Biosynthesis system for high yield of 1, 6-hexamethylenediamine and application of biosynthesis system

Abstract

The invention discloses a biosynthesis system for high-yield 1, 6-hexamethylenediamine and application of the biosynthesis system, and belongs to the technical field of biology. According to the method, key genes which are excavated and optimized in a 1, 6-hexamethylenediamine biosynthetic pathway are co-expressed in a single cell module or a multi-cell module, and different enzyme elements expressed by the cell module are subjected to enzyme cascade reaction, so that 1, 6-hexamethylenediamine can be efficiently synthesized by taking caprolactam or 6-aminocaproic acid as a substrate; furthermore, after the carboxylic acid reductase MaCAR is mutated, the catalytic activity of the carboxylic acid reductase MaCAR can be remarkably improved, and the yield of the 1, 6-hexamethylenediamine is further improved; in addition, the biosynthesis system disclosed by the invention directly takes the cell module as a whole-cell catalyst, and an enzyme purification step is avoided, so that the biosynthesis system has the advantage of low cost.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biotechnology, and particularly relates to a biosynthesis system for high-yield 1,6-hexanediamine and application thereof. BACKGROUND

[0002] 1,6-hexanediamine (HMD) is an important bulk chemical, which is synthesized by polycondensation with adipic acid to nylon 66, and plays an indispensable role in the polymer material industry. The current industrial production of HMD relies on a two-step chemical route: 1,3-butadiene is used to synthesize adiponitrile, which is then catalytically hydrogenated to obtain HMD. However, the traditional process has inherent defects, including high energy consumption, serious environmental pollution, and dependence on highly toxic hydrogen cyanide (HCN), resulting in cyanide-containing wastewater discharge. These challenges have prompted an urgent need to develop a cyanide-free, sustainable HMD biosynthesis strategy.

[0003] Enzymatic catalysis, as a promising green alternative method, has advantages such as high selectivity, atom economy, and operational safety. Multi-enzyme cascade catalysis is an especially effective means, which can seamlessly integrate consecutive enzymatic reactions to synthesize target molecules from simple, low-cost substrates. This strategy eliminates the need for intermediate product isolation and purification, reducing solvent use and waste generation, and providing a powerful tool for research in the fields of pharmaceuticals, fine chemicals, nylon monomer synthesis, and biofuels. In recent years, the development of multi-enzyme cascade systems has realized several green biosynthesis routes for HMD, including in vivo and in vitro methods. In terms of in vitro cascade reaction synthesis of HMD, there have been several research reports. For example, an in vitro pathway for synthesizing HMD from adipic acid (AA) was developed through two rounds of reduction / amination reactions catalyzed by carboxylic acid reductases (CARs) and transaminases (TAs), resulting in only 3 mM HMD with 70% 6-aminohexanoic acid (ACA) byproduct accumulation. Through engineering of key enzymes, the yield of HMD biosynthesized from AA in vitro was significantly improved to 59.4 mM. However, in vitro systems are costly due to the need for enzyme purification, expensive substrates, and coenzyme supplementation.

[0004] In contrast, in vivo multi-enzyme cascade reactions have obvious advantages, as they can avoid the expensive steps (such as enzyme purification and addition of expensive cofactors) in in vitro methods. Therefore, researchers have turned to in vivo multi-enzyme cascade reactions for the synthesis of HMD. There have been reports of in vivo biosynthesis systems using adipic acid as a substrate, with HMD yields of 2.1 mM. The inventors previously constructed a pathway for in vivo synthesis of HMD from cyclohexane (CH) or cyclohexanol through a redesigned cascade reaction, resulting in 7.6 mM and 16.5 mM of the product, respectively. Meanwhile, other researchers have also reported the biosynthesis of HMD from cyclohexanol, with a yield of 4.2 mM. Although in vivo biosynthesis has advantages in terms of avoiding the high cost of purifying enzymes and substrates, the yield is still not ideal. Therefore, it is imperative to develop an efficient in vivo biosynthesis method. SUMMARY

[0005] The present application aims to provide a biosynthesis system for high-yield 1,6-hexanediamine and its application. The present application aims to solve the problems of low yield and high cost in the existing in vivo biosynthesis of HMD.

[0006] In a first aspect, the present application provides a biosynthesis system for high-yield 1,6-hexanediamine, comprising a cell module 2 co-expressing a carboxylate reductase MaCAR or mutant gene thereof and a phosphopantetheinyl transferase SFP gene, a cell module 3 co-expressing an alcohol dehydrogenase ChnD gene and a transaminase CV gene, or a cell module 4 co-expressing a carboxylate reductase MaCAR or mutant gene thereof, a phosphopantetheinyl transferase SFP gene, and a transaminase PatA gene; wherein the nucleotide sequence of the carboxylate reductase MaCAR gene (derived from ATCC 19977) is shown in SEQ ID NO. 3, the phosphopantetheinyl transferase SFP gene is derived from Mycobacterium abscessus , the alcohol dehydrogenase ChnD gene is derived from Bacillus subtilis , the transaminase CV gene is derived from Acinetobacter sp. NCIMB 9871, and the nucleotide sequence of the transaminase PatA gene (derived from Chromobacterium violaceum ) is shown in SEQ ID NO. 5. Escherichia coli

