP tube shaker (1000 rpm) or in a reaction flask (8 mL or 200 mL) with 2M NaOH added using a pH stat to maintain a constant pH. The reaction time was determined by complete substrate conversion or when product concentration stopped increasing. During the reaction, 100 μL of the reaction solution was sampled and terminated with 5 μL of 6M HCl.

[0246] 2. Detect the product ω-amino fatty acid using high performance liquid chromatography (HPLC)

[0247] The sample treatment method is as follows: take 20 μL of sample, add 20 μL of acetonitrile, mix well and place in a -20℃ refrigerator for more than half an hour, then centrifuge at 10,000 rpm for 1 min, take 20 μL of supernatant, add 20 μL of 28 mM Marfey and 100 μL of DMSO, mix well, and stand at 40℃ for derivatization for 1 hour. Finally, add 40 μL of 1M HCl to quench, mix well and filter with an organic nylon membrane.

[0248] The specific analysis conditions are as follows:

[0249] The chromatographic column was Ilite C18, the mobile phase was methanol: water (0.1% trifluoroacetic acid) = 50:50 (v / v), the flow rate was 1 mL / min; the column temperature was 35° C., and the UV detection wavelength was 340 nm.

[0250] 3. Detect the product α, ω-diamine using high performance liquid chromatography (HPLC)

[0251] The sample treatment method is as follows: take 42 μL of sample, add 250 μL of dansyl chloride (5 mg / mL) and 208 μL of Hepes-Na buffer (0.1 M, pH 6.5), mix well and let stand for 10 minutes, finally add 500 μL of methanol, mix well and filter with an organic nylon membrane.

[0252] The specific analysis conditions are as follows:

[0253] The chromatographic column was Ilite C18, the mobile phase was methanol: water (0.1% trifluoroacetic acid) = 83:17 (v / v), the flow rate was 1 mL / min; the column temperature was 35° C., and the UV detection wavelength was 254 nm.

[0254] 4. Detection of substrate α, ω-dicarboxylic acid using gas chromatography (GC)

[0255] Take 30 μL of sample and add 300 μL of ethyl acetate (containing internal standard), shake thoroughly for 5-10 minutes, then centrifuge at 12000 rpm for 1 minute, take 150 μL of organic phase, add anhydrous sodium sulfate and shake to dry for more than 1 hour, centrifuge at 12000 rpm for 1 minute, take 50 μL of organic phase and add it to the memory tube, add 10 μL of trimethylsilylated diazomethane and 50 μL of methanol-ethyl ether (v / v=1:1).

[0256] The specific analysis conditions are as follows:

[0257] The chromatographic column was an SH-Rxi-5Sil MS column (30 m × 0.25 mm × 0.25 μm), and nitrogen was used as the carrier gas at a flow rate of 3 mL / min. The injector and detector temperatures were 250°C and 280°C, respectively. The GC detection program was 85°C for 3 minutes, then heated to 100°C at a rate of 5°C / min, then to 160°C at a rate of 10°C / min, and finally to 280°C at a rate of 20°C / min, where it was held at 280°C for 2 minutes.

[0258] 5. Preparation method of ω-amino fatty acid

[0259] A 1000 mL three-necked flask was used as the reactor, and a 200 mL scale reaction was performed according to the above reaction system. After completion of the reaction, the reaction solution was centrifuged at 12,000 rpm for 40 min to remove cells. The reaction solution was acidified to pH 3.0 by adding 6 M HCl solution, and the precipitated protein was removed by further centrifugation. The reaction solution was concentrated to reduce the reaction volume and increase the product concentration. Unreacted substrate and intermediates were then extracted with ethyl acetate (3 × 20 mL). The remaining aqueous phase primarily contained the product and salts from the buffer. After the aqueous phase was carefully neutralized by the addition of 6 M NaOH, an equal volume of tert-butyl alcohol, 1.1 equiv. NaOH, and 2.0 equiv. di-tert-butyl dicarbonate (Boc-anhydride) were added. The reaction mixture was stirred at room temperature for 16 h (see reaction scheme in Figure 1). After the reaction, 2M HCl was slowly added to neutralize the mixture in an ice-water bath, and then extracted with ethyl acetate (1×50 mL+2×20 mL). The ethyl acetate phases were combined, washed with a saturated NaCl solution, dried over anhydrous sodium sulfate, and then concentrated in vacuo by a rotary evaporator to obtain a crude product. The crude product was further purified by silica gel column chromatography using a mobile phase of ethyl acetate: petroleum ether: acetic acid = 75:200:1. The purer mobile phase was collected and concentrated in vacuo by a rotary evaporator to obtain a purer product. The product was analyzed by GC-MS or H 1 The structure was further characterized and identified by NMR spectroscopy.

[0260] Example 1: Preparation of carboxylic acid reductase MabCAR3 mutants

[0261] In this example, a gene encoding a carboxylic acid reductase from Mycobacteroides abscessus (NCBI Reference Sequence: WP_052537360.1) was first obtained through gene mining. The recombinant expression vector pET30b containing this gene and the recombinant expression transformant E. coli BL21(DE3) were used. Site-directed mutagenesis or multi-site combination mutagenesis was performed on positions 284, 303, 306, 342, 344, 393, 395, 418, and 426 of the amino acid sequence shown in SEQ ID No. 2. Site-directed mutagenesis was performed using the protocol described in the II Site-Directed Mutegenesis Kit (Stratagenem Catalog #200502). First, mutagenic primers containing mutation points were designed. The mutagenic primers required for the present invention are commonly used in the art. The amino acid residues in SEQ ID No. 2 are simply mutated to the desired amino acid residues using standard genetic engineering techniques, and the specific mutation code is not limited. Site-directed mutagenic primers for introducing mutations at positions 284, 303, 306, 342, 344, 393, 395, 418, and 426 in the sequence shown are all site-directed mutagenic primers.

[0262] PCR reaction system (10 μL): 20-50 ng template, 0.5 μL each of a pair of mutation primers (10 μM), 5 μL PrimeStarmix, and sterile double-distilled water to make up the system to 10 μL. The template is carboxylic acid reductase (MabCAR3, WP_198151610.1, SEQ ID No. 2).

[0263] PCR reaction procedure: (1) denaturation at 95°C for 3 min; (2) denaturation at 98°C for 10 s; (3) annealing at 55°C for 5 s; (4) extension at 72°C for 9 min; steps (2) to (4) were extended for 25 cycles, with a final extension at 72°C for 10 min, and the product was stored at 4°C.

[0264] For multi-point combination mutations, in order to further improve the mutation efficiency, a short gene fragment containing multiple combination mutation sites is designed, and the gene fragment corresponding to the wild-type carboxylic acid reductase is replaced by a one-step cloning method.

[0265] The resulting PCR product was digested with the restriction endonuclease Dpn I at 37°C for 0.5-2 hours, then transformed into competent E. coli BL21(DE3) cells and evenly plated onto agar plates containing LB medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 20 g / L agar powder) supplemented with 50 μg / mL kanamycin. After incubation at 37°C for 10-16 hours, single colonies were selected, resulting in the E. coli BL21(DE3) strain containing the mutant expression plasmid. The strains were then sent to Shanghai Qingke Biotechnology Co., Ltd. for sequencing analysis. The sequencing results were aligned with the wild-type carboxylic acid reductase gene sequence using SnapGene software to confirm differences in the gene sequence and corresponding amino acid sequence before and after the mutation.

[0266] The gene sequences of other biological enzyme catalysts required for the multi-enzyme cascade reaction of the present invention, such as transaminase, glucose dehydrogenase, and polyphosphate kinase, were synthesized by Shanghai Qingke Biotechnology Co., Ltd. after codon optimization based on the Escherichia coli expression system, introduced into the corresponding vector plasmids, and finally transformed into Escherichia coli BL21 (DE3).

[0267] Example 2: Co-expression of the carboxylic acid reductase MabCAR3 mutant gene and the phosphopantetheinyl transferase sfp gene

[0268] Plasmids were extracted from E. coli BL21(DE3) strains harboring the mutant plasmids using a Qiagen mini-plasmid extraction kit. Simultaneously, the sfp-expressing plasmid pCDFDuet was extracted in bulk from transformants harboring sfp. The resulting dual plasmids were chemically transformed into competent E. coli BL21(DE3) cells and evenly plated onto LB agar plates (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 20 g / L agar powder) containing 50 μg / mL kanamycin and streptomycin. Cultures were performed at 37°C for 12-18 hours, and single colonies were isolated to obtain strains expressing the different carboxylic acid reductase mutants. These strains also co-expressed sfp, which modifies the terminal end of the carboxylic acid reductase to enhance catalytic efficiency.

[0269] Example 3: Preparation of MabCAR3 and its mutants, transaminase, glucose dehydrogenase / formate dehydrogenase and polyphosphate kinase enzyme catalysts

[0270] Preparation of crude enzyme: Monoclonal colonies obtained in Examples 1 and 2 were transferred to 4 mL of LB medium (containing streptomycin and kanamycin) and cultured in a shaker at 37°C and 180 rpm for 12-16 hours. Subsequently, a 1% (v / v) inoculum was inoculated into 100 mL of LB medium (containing streptomycin, kanamycin, or ampicillin) and cultured in a shaker at 37°C and 180 rpm. When the OD value of the culture reached 0.6-0.8, isopropyl-β-D-thiogalactopyranoside (IPTG) was added as an inducer at a final concentration of 0.2 mM and induced at 16°C for 16-24 hours. The culture was centrifuged at 10,000 rpm for 3 minutes, and the precipitate was washed twice with physiological saline to obtain recombinant expression transformant cells. The harvested recombinant cells were then suspended in 10 mL of Buffer A, ultrasonically disrupted, and the supernatant was collected by centrifugation to obtain the crude enzyme solutions of the recombinant carboxylic acid reductase mutant, transaminase, glucose dehydrogenase, and polyphosphate kinase. The obtained crude enzyme solutions can also be further concentrated 10-20 times using a 100 kDa or 10 kDa ultrafiltration tube, and then re-dissolved and stored in a -80°C refrigerator.

[0271] Preparation of Pure Enzymes: The crude enzyme solutions of the recombinant carboxylic acid reductase mutant, transaminase, glucose dehydrogenase, and polyphosphate kinase were further purified. The following protein purification buffer recipes are as follows: Buffer A: 25 mM Hepes-Na, pH 7.5, 500 mM NaCl, 20 mM imidazole; Buffer B: 25 mM Hepes-Na, pH 7.5, 500 mM NaCl, 500 mM imidazole; Buffer C: 25 mM Hepes-Na, pH 7.5, 150 mM NaCl, 1 mM DTT, 5% glycerol. The crude enzyme solution of the crushed carboxylic acid reductase was loaded onto a nickel column equilibrated with solution A. After loading, the impurities in the column were eluted with 10% solution B, and then the target protein was eluted with 50% solution B. SDS-PAGE was used to verify whether the target protein was present. The eluate containing the target protein was then collected and concentrated by ultrafiltration using an ultrafiltration tube (10 kDa or 30 kDa). The eluate was then replaced twice with solution C to remove the imidazole in the protein solution. The solution was quickly frozen in liquid nitrogen and stored at -80°C for later use.

[0272] Preparation of lyophilized enzyme powder: After collecting the wet cells by centrifugation, resuspend the cells (150-200 g / L) in sodium phosphate buffer (100 mM, pH 7.5), sieve, and disrupt the cells three times using a high-pressure homogenizer (700-800 MPa). The supernatant was collected by centrifugation at 8000 rpm for 40 min at 4°C and frozen overnight at -80°C. The frozen enzyme solution was lyophilized using a freeze dryer, and the resulting lyophilized enzyme powder was collected and stored sealed at 4°C.

[0273] Example 4: Specific activity assay of MabCAR3 and its mutants against model substrates adipic acid and 6-aminohexanoic acid

[0274] Since the reaction of MabCAR3 catalytic mode substrate adipic acid requires the consumption of cofactor NADPH, and NADPH has a higher absorption value at 340nm, the corresponding product NADP + There is no absorbance value, so the ΔOD is detected by spectrophotometer 340 The specific activity of MabCAR3 was calculated based on the reduction in activity within 1 min.

[0275] The specific activity assay system of MabCAR3 and its mutants for adipic acid and 6-aminohexanoic acid was as follows: the total reaction system was 1 mL, the buffer used was 100 mM Hepes-Na buffer (pH 7.5), the reaction was carried out at 30°C, the reaction system included adipic acid or 6-aminohexanoic acid at a final concentration of 10 mM, 0.15 mM NADPH, 1 mM ATP and 10 mM MgCl2, and an appropriate amount of pure enzyme catalyst of the recombinant carboxylic acid reductase mutant to ensure ΔOD 340 Between 0.05-0.2, the enzyme activity measurement data is more accurate.

[0276] First, we performed site-directed saturation mutagenesis on some key amino acid sites based on Rosetta energy scores and NAC frequencies, and conducted virtual screening of a total of more than 8,000 mutants. We then selected a small number of mutants for wet experiment verification, and then selected the advantageous single-point mutants after experimental verification for virtual screening calculations of combination mutations. Finally, we selected the advantageous multi-point combination mutants after virtual screening for wet experiment verification.

[0277] When synthesizing 6-aminohexanoic acid, our optimal carboxylic acid reductase mutants exhibit increased specific activity toward the substrate adipic acid, while simultaneously exhibiting decreased or even no specific activity toward the product 6-aminohexanoic acid. Results showed that the wild-type MabCAR3 pure enzyme exhibited a specific activity of 0.20 U / mg toward the substrate adipic acid. Furthermore, a series of single- or multi-point advantageous mutants with enhanced specific activity were also obtained. The mutation sequences and the changes in catalytic activity toward adipic acid are listed in Table 1. In the table in Table 1, the sequence numbers refer to the corresponding series of sequences at the end of Table 1. In the mutant specific activity improvement factor, one plus sign "+" indicates that the mutant protein has a 0.1-1.5-fold improvement in specific activity against adipic acid compared to the protein consisting of the amino acid sequence of SEQ ID No. 2; two plus signs "++" indicate that the mutant protein has a 1.6-3.0-fold improvement in specific activity against adipic acid compared to the protein consisting of the amino acid sequence of SEQ ID No. 2; three plus signs "+++" indicate that the mutant protein has a 3.1-5.0-fold improvement in specific activity against adipic acid compared to the protein consisting of the amino acid sequence of SEQ ID No. 2. Four plus signs "++++" indicate that the mutant protein has a 5.0-fold improvement in specific activity against adipic acid compared to the protein consisting of the amino acid sequence of SEQ ID No. 2.

[0278] The wild-type MabCAR3 pure enzyme exhibited a specific activity of 0.017 U / mg for the product 6-aminohexanoic acid. A series of single-point dominant mutants with reduced specific activity were also obtained. The mutant sequences and the changes in their catalytic activity for 6-aminohexanoic acid are listed in Table 1. In Table 1, the sequence numbers refer to the corresponding sequences following Table 1. In the fold reduction of specific activity for a mutant, a single plus sign "+" indicates that the specific activity of the mutant protein for 6-aminohexanoic acid is reduced by more than 40% compared to the protein consisting of the amino acid sequence of SEQ ID No. 2; two plus signs "++" indicate that the specific activity of the mutant protein for 6-aminohexanoic acid is reduced by 20%-40% compared to the protein consisting of the amino acid sequence of SEQ ID No. 2; and three plus signs "+++" indicate that the specific activity of the mutant protein for 6-aminohexanoic acid is reduced by 10%-20% compared to the protein consisting of the amino acid sequence of SEQ ID No. 2. Four plus signs "++++" indicate that the specific activity of the mutant protein toward 6-aminohexanoic acid is reduced to less than 10% of the original activity compared with the protein consisting of the amino acid sequence shown in SEQ ID No. 2.