[0007] In the present application, the inventors have found that the key genes in the 1,6-hexanediamine biosynthesis pathway, which are optimized by mining, are co-expressed in a single cell module or multiple cell modules. The different enzyme elements expressed by the above cell modules can efficiently synthesize 1,6-hexanediamine from 6-aminohexanoic acid through enzyme cascade reaction. Further, after mutation of the carboxylate reductase MaCAR, the catalytic activity of the carboxylate reductase MaCAR can be significantly improved, thereby improving the yield of 1,6-hexanediamine. In addition, the biosynthesis system of the present application directly uses the cell module as a whole cell catalyst, avoiding the purification step of the enzyme, and thus has the advantage of low cost.

[0008] In some embodiments, when caprolactam is used as the substrate, the biosynthesis system further comprises a cell module 1 co-expressing a cyclooligopeptide hydrolase 4270 gene and a cyclooligopeptide hydrolase 4271 gene (both derived from Pseudomonas jessenii ); wherein the nucleotide sequence of the cyclooligopeptide hydrolase 4270 gene is shown in SEQ ID NO. 1, and the nucleotide sequence of the cyclooligopeptide hydrolase 4271 gene is shown in SEQ ID NO. 2.

[0009] ​In the present application, the inventors further found that the higher catalytic activity of the cycloamidohydrolase 4270 gene and the cycloamidohydrolase 4271 gene obtained by gene mining can be used to synthesize 6-aminohexanoic acid from caprolactam through co-expression in a single cell module; therefore, the biosynthetic system of the present application can realize the efficient synthesis of 1,6-hexanediamine from cheap and readily available caprolactam through enzyme cascade reaction between different enzyme elements expressed by multiple cell modules.

[0010] In some embodiments, the carboxylate reductase MaCAR mutant is a mutation at at least one of the following positions of the amino acid sequence shown in SEQ ID NO. 4: A1) L342E, A2) L284T, A3) S986A, A4) D987N, A5) D987C.

[0011] In the present application, the structure of the carboxylate reductase MaCAR is analyzed, and the carboxylate reductase MaCAR mutant with significantly improved catalytic activity is obtained by using the method of site-directed saturation mutation, thereby significantly improving the yield of 6-amino-1-hexanol and / or 1,6-hexanediamine.

[0012] In some embodiments, the carboxylate reductase MaCAR mutant is selected from at least one of L342E, L342E / L284T, L342E / L284T / D987C, L342E / L284T / S986A, L342E / L284T / S986A / D987N.

[0013] In the present application, the catalytic activity of the carboxylate reductase MaCAR combination mutant is significantly increased compared to the catalytic activity of the carboxylate reductase MaCAR single-point mutant, and in the carboxylate reductase MaCAR combination mutant, the catalytic activity of the carboxylate reductase MaCAR combination mutant gradually increases with the increase of the mutation site.

[0014] In some embodiments, in addition to the mutations at at least one of positions 284, 342, 986, 987 in the above carboxylate reductase MaCAR mutants, further conservative substitution of amino acids at other positions can be made such that the mutated carboxylate reductase MaCAR has higher catalytic efficiency than the wild type carboxylate reductase MaCAR as shown in SEQ ID NO. 4. Preferably, the conservative substitution of amino acids retains the higher catalytic efficiency of the carboxylate reductase MaCAR mutants of the present application. It is obvious to those skilled in the art that such substitution can occur at regions other than the above-mentioned positions while retaining the corresponding activity. Preferably, the conservative substitution variant has conservative substitution of amino acids at at least one position. Examples of conservative substitution are substitution within the following groups of amino acids: basic amino acids (such as arginine, lysine and histidine), acidic amino acids (such as glutamic acid and aspartic acid), polar amino acids (such as glutamine, asparagine), hydrophobic amino acids (such as leucine, isoleucine and valine), aromatic amino acids (such as phenylalanine, tryptophan and tyrosine), and small amino acids (such as glycine, alanine, serine, threonine and methionine). The most common amino acid exchanges are the exchanges of amino acids G to A; A to G, S; V to I, L, A, T, S; I to V, L, M; L to I, M, V; M to L, I, V; P to A, S, N; F to Y, W, H; Y to F, W, H; W to Y, F, H; R to K, E, D; K to R, E, D; H to Q, N, S; D to N, E, K, R, Q; E to Q, D, K, R, N; S to T, A; T to S, V, A; C to S, T, A; N to D, Q, H, S; Q to E, N, H, K, R, and their inverse exchanges.

[0015] Carboxylate reductase MaCAR mutants having a certain amino acid homology to the amino acid sequence of the above carboxylate reductase MaCAR mutants, preferably a homology of between 70-99%, such as 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, or a value or a range between any two of these values; more preferably a homology of between 80-99%, such as 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, or a value or a range between any two of these values; further more preferably a homology of between 90-99%, such as 90%, 92%, 94%, 96%, 98%, 99%, or a value or a range between any two of these values; most preferably a homology of 99%, also belong to the protection scope of the present application.