[0279] Table 1 The ratio of the increased specific activity of MaCAR3 and its mutants for substrate adipic acid (AA) and the decreased specific activity of product 6-aminocaproic acid (6-ACA)

[0280] To further enhance the efficient synthesis of hexamethylenediamine, we sought to identify mutants with enhanced catalytic efficiency for 6-aminohexanoic acid. We first attempted to determine the specific activity of single-point mutants screened after virtual screening. The results showed that the wild-type MabCAR3 pure enzyme had a specific activity of 0.017 U / mg for 6-aminohexanoic acid. The mutant sequences and the fold increase in catalytic activity for 6-aminohexanoic acid are listed in Table 2. In Table 2, the sequence numbers refer to the corresponding sequences following Table 2. In the fold increase in specific activity, a single plus sign ("+") indicates that the mutant protein's specific activity for 6-aminohexanoic acid is 0.1-1.5 times higher than that of the protein consisting of the amino acid sequence of SEQ ID No. 2; two plus signs ("++") indicate that the mutant protein's specific activity for 6-aminohexanoic acid is 1.6-3.0 times higher than that of the protein consisting of the amino acid sequence of SEQ ID No. 2; and three plus signs ("+++") indicate that the mutant protein's specific activity for 6-aminohexanoic acid is 3.1-5.0 times higher than that of the protein consisting of the amino acid sequence of SEQ ID No. 2. Four plus signs "++++" indicate that the specific activity of the mutant protein toward 6-aminohexanoic acid is more than 5.0 times higher than that of the protein consisting of the amino acid sequence shown in SEQ ID No. 2.

[0281] Table 2 Increased folds of specific activity of MaCAR3 and its mutants against 6-aminohexanoic acid (6-ACA)

[0282] Single-point mutations yielded promising results, successfully generating mutants with significantly improved specific activity against 6-ACA. Therefore, further combinations of the six dominant mutations, L284 / L306 / L342 / G393 / G418 / A303, were evaluated, yielding a cumulative total of over 8,000 mutants. Finally, 18 combined mutants, shown in Table 3, were selected for determination of their specific activity against adipic acid and 6-aminocaproic acid. The fold increase values ​​for the mutants in Table 3 are indicated as described in Table 1, differing from Table 1 in that Table 3 uses a "+" to indicate a fold increase in 6-ACA activity, rather than a fold decrease. As shown in Table 3, multiple multi-point combined mutants significantly improved the specific activity against both adipic acid and 6-aminocaproic acid, with increases exceeding 5-fold simultaneously and a maximum increase exceeding 65-fold for 6-aminocaproic acid. These dominant combined mutants can be used for the efficient synthesis of α,ω-diamines in subsequent examples.

[0283] Table 3 Increased folds of specific activity of MaCAR3 and its mutants towards substrate adipic acid (AA) and product 6-aminocaproic acid (6-ACA)

[0284] Example 6: Wild-type carboxylic acid reductase MabCAR3 WTApplication in the synthesis of 6-aminocaproic acid

[0285] The present invention is not limited to the conversion of α,ω-dicarboxylic acids into ω-amino fatty acids and α,ω-diamines, but is also applicable to the conversion of other similar α,ω-dicarboxylic acids into ω-amino fatty acids and α,ω-diamines.

[0286] In this example, glucose dehydrogenase GDH is used to regenerate NADPH, polyphosphate kinase PPK is used to regenerate ATP, and CAR and TA are used to cascade catalyze the synthesis of 6-aminocaproic acid from adipic acid. The reaction system is 8 mL and is carried out in a Hepes-Na buffer solution with a pH of 6.5 at 30°C (temperature controlled by water circulation) and 800 rpm. Since a large amount of acid is produced during the cascade reaction, 2M NaOH is added through a pH Stat flow to control the pH of the reaction solution. The reaction system includes a final concentration of 100 mM AA, 1 mM NADP (H), 10 mM ATP, 120 mM MgCl2, 1 mM PLP, 120 mM glucose, 100 mM Poly P6, and 200 mM isopropylamine, as well as 50 mg wet cell / mL recombinant carboxylic acid reductase MabCAR3 WT Crude cell extract, 9U transaminase SpTA, 50U glucose dehydrogenase BmGDH and 10U polyphosphate kinase PPK12, sample was taken after 18h reaction.

[0287] The production of 6-aminocaproic acid was detected by HPLC, and the consumption of substrate adipic acid and possible by-products 6-hydroxycaproic acid and hexanediol were detected by GC. The yield of 6-aminocaproic acid reached 24.8% (titer: 24.8mM), and no by-products 6-hydroxycaproic acid and hexanediol were detected. At the same time, a small amount of hexanediamine was detected, indicating that the wild-type carboxylic acid reductase MabCAR3 WT It has catalytic activity towards 6-aminohexanoic acid, but its substrate specificity is poor and needs further improvement.

[0288] Example 7: Carboxylic acid reductase mutant MabCAR3 M9 Application in the synthesis of 6-aminocaproic acid

[0289] In this example, glucose dehydrogenase GDH is used to regenerate NADPH, polyphosphate kinase PPK is used to regenerate ATP, and CAR and TA are used to cascade catalyze the synthesis of 6-aminocaproic acid from adipic acid. The reaction system is 8 mL and is carried out in a Hepes-Na buffer solution with a pH of 6.5 at 30°C (temperature controlled by water circulation) and 800 rpm. Since a large amount of acid is produced during the cascade reaction, 2M NaOH is added through a pH Stat flow to control the pH of the reaction solution. The reaction system includes a final concentration of 100 mM AA, 1 mM NADP (H), 10 mM ATP, 120 mM MgCl2, 1 mM PLP, 120 mM glucose, 100 mM Poly P6, and 200 mM isopropylamine, as well as 50 mg wet cell / mL recombinant carboxylic acid reductase MabCAR3 M9 Crude cell extract, 9U transaminase SpTA, 50U glucose dehydrogenase BmGDH and 10U polyphosphate kinase PPK12, sample was taken after 18h reaction.

[0290] The production of the product 6-aminohexanoic acid was detected by HPLC, and the consumption of the substrate adipic acid and possible by-products 6-hydroxyhexanoic acid and hexanediol were detected by GC. The yield of 6-aminohexanoic acid reached 65.1% (titer: 65.1mM), and no by-products 6-hydroxyhexanoic acid and hexanediol were detected. At the same time, no hexanediamine was detected, indicating that the carboxylic acid reductase mutant MabCAR3 M9 It has no catalytic activity towards 6-aminohexanoic acid and has strong substrate specificity. This mutant successfully reduces the catalytic efficiency towards 6-ACA while improving the catalytic efficiency towards AA.

[0291] Example 8: Carboxylic acid reductase mutant MabCAR3 M9 Application of formate dehydrogenase cascade in the synthesis of 6-aminocaproic acid

[0292] In this example, formate dehydrogenase FDH is used to regenerate NADPH, polyphosphate kinase PPK is used to regenerate ATP, and CAR and TA are used to cascade catalyze the synthesis of 6-aminocaproic acid from adipic acid. The reaction system is 8 mL and is carried out in a Hepes-Na buffer solution with a pH of 6.5 at 30°C (temperature controlled by water circulation) and 800 rpm. Since a large amount of acid is produced during the cascade reaction, 2M NaOH is added through a pH Stat flow to control the pH of the reaction solution. The reaction system includes a final concentration of 100 mM AA, 1 mM NADP (H), 10 mM ATP, 120 mM MgCl2, 1 mM PLP, 120 mM sodium formate, 100 mM Poly P6, and 200 mM isopropylamine, as well as 50 mg wet cell / mL recombinant carboxylic acid reductase MabCAR3 M9Crude cell extract, 9U transaminase SpTA, 50U formate dehydrogenase BstFDH and 10U polyphosphate kinase PPK12, sample was taken after 18h reaction.

[0293] The production of the product 6-aminocaproic acid was detected by HPLC, and the consumption of the substrate adipic acid and possible by-products such as 6-hydroxycaproic acid and hexanediol were detected by GC. The yield of 6-aminocaproic acid reached 62.4% (titer: 62.4 mM), and no by-products such as 6-hydroxycaproic acid and hexanediol were detected. The titer of the 6-aminocaproic acid product in this example was almost the same as that in Example 7 when glucose dehydrogenase GDH was used to regenerate NADPH.

[0294] Example 9: Application of carboxylic acid reductase MabCAR3 mutants in the synthesis of short-chain ω-amino fatty acids (C3-C5)

[0295] In this embodiment, glucose dehydrogenase GDH is used to regenerate NADPH, polyphosphate kinase PPK is used to regenerate ATP, and CAR and TA are used to cascade catalyze the synthesis of ω-amino fatty acids (C3-C5) from α, ω-dicarboxylic acids (C3-C5). Other bio-enzyme catalysts used in this route to regenerate NADPH and ATP are also protected by this patent. The reaction system is 8 mL, and is carried out in Hepes-Na buffer at pH 6.5 at 30°C (temperature controlled by water circulation) and 800 rpm. Since a large amount of acid is produced during the cascade reaction, 2M NaOH is added through pH Stat to control the pH of the reaction solution. The reaction system includes a final concentration of 50 mM malonic acid / succinic acid / glutaric acid, 0.5 mM NADP (H), 5 mM ATP, 60 mM MgCl2, 1 mM PLP, 60 mM glucose, 50 mM Poly P6 and 100 mM isopropylamine, as well as 25 mg wet cell / mL recombinant carboxylic acid reductase MabCAR3 mutant crude cell extract, 4.5U transaminase SpTA, 25U glucose dehydrogenase BmGDH and 5U polyphosphate kinase PPK12 lyophilized enzyme powder, the reaction was carried out for 18h and the sample was taken.

[0296] The production of the products 3-aminopropionic acid, 4-aminobutyric acid, and 5-aminovaleric acid was determined by HPLC, and the consumption of the substrates malonate, succinate, and glutarate, as well as possible byproducts such as 3-hydroxypropionic acid, 4-hydroxybutyrate, 5-hydroxyvaleric acid, and propylene glycol, butylene glycol, and pentanediol, was determined by GC. As shown in Table 4, the product titer of 5-aminovaleric acid reached 20.3 mM, a significant decrease compared to 6-aminohexanoic acid. This was primarily due to a decrease in the number of carbon atoms to shorter chain carbons, which significantly reduced the CAR specific activity. Furthermore, the product titer also showed a gradually decreasing trend with decreasing carbon number. The product titers of 3-aminopropionic acid and 4-aminobutyric acid were 11.6 mM and 15.8 mM, respectively.

[0297] Table 4 MabCAR3 mutants and transaminase cascade catalyze the synthesis of short-chain α,ω-dicarboxylic acids (C3-C5) into ω-amino fatty acids

[0298] Example 10: Application of carboxylic acid reductase MabCAR3 mutants in the synthesis of medium-chain ω-amino fatty acids (C7-C9)

[0299] In this embodiment, glucose dehydrogenase GDH is used to regenerate NADPH, polyphosphate kinase PPK is used to regenerate ATP, and CAR and TA are used to cascade catalyze the synthesis of ω-amino fatty acids (C7-C9) from α, ω-dicarboxylic acids (C7-C9). Other bio-enzyme catalysts used in this route to regenerate NADPH and ATP are also protected by this patent. The reaction system is 8 mL, and is carried out in Hepes-Na buffer at pH 6.5 at 30°C (temperature controlled by water circulation) and 1000 rpm. Since a large amount of acid is produced during the cascade reaction, 2M NaOH is added through pH Stat to control the pH of the reaction solution. The reaction system includes a final concentration of 100 mM pimelic acid / suberic acid / zelaic acid, 1 mM NADP (H), 10 mM ATP, 120 mM MgCl2, 1 mM PLP, 120 mM glucose, 100 mM Poly P6 and 200 mM isopropylamine, as well as 50 mg wet cell / mL recombinant carboxylic acid reductase MabCAR3 mutant crude cell extract, 9U transaminase SpTA, 50U glucose dehydrogenase BmGDH and 10U polyphosphate kinase PPK12, and the sample was taken after 18h of reaction.

[0300] The production of the products 7-aminoheptanoic acid, 8-aminooctanoic acid, and 9-aminononanoic acid was determined by HPLC, and the consumption of the substrates pimelic acid, suberic acid, and nonanedioic acid, as well as the possible byproducts 7-hydroxyheptanoic acid, 8-hydroxyoctanoic acid, 9-hydroxynonanoic acid, and heptanediol, octanediol, and nonanediol, were determined by GC. As shown in Table 5, the yield of 7-aminoheptanoic acid was higher than that of 6-aminohexanoic acid, reaching 74.3% (74.3 mM titer). The titers of 8-aminooctane and 9-aminononanoic acid were further increased, reaching 82.5 mM and 90.3 mM, respectively. No byproducts were detected.

[0301] Table 5 MabCAR3 mutants and transaminase cascade catalyze the synthesis of ω-amino fatty acids from medium-chain α,ω-dicarboxylic acids (C7-C9)

[0302] Example 11: Application of carboxylic acid reductase MabCAR3 mutants in the synthesis of medium- and long-chain ω-amino fatty acids (C10-C12)

[0303] In this embodiment, glucose dehydrogenase GDH is used to regenerate NADPH, polyphosphate kinase PPK is used to regenerate ATP, and CAR and TA are used to cascade catalyze the synthesis of ω-amino fatty acids (C10-C12) from α, ω-dicarboxylic acids (C10-C12). Other bio-enzyme catalysts used in this route to regenerate NADPH and ATP are also protected by this patent. The reaction system is 8 mL, and is carried out in Hepes-Na buffer at pH 6.5 at 30°C (temperature controlled by water circulation) and 1000 rpm. Since a large amount of acid is produced during the cascade reaction, 3M NaOH is added through pH Stat to control the pH of the reaction solution. The reaction system includes a final concentration of 200 mM sebacic acid / undecanedioic acid / dodecanedioic acid, 1 mM NADP (H), 15 mM ATP, 240 mM MgCl2, 1 mM PLP, 240 mM glucose, 200 mM Poly P6 and 400 mM isopropylamine, as well as 100 mg wet cell / mL recombinant carboxylic acid reductase MabCAR3 mutant crude cell extract, 18U transaminase SpTA, 100U glucose dehydrogenase BmGDH and 20U polyphosphate kinase PPK12, and the sample was taken after 18h of reaction.

[0304] The production of the products 10-aminodecanoic acid, 11-aminoundecanoic acid, and 12-aminododecanoic acid was determined by HPLC, and the consumption of the substrates sebacic acid, undecanoic acid, and dodecanedioic acid, as well as the possible byproducts 10-hydroxydecanoic acid, 11-hydroxyundecanoic acid, 12-hydroxydodecanoic acid, and decanediol, undecanediol, and dodecanediol, was determined by GC. The CAR specific activity tended to increase with increasing carbon chain length, so the substrate loading was further increased to 200 mM. The results, shown in Table 6, showed a 10-aminodecanoic acid titer of 110.4 mM. The titers of the products 11-aminoundecanoic acid (122.3 mM titer) and 12-aminododecanoic acid (103.9 mM titer) were comparable to those of 10-aminodecanoic acid, and no byproducts were detected.

[0305] Table 6 MabCAR3 mutants and transaminase cascade catalyze the synthesis of ω-amino fatty acids from medium- and long-chain α,ω-dicarboxylic acids (C10-C12)

[0306] Example 12: Carboxylic acid reductase mutant MabCAR3 M9 Scale-up of the 6-aminocaproic acid synthesis system and product preparation

[0307] 6-aminocaproic acid is used as a monomer of nylon 6. In this embodiment, the reaction system is further expanded to a 300 mL system, and glucose dehydrogenase GDH is used to regenerate NADPH, polyphosphate kinase PPK is used to regenerate ATP, and CAR and TA are used to cascade catalyze the synthesis of 6-aminocaproic acid from adipic acid. The reaction is carried out in a Hepes-Na buffer solution with a pH of 6.5, at 30°C (temperature controlled by water circulation), and at 800 rpm. Since a large amount of acid is produced during the cascade reaction, 2M NaOH is added by pH Stat to control the pH of the reaction solution. The reaction system includes a final concentration of 100 mM AA, 1 mM NADP + , 10mM ATP, 120mM MgCl2, 1mM PLP, 120mM glucose, 100mM Poly P6 and 200mM isopropylamine, and 50mg wet cell / mL recombinant carboxylic acid reductase MabCAR3 M9 Crude cell extract, 225 U transaminase SpTA, 500 U glucose dehydrogenase BmGDH, and 250 U polyphosphate kinase PPK12 were added after 18 hours of reaction. Due to the large reaction system and the high consumption of freeze-dried enzyme powder, all four component enzymes were directly crushed into the crude enzyme solution after centrifugation using a high-pressure homogenizer (as prepared in Example 3). A high-pressure homogenizer can crush highly concentrated bacterial cells, which is equivalent to crushing the concentrated crude enzyme solution using an ultrasonic crusher.

[0308] The production of the product 6-aminocaproic acid was determined by HPLC, while the consumption of the substrate adipic acid and possible byproducts such as 6-hydroxycaproic acid and hexanediol were measured by GC. The yield of 6-aminocaproic acid reached 55.1% (55.1 mM), with no detectable byproducts. Finally, 6-aminocaproic acid was isolated from the reaction mixture according to the ω-amino fatty acid preparation method. GC-MS verification revealed a yield of 42.1%, successfully achieving gram-scale production of nylon monomer.

[0309] Example 13: Wild-type carboxylic acid reductase MabCAR3 WT Application in the synthesis of 1,6-hexanediamine

[0310] In this embodiment, glucose dehydrogenase GDH is used to regenerate NADPH, polyphosphate kinase PPK is used to regenerate ATP, and CAR and TA cascade catalyze adipic acid to first synthesize 6-aminohexanoic acid, and then CAR and TA cascade catalyze 6-aminohexanoic acid to synthesize 1,6-hexanediamine. Other bio-enzyme catalysts that regenerate NADPH and ATP can also be applied to this route. The reaction system is 8 mL, and is carried out in Hepes-Na buffer at pH 6 at 30°C (temperature controlled by water circulation) and 800 rpm. Since a large amount of acid is produced during the cascade reaction, 2M NaOH is added through pH Stat to control the pH of the reaction solution. The reaction system includes a final concentration of 100 mM AA, 2 mM NADP (H), 10 mM ATP, 240 mM MgCl2, 1 mM PLP, 240 mM glucose, 200 mM Poly P6 and 400 mM isopropylamine, as well as 100 mg wet cell / mL recombinant carboxylic acid reductase MabCAR3 WT Crude cell extract, 18U transaminase SpTA, 50U glucose dehydrogenase BmGDH and 20U polyphosphate kinase PPK12 freeze-dried enzyme powder, sampled after 18h of reaction.

[0311] The concentrations of the product 1,6-hexanediamine and the intermediate 6-aminocaproic acid were detected by HPLC, and the consumption of the substrate adipic acid and possible by-products 6-hydroxycaproic acid and hexanediol were detected by GC. The concentration of the product 1,6-hexanediamine was 1.4 mM, and most of it accumulated in the intermediate 6-aminocaproic acid. No by-products 6-hydroxycaproic acid and hexanediol were detected. This result indicates that the wild-type carboxylic acid reductase MabCAR3 WT The catalytic activity for 6-aminocaproic acid is too low, which leads to the accumulation of the intermediate product 6-aminocaproic acid and the low concentration of the final product 1,6-hexanediamine.

[0312] Example 14: Carboxylic acid reductase mutant MabCAR3 M39 and MabCAR3 M42 Application in the synthesis of 1,6-hexanediamine

[0313] In this example, glucose dehydrogenase GDH was used to regenerate NADPH, polyphosphate kinase PPK was used to regenerate ATP, and MabCAR3 M39 and TA cascade catalyzed adipic acid to synthesize 6-aminocaproic acid, and then MabCAR3 M42The TA cascade catalyzed synthesis of 1,6-hexanediamine from 6-aminocaproic acid was carried out in an 8 mL reaction system in a pH 6.5 Hepes-Na buffer at 30°C (controlled by water circulation) and 800 rpm. Since the cascade reaction produces a large amount of acid, the pH of the reaction solution was controlled by adding 2M NaOH via a pH Stat flow control. The reaction system included a final concentration of 100 mM AA, 1 mM NADP(H), 10 mM ATP, 240 mM MgCl2, 1 mM PLP, 240 mM glucose, 200 mM Poly P6, and 400 mM isopropylamine, as well as 8.3 mg wet cell / mL recombinant carboxylic acid reductase MabCAR3 M39 Crude cell extract, 91.7 mg wet cell / mL recombinant carboxylic acid reductase MabCAR3 M42 Crude cell extract, 18U transaminase SpTA, 50U glucose dehydrogenase BmGDH and 20U polyphosphate kinase PPK12, sampled after 18h reaction. M39 The mutant MabCAR3 has higher catalytic efficiency for AA. M42 The catalytic efficiency for 6-ACA is higher. In this cascade reaction, the ratio of the two carboxylic acid reductase mutants can be adjusted to control the specific activity for 6-ACA to be slightly higher than that for AA. The subsequent cascade reaction promotes the progress of the previous cascade reaction, thereby increasing the yield of the final product 1,6-hexanediamine.

[0314] The concentrations of the product 1,6-hexanediamine and the intermediate 6-aminocaproic acid were detected by HPLC, and the consumption of the substrate adipic acid and possible by-products 6-hydroxycaproic acid and hexanediol were detected by GC. The concentration of the product 1,6-hexanediamine increased to 49.5mM, and some of the remaining accumulated in the intermediate 6-aminocaproic acid (25.1mM) and the substrate adipic acid (15.3mM) in a small amount and was not completely consumed. No by-products 6-hydroxycaproic acid and hexanediol were detected. The results showed that compared with the wild-type carboxylic acid reductase MabCAR3 WT , mutant MabCAR3 M39 、MabCAR3 M42 The catalytic activity for adipic acid and 6-aminocaproic acid was significantly improved, resulting in a 35-fold increase in the final product 1,6-hexanediamine, and a significant reduction in the accumulation of the intermediate product 6-aminocaproic acid, with the substrate adipic acid almost completely consumed.

[0315] Example 15: Application of Carboxylic Acid Reductase MabCAR3 Mutants in the Synthesis of Short-Chain α,ω-Diamines (C3-C5)

[0316] In this embodiment, glucose dehydrogenase GDH is used to regenerate NADPH, polyphosphate kinase PPK is used to regenerate ATP, and CAR and TA are used to cascade the continuous carboxylic acid reductive amination to catalyze the synthesis of α, ω-diamine (C3-C5) from α, ω-dicarboxylic acid (C3-C5). Other bio-enzyme catalysts used in this route to regenerate NADPH and ATP are also protected by this patent. The system is 8 mL and is carried out in Hepes-Na buffer at pH 6.5 at 25°C (temperature controlled by water circulation) and 800 rpm. Since a large amount of acid is produced during the cascade reaction, 2M NaOH is added through pH Stat to control the pH of the reaction solution. The reaction system includes a final concentration of 50 mM malonic acid / succinic acid / glutaric acid, 0.5 mM NADP (H), 5 mM ATP, 120 mM MgCl2, 1 mM PLP, 120 mM glucose, 100 mM Poly P6 and 200 mM isopropylamine, as well as 4.2 mg wet cell / mL recombinant carboxylic acid reductase Crude cell extract, 45.8 mg wet cell / mL recombinant carboxylic acid reductase Crude cell extract, 9U transaminase SpTA, 25U glucose dehydrogenase BmGDH and 10U polyphosphate kinase PPK12 freeze-dried enzyme powder, sampled after 18h of reaction.

[0317] The concentrations of the products 1,3-propylenediamine / 1,4-butanediamine / 1,5-pentanediamine and the intermediates 3-aminopropionic acid / 4-aminobutyric acid / 5-aminovaleric acid were determined by HPLC. The consumption of the substrates malonic acid / succinic acid / glutaric acid, as well as possible byproducts such as ω-hydroxy fatty acids and α,ω-diols, were determined by GC. As shown in Table 7, the product concentrations of 1,3-propylenediamine, 1,4-butanediamine, and 1,5-pentanediamine were 16.2 mM, 26.1 mM, and 31.3 mM, respectively. No byproducts were detected, and a small amount of the intermediate ω-amino fatty acid accumulated.

[0318] Table 7 MabCAR3 mutants and transaminase cascade catalyze the synthesis of short-chain α,ω-dicarboxylic acids (C3-C5) into α,ω-diamines

[0319] Example 16: Application of Carboxylic Acid Reductase MabCAR3 Mutants in the Synthesis of Medium-Chain α,ω-Diamines (C7-C9)

[0320] In this embodiment, glucose dehydrogenase GDH is used to regenerate NADPH, polyphosphate kinase PPK is used to regenerate ATP, and CAR and TA are used in a cascade to continuously reduce and aminize α, ω-dicarboxylic acids (C7-C9) to synthesize α, ω-diamines (C7-C9). Other bio-enzyme catalysts used in this route to regenerate NADPH and ATP are also protected by this patent. The reaction system is 8 mL, and is carried out in Hepes-Na buffer at pH 6.5 at 30°C (temperature controlled by water circulation) and 1000 rpm. Since a large amount of acid is produced during the cascade reaction, 2M NaOH is added through pH Stat to control the pH of the reaction solution. The reaction system includes a final concentration of 100mM pimelic acid / suberic acid / zelaic acid, 1mM NADP(H), 10mM ATP, 240mM MgCl2, 1mM PLP, 240mM glucose, 200mM Poly P6 and 400mM isopropylamine, as well as 8.4mg wet cell / mL recombinant carboxylic acid reductase Crude cell extract, 91.6 mg wet cell / mL recombinant carboxylic acid reductase Crude cell extract, 18U transaminase SpTA, 50U glucose dehydrogenase BmGDH and 20U polyphosphate kinase PPK12, sample was taken after 18h reaction.

[0321] The concentrations of the products 1,7-heptanediamine / 1,8-octanediamine / 1,9-nonanediamine and the intermediates 7-aminoheptanoic acid / 8-aminooctanoic acid / 9-aminononanediamine were determined by HPLC. The consumption of the substrates pimelic acid / octanediamine / nonanediamine and possible byproducts, such as 7-hydroxyheptanediamine / 8-hydroxyoctanoic acid / 9-hydroxynonanediamine and heptanediol / octanediol / nonanediol, was determined by GC. As shown in Table 8, the yield of 1,7-heptanediamine reached 60.2% (60.2 mM), with no detectable byproducts. This yield was higher than the 1,6-hexanediamine concentration in Example 14. Furthermore, the titer of the α,ω-diamine product increased slightly with increasing carbon chain length, reaching 71.8 mM for 1,8-octanediamine and 80.2 mM for 1,9-nonanediamine, respectively.

[0322] Table 8 MabCAR3 mutants and transaminase cascade catalyze the synthesis of medium-chain α,ω-dicarboxylic acids (C7-C9) into α,ω-diamines

[0323] Example 17: Application of Carboxylic Acid Reductase MabCAR3 Mutants in the Synthesis of Medium- and Long-Chain α,ω-Diamines (C10-C12)

[0324] In this embodiment, glucose dehydrogenase GDH is used to regenerate NADPH, polyphosphate kinase PPK is used to regenerate ATP, and CAR and TA are used in a cascade to continuously reduce and aminize α, ω-dicarboxylic acids (C10-C12) to synthesize α, ω-diamines (C10-C12). Other bio-enzyme catalysts used in this route to regenerate NADPH and ATP are also protected by this patent. The reaction system is 8 mL, and is carried out in Hepes-Na buffer at pH 6.5 at 25°C (temperature controlled by water circulation) and 1000 rpm. Since a large amount of acid is produced during the cascade reaction, 3M NaOH is added by pH Stat to control the pH of the reaction solution. The reaction system includes a final concentration of 200mM sebacic acid / undecanedioic acid / dodecanedioic acid, 2mM NADP(H), 15mM ATP, 480mM MgCl2, 1mM PLP, 480mM glucose, 400mM Poly P6 and 800mM isopropylamine, as well as 16.8mg wet cell / mL recombinant carboxylic acid reductase Crude cell extract, 183.2 mg wet cell / mL recombinant carboxylic acid reductase Crude cell extract, 36U transaminase SpTA, 100U glucose dehydrogenase BmGDH and 40U polyphosphate kinase PPK12, sampled after 18h reaction.

[0325] HPLC was used to determine the concentrations of the products 1,10-decanediamine / 1,11-undecanediamine / 1,12-dodecanediamine and the intermediates 10-aminodecanoic acid / 11-aminoundecanoic acid / 12-aminododecanoic acid. GC was used to determine the consumption of the substrates sebacic acid / undecanediamine / dodecanediol, as well as the possible byproducts 10-hydroxydecanoic acid / 11-hydroxyundecanoic acid / 12-hydroxydodecanoic acid and decanediol / undecanediol / dodecanediol. As the chain length increased, the CAR specific activity increased, and the substrate loading was increased to 200 mM. The results are shown in Table 9. The product titer of 1,10-decanediamine was 105.3 mM, and no byproducts were detected. In this example, bio-based α,ω-dicarboxylic acids are used as substrates, and the titer of the α,ω-diamine product is the highest among all biosynthesized methods reported in the literature and patents. This is especially true for medium- and long-chain substrates (C10-C12), where substrate steric hindrance is generally greater. This further demonstrates the innovativeness and advancement of the carboxylic acid reductase mutants in this example.

[0326] Table 9 MabCAR3 mutants and transaminase cascade catalyze the synthesis of medium-chain α,ω-dicarboxylic acids (C10-C12) into α,ω-diamines

[0327] Example 18: Amplification of the Carboxylic Acid Reductase MabCAR3 Mutant Synthesis of 1,6-Hexanediamine System and Product Preparation

[0328] 1,6-Hexanediamine is one of the two monomers of nylon 66. In this embodiment, the reaction system is further expanded to a 300mL system, using glucose dehydrogenase GDH to regenerate NADPH, polyphosphate kinase PPK to regenerate ATP, CAR and TA cascade catalyzed adipic acid to synthesize 1,6-hexanediamine, in Hepes-Na buffer at pH 6.5, 30°C (temperature controlled by water circulation), 1000rpm conditions. Since a large amount of acid is generated during the cascade reaction, 3M NaOH is added by pH Stat to control the pH of the reaction solution. The reaction system includes a final concentration of 100mM AA, 1mM NADP + , 10mM ATP, 240mM MgCl2, 1mM PLP, 240mM glucose, 200mM Poly P6 and 400mM isopropylamine, and 8.3mg wet cell / mL recombinant carboxylic acid reductase MabCAR3 M39 Crude cell extract, 91.7 mg wet cell / mL recombinant carboxylic acid reductase MabCAR3 M42 Crude cell extract, 450 U transaminase SpTA, 1000 U glucose dehydrogenase BmGDH, and 500 U polyphosphate kinase PPK12 were added after 18 hours of reaction. All five component enzymes were directly crushed using a high-pressure homogenizer (prepared as described in Example 3) to break up the crude enzyme solution after centrifugation. A high-pressure homogenizer can break up highly concentrated bacterial cells, equivalent to using an ultrasonic disruptor to break up a concentrated crude enzyme solution.

[0329] HPLC analysis determined the production of the product 1,6-hexanediamine and the concentration of the intermediate 6-aminocaproic acid. GC analysis determined the consumption of the substrate adipic acid and possible byproducts, such as 6-hydroxycaproic acid and hexanediol. The yield of 1,6-hexanediamine reached 42.1%, with no detectable byproducts. Finally, 1,6-hexanediamine was extracted from the reaction mixture using n-butanol and then dried to a powder using a rotary evaporator. GC-MS analysis confirmed the isolated yield of 31.2%, successfully achieving gram-scale production of 1,6-hexanediamine.

[0330] The above-described embodiments merely represent several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art may make several modifications and improvements without departing from the scope of the present invention, and these modifications and improvements fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be based on the appended claims. At the same time, all documents mentioned in this application are cited as references in this application, just as if each document was cited as a reference individually.

[0331] Sequence Listing

Claims

1. A method for efficiently synthesizing α,ω-diamine, comprising the following steps: (1) using α,ω-dicarboxylic acid as a substrate and a catalyst containing a first carboxylic acid reductase mutant to catalyze the synthesis of ω-amino fatty acid; (2) using α,ω-dicarboxylic acid and ω-amino fatty acid as substrates and using a catalyst containing a second carboxylic acid reductase mutant to catalyze the synthesis of α,ω-diamine; wherein the first carboxylic acid reductase mutant has enhanced catalytic activity for substrate α,ω-dicarboxylic acid and reduced catalytic activity for ω-amino fatty acid; and / or, The second carboxylic acid reductase mutant has enhanced catalytic activity on substrates α, ω-dicarboxylic acid and ω-amino fatty acid.

2. The method according to claim 1, characterized in that The first carboxylic acid reductase mutant is: (a1) a protein having an amino acid sequence corresponding to SEQ ID No. 2, with one or more amino acids at positions 303, 306, 342, 344, 395, 418, and 426 mutated; (b1) a protein derived from (a1) which is formed by replacing, deleting or adding one or more amino acid residues in the amino acid sequence of protein (a1) and has the function of protein (a1), but one or more amino acids at positions 303, 306, 342, 344, 395, 418 and 426 corresponding to SEQ ID No.2 are the same as the amino acids at the corresponding positions of protein (a1) after mutation; (c1) a protein derived from (a1) that has an amino acid sequence homology of more than 80% with that of the protein (a1) and has the function of the protein (a1), but one or more of the amino acids at positions 303, 306, 342, 344, 395, 418 and 426 corresponding to SEQ ID No. 2 are identical to the amino acids at the corresponding positions of the protein (a) after mutation; (d1) an active fragment of the protein (a1), which comprises a structure that interacts with a carboxylic acid substrate in the spatial structure of a carboxylic acid reductase, and one or more amino acids at positions 303, 306, 342, 344, 395, 418 and 426 corresponding to SEQ ID No. 2 are identical to the amino acids at the corresponding positions of the protein (a1) after mutation; Preferably, the first carboxylic acid reductase mutant is selected from the following group: (1) Alanine (Ala) at position 303 was mutated to lysine (Lys); (2) mutation of leucine (Leu) at position 306 to lysine (Lys) or arginine (Arg); (3) Leucine (Leu) at position 342 mutated to lysine (Lys) or arginine (Arg); (4) Valine (Val) at position 344 was mutated to lysine (Lys); (5) Glycine (Gly) at position 395 was mutated to lysine (Lys); (6) Glycine (Gly) at position 418 is mutated to histidine (His), lysine (Lys), asparagine (Asn), or serine (Ser); (7) Glycine (Gly) at position 426 was mutated to histidine (His).

3. The method according to claim 1, characterized in that The second carboxylic acid reductase mutant is: (a2) a protein having an amino acid sequence corresponding to SEQ ID No. 2, with one or more amino acids at positions 284, 303, 306, 342, 393, 395, 418, and 426 mutated; (b2) a protein derived from (a2) which is formed by replacing, deleting or adding one or more amino acid residues in the amino acid sequence of protein (a2) and has the function of protein (a2), but one or more amino acids at positions 284, 303, 306, 342, 393, 395, 418 and 426 corresponding to SEQ ID No. 2 are the same as the amino acids at the corresponding positions of protein (a2) after mutation; (c2) a protein derived from (a2) that has an amino acid sequence homology of more than 80% with that of (a2) and has the function of (a2), but One or more of the amino acids at positions 284, 303, 306, 342, 393, 395, 418 and 426 of SEQ ID No. 2 are identical to the amino acids at the corresponding positions of the protein (a) after mutation; (d2) an active fragment of the (a2) protein, which comprises a structure that interacts with a carboxylic acid substrate in the spatial structure of a carboxylic acid reductase, and one or more amino acids at positions 284, 303, 306, 342, 393, 395, 418, and 426 corresponding to SEQ ID No. 2 are identical to the amino acids after mutation at the corresponding positions of the (a2) protein; Preferably, the second carboxylic acid reductase mutant is selected from the following group, or a combination of two or more of the following groups: (8) Leucine (Leu) at position 284 is mutated to aspartic acid (Asp), glutamic acid (Glu), glycine (Gly), asparagine (Asn), threonine (Thr), valine (Val) or tryptophan (Trp); (9) Alanine (Ala) at position 303 mutated to glutamic acid (Glu), methionine (Met) or valine (Val); (10) Leucine (Leu) at position 306 mutated to methionine (Met) or valine (Val); (11) Leucine (Leu) at position 342 is mutated to aspartic acid (Asp), glutamic acid (Glu), lysine (Lys), methionine (Met), asparagine (Asn), glutamine (Gln), valine (Val) or tryptophan (Trp); (12) Glycine (Gly) at position 393 was mutated to aspartic acid (Asp) or glutamic acid (Glu); (13) Glycine (Gly) at position 395 was mutated to glutamic acid (Glu); (14) Glycine (Gly) at position 418 is mutated to aspartic acid (Asp) or glutamic acid (Glu); (15) Glycine (Gly) at position 426 was mutated to serine (Ser); More preferably, the second carboxylic acid reductase mutant is selected from the following group: (16) Leucine (Leu) at position 284 mutated to aspartic acid (Asp), alanine (Ala) at position 303 mutated to methionine (Met), leucine (Leu) at position 306 mutated to isoleucine (Ile), leucine (Leu) at position 342 mutated to glutamic acid (Glu), and glycine (Gly) at position 393 mutated to alanine (Ala); (17) Leucine (Leu) at position 284 mutated to aspartic acid (Asp), Leucine (Leu) at position 306 mutated to methionine (Met), and Leucine (Leu) at position 342 mutated to glutamic acid (Glu); (18) Leucine (Leu) at position 284 mutated to aspartic acid (Asp), alanine (Ala) at position 303 mutated to isoleucine (Ile), leucine (Leu) at position 306 mutated to isoleucine (Ile), leucine (Leu) at position 342 mutated to glutamic acid (Glu), and glycine (Gly) at position 418 mutated to aspartic acid (Asp); (19) Leucine (Leu) at position 284 mutated to isoleucine (Ile), Leucine (Leu) at position 306 mutated to methionine (Met), Leucine (Leu) at position 342 mutated to glutamic acid (Glu), and glycine (Gly) at position 393 mutated to glutamic acid (Glu); (20) Leucine (Leu) at position 284 mutated to aspartic acid (Asp), Leucine (Leu) at position 306 mutated to methionine (Met), Leucine (Leu) at position 342 mutated to glutamic acid (Glu), glycine (Gly) at position 393 mutated to alanine (Ala), and glycine (Gly) at position 418 mutated to glutamic acid (Glu); (21) Leucine (Leu) at position 284 mutated to aspartic acid (Asp), Leucine (Leu) at position 306 mutated to methionine (Met), Leucine (Leu) at position 342 mutated to glutamic acid (Glu), and glycine (Gly) at position 393 mutated to glutamic acid (Glu); (22) Leucine (Leu) at position 284 mutated to glutamic acid (Glu), Leucine (Leu) at position 306 mutated to methionine (Met), Leucine (Leu) at position 342 mutated to glutamic acid (Glu), glycine (Gly) at position 393 mutated to alanine (Ala), and glycine (Gly) at position 426 mutated to serine (Ser); (23) Leucine (Leu) at position 284 mutated to phenylalanine (Phe), Leucine (Leu) at position 306 mutated to isoleucine (Ile), Leucine (Leu) at position 342 mutated to aspartic acid (Asp), and glycine (Gly) at position 393 mutated to glutamic acid (Glu); (24) The 284th leucine (Leu) mutated to phenylalanine (Phe), the 306th leucine (Leu) mutated to methionine (Met), Leucine (Leu) at position 342 mutated to aspartic acid (Asp), and glycine (Gly) at position 393 mutated to glutamic acid (Glu); (25) Alanine (Ala) at position 303 mutated to methionine (Met), leucine (Leu) at position 306 mutated to isoleucine (Ile), leucine (Leu) at position 342 mutated to aspartic acid (Asp), and glycine (Gly) at position 393 mutated to glutamic acid (Glu); (26) Alanine (Ala) at position 303 mutated to methionine (Met), leucine (Leu) at position 306 mutated to methionine (Met), leucine (Leu) at position 342 mutated to aspartic acid (Asp), and glycine (Gly) at position 393 mutated to glutamine (Gln); (27) Leucine (Leu) at position 284 mutated to aspartic acid (Asp), Leucine (Leu) at position 306 mutated to methionine (Met), Leucine (Leu) at position 342 mutated to aspartic acid (Asp), and glycine (Gly) at position 393 mutated to glutamic acid (Glu); (28) Leucine (Leu) at position 306 mutated to isoleucine (Ile), Leucine (Leu) at position 342 mutated to aspartic acid (Asp), and glycine (Gly) at position 418 mutated to aspartic acid (Asp); (29) Leucine (Leu) at position 284 mutated to glutamic acid (Glu), alanine (Ala) at position 303 mutated to isoleucine (Ile), leucine (Leu) at position 306 mutated to isoleucine (Ile), leucine (Leu) at position 342 mutated to glutamic acid (Glu), and glycine (Gly) at position 393 mutated to glutamic acid (Glu); (30) Leucine (Leu) at position 284 mutated to aspartic acid (Asp), alanine (Ala) at position 303 mutated to methionine (Met), leucine (Leu) at position 306 mutated to isoleucine (Ile), leucine (Leu) at position 342 mutated to glutamic acid (Glu), glycine (Gly) at position 393 mutated to alanine (Ala), and glycine (Gly) at position 418 mutated to aspartic acid (Asp); (31) Leu at position 284 mutated to glutamine (Gln), Leu at position 306 mutated to methionine (Met), Leu at position 342 mutated to glutamic acid (Glu), Gly at position 418 mutated to glutamic acid (Glu), and Gly at position 426 mutated to serine (Ser); (32) Leucine (Leu) at position 284 mutated to aspartic acid (Asp), alanine (Ala) at position 303 mutated to isoleucine (Ile), leucine (Leu) at position 306 mutated to isoleucine (Ile), leucine (Leu) at position 342 mutated to aspartic acid (Asp), glycine (Gly) at position 393 mutated to glutamic acid (Glu), and glycine (Gly) at position 418 mutated to glutamic acid (Glu); (33) Leucine (Leu) at position 284 mutated to phenylalanine (Phe), Leucine (Leu) at position 306 mutated to isoleucine (Ile), Leucine (Leu) at position 342 mutated to glutamic acid (Glu), and glycine (Gly) at position 393 mutated to glutamine (Gln).

4. The method according to claim 1, characterized in that The α,ω-dicarboxylic acids include malonic acid (C3), succinic acid (C4), glutaric acid (C5), adipic acid (C6), pimelic acid (C7), suberic acid (C8), azelaic acid (C9), sebacic acid (C10), undecanedioic acid (C11) and dodecanedioic acid (C12); or, The ω-amino fatty acid includes: 3-aminopropionic acid (C3), 4-aminobutyric acid (C4), 5-aminopentanoic acid (C5), 6-aminocaproic acid (C6), 7-aminoheptanoic acid (C7), 8-aminooctanoic acid (C8), 9-aminononanoic acid (C9), 10-aminodecanoic acid (C10), 11-aminoundecanoic acid (C11), 12-aminododecanoic acid (C12) or their derivatives; or, The α,ω-diamine is a short-chain α,ω-diamine, a medium-chain α,ω-diamine, a medium-long chain α,ω-diamine or a long-chain α,ω-diamine; preferably, the short chain refers to a carbon chain with a length of less than 5C, the medium chain refers to a carbon chain with a length of 6C-9C, the medium-long chain refers to a carbon chain with a length of 10C-12C, and the long chain refers to a carbon chain with a length of more than 13C; more preferably, the α,ω-diamine includes: 1,3-propylenediamine (C3), 1,4-butylenediamine (C4), 1,5-pentanediamine (C5), 1,6-hexanediamine (C6), 1,7-heptanediamine (C7), 1,8-octanediamine (C8), 1,9-nonanediamine (C9), 1,10-decanediamine (C10), 1,11-undecanediamine (C11), 1,12-dodecanediamine (C12) or a derivative thereof; or, The catalyst also includes: one or more of transaminase, glucose dehydrogenase and polyphosphate kinase; preferably, the transaminase uses organic ammonia as an amino donor; more preferably, the amino donor includes: an aliphatic amine donor and an aromatic amino donor; preferably, the glucose dehydrogenase uses glucose and NADP + As substrate, it catalyzes the oxidation of glucose and NADP + is reduced to NADPH; more preferably, the glucose dehydrogenase is glucose dehydrogenase BmGDH; preferably, the polyphosphate kinase is hexametaphosphorus Preferably, sodium phosphate is used as a substrate to catalyze AMP to generate ATP; more preferably, the polyphosphate kinase is polyphosphate kinase PPK12.

5. A method for efficiently synthesizing ω-amino fatty acids, comprising: Using α,ω-dicarboxylic acid as a substrate and a first carboxylic acid reductase mutant as a catalyst, catalytic synthesis of ω-amino fatty acid; wherein the first carboxylic acid reductase mutant has enhanced catalytic activity for the substrate α,ω-dicarboxylic acid and reduced catalytic activity for the ω-amino fatty acid; Preferably, the first carboxylic acid reductase mutant is: (a1) a protein having an amino acid sequence corresponding to SEQ ID No. 2, with one or more amino acids at positions 303, 306, 342, 344, 395, 418, and 426 mutated; (b1) a protein derived from (a1) which is formed by replacing, deleting or adding one or more amino acid residues in the amino acid sequence of protein (a1) and has the function of protein (a1), but one or more amino acids at positions 303, 306, 342, 344, 395, 418 and 426 corresponding to SEQ ID No.2 are the same as the amino acids at the corresponding positions of protein (a1) after mutation; (c1) a protein derived from (a1) that has an amino acid sequence homology of more than 80% with that of the protein (a1) and has the function of the protein (a1), but one or more of the amino acids at positions 303, 306, 342, 344, 395, 418 and 426 corresponding to SEQ ID No. 2 are identical to the amino acids at the corresponding positions of the protein (a) after mutation; (d1) an active fragment of the protein (a1), which comprises a structure that interacts with a carboxylic acid substrate in the spatial structure of a carboxylic acid reductase, and one or more amino acids at positions 303, 306, 342, 344, 395, 418 and 426 corresponding to SEQ ID No. 2 are identical to the amino acids at the corresponding positions of the protein (a1) after mutation; More preferably, the first carboxylic acid reductase mutant is selected from the following group: (1) Alanine (Ala) at position 303 was mutated to lysine (Lys); (2) mutation of leucine (Leu) at position 306 to lysine (Lys) or arginine (Arg); (3) Leucine (Leu) at position 342 mutated to lysine (Lys) or arginine (Arg); (4) Valine (Val) at position 344 was mutated to lysine (Lys); (5) Glycine (Gly) at position 395 was mutated to lysine (Lys); (6) Glycine (Gly) at position 418 is mutated to histidine (His), lysine (Lys), asparagine (Asn), or serine (Ser); (7) Glycine (Gly) at position 426 was mutated to histidine (His).

6. A carboxylic acid reductase mutant, wherein the catalytic activity of the mutant towards a substrate is altered relative to that of a wild-type carboxylic acid reductase; Preferably, when the substrate is an α,ω-dicarboxylic acid, the carboxylic acid reductase mutant has an enhanced catalytic activity for the substrate α,ω-dicarboxylic acid and a reduced catalytic activity for ω-amino fatty acid; more preferably, the carboxylic acid reductase mutant is as defined in claim 2; Preferably, when the substrate is α, ω-dicarboxylic acid and ω-amino fatty acid, the catalytic activity of the carboxylic acid reductase mutant on the substrate α, ω-dicarboxylic acid and ω-amino fatty acid is enhanced; more preferably, the carboxylic acid reductase mutant is as defined in claim 3.

7. An isolated polynucleotide, characterized in that The nucleic acid encodes the carboxylic acid reductase mutant according to claim 6, or the nucleic acid encodes the carboxylic acid reductase mutant as defined in claim 2 or 3.

8. A carrier, characterized in that It contains the polynucleotide according to claim 7; preferably, the vector is an expression vector or an expression transformant; more preferably, the expression transformant comprises an expression vector.

9. A genetically engineered host cell, characterized in that It contains the vector according to claim 8, or the polynucleotide according to claim 7 is integrated into its genome.

10. Select from the following applications: (a) Use of the carboxylic acid reductase mutant according to claim 6, or the carboxylic acid reductase mutant as defined in claim 2 or 3, in catalyzing the synthesis of α,ω-diamines; (b) Use of the carboxylic acid reductase mutant according to claim 6 or the carboxylic acid reductase mutant as defined in claim 2 in catalyzing the synthesis of ω-amino fatty acids; (c) Use of the carboxylic acid reductase mutant according to claim 6 or the carboxylic acid reductase mutant as defined in claim 2 or 3 in the preparation of a catalyst for catalytic synthesis of ω-amino fatty acids and / or α,ω-diamines.

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