[0016] In some embodiments, the biosynthetic system comprises a cell module 4 co-expressing a carboxylate reductase MaCAR or a mutant gene thereof, a phosphopantetheinyl transferase SFP gene, a transaminase PatA gene, and the genome of the cell module 4 comprises ldhA a site integration of an alcohol dehydrogenase ChnD gene.

[0017] In the present application, when the biosynthetic system only comprises the cell module 4, it can synthesize 1,6-hexanediamine with 6-aminohexanoic acid as the substrate, but the endogenous aldehyde ketone reductase in the cell module 4 can partially reduce 6-aminohexanal to the corresponding 6-amino-1-hexanol, which limits the production of 1,6-hexanediamine; based on this, the inventors integrate the alcohol dehydrogenase ChnD gene into the genome of the cell module 4 at the ldhA site, which can convert 6-amino-1-hexanol to 6-aminohexanal, and under the action of the transaminase PatA, the yield of 1,6-hexanediamine is improved.

[0018] In some embodiments, the biosynthetic system further comprises a cell module 3 co-expressing an alcohol dehydrogenase ChnD gene and a transaminase CV gene.

[0019] In the present application, the inventors further found that after the alcohol dehydrogenase ChnD gene is integrated into the genome of the cell module 4 at the ldhA site, although the yield of 1,6-hexanediamine is improved, a large amount of 6-amino-1-hexanol byproduct is accumulated, therefore, by combining the cell module 4 with the integrated alcohol dehydrogenase ChnD gene and the cell module 3 in the genome, high-yield 1,6-hexanediamine can be achieved with 6-aminohexanoic acid as the substrate.

[0020] In some embodiments, the cells in the cell module 1, the cell module 2, the cell module 3, and the cell module 4 comprise Escherichia coli.

[0021] In the present application, the cells can be routinely selected according to actual needs, as long as they can efficiently express exogenous genes and contain endogenous aldehyde ketone reductase genes. For example, in the present application, the cells preferably comprise Escherichia coli, and the Escherichia coli preferably comprises Escherichia coli BL21 (DE3).

[0022] In a second aspect, the present application provides the use of any of the above biosynthetic systems in the high-yield production of 1,6-hexanediamine.

[0023] The biosynthetic system provided by the present application can obtain 1,6-hexanediamine with significantly improved yield with caprolactam and / or 6-aminohexanoic acid as the substrate, through enzyme cascade reactions between different enzyme elements expressed by different cell modules in the biosynthetic system.

[0024] In a third aspect, the present application provides a method for one-pot synthesis of 1,6-hexanediamine by using the biosynthesis system as described above, comprising the following steps: taking 6-aminohexanoic acid as a substrate, adding a mixture of cell module 2 and cell module 3, glycerol and isopropylamine, or adding cell module 4, glycerol and monosodium glutamate, and then performing a reaction to obtain 1,6-hexanediamine; or taking caprolactam as a substrate, adding a mixture of cell module 1, cell module 2 and cell module 3, glycerol and isopropylamine, or adding a mixture of cell module 1 and cell module 4, glycerol and monosodium glutamate, and then performing a reaction to obtain 1,6-hexanediamine.

[0025] In some embodiments, in the step of taking 6-aminohexanoic acid as a substrate, adding a mixture of cell module 2 and cell module 3, glycerol and isopropylamine, and then performing a reaction to obtain 1,6-hexanediamine, the concentration of 6-aminohexanoic acid is 50-70 mM (preferably 60 mM), the ratio of cell module 2 and cell module 3 in the mixture is 1:1, the total cell density is 14-18 g CDW·L -1 (preferably 16 g CDW·L -1 ), the concentration of glycerol is 65-70 mM (preferably 68 mM), the concentration of isopropylamine is 180-220 mM (preferably 200 mM), the temperature of the reaction is 28-35℃ (preferably 30℃), the rotation speed is 200-240 rpm (preferably 220 rpm), and the time is at least 30 h.

[0026] In some embodiments, in the step of taking 6-aminohexanoic acid as a substrate, adding a mixture of cell module 2 and cell module 3, glycerol and isopropylamine, and then performing a reaction to obtain 1,6-hexanediamine, the concentration of 6-aminohexanoic acid is 50-70 mM (preferably 60 mM), the ratio of cell module 2 and cell module 3 in the mixture is 1:1, the total cell density is 14-18 g CDW·L -1 (preferably 16 g CDW·L -1 ), the concentration of glycerol is 65-70 mM (preferably 68 mM), the concentration of isopropylamine is 180-220 mM (preferably 200 mM), the temperature of the reaction is 28-35℃ (preferably 30℃), the rotation speed is 200-240 rpm (preferably 220 rpm), and the time is at least 30 h.

[0027] In some embodiments, in the step of taking 6-aminohexanoic acid as a substrate, adding a mixture of cell module 2 and cell module 3, glycerol and isopropylamine, and then performing a reaction to obtain 1,6-hexanediamine, the concentration of 6-aminohexanoic acid is 50-70 mM (preferably 60 mM), the ratio of cell module 2 and cell module 3 in the mixture is 1:1, the total cell density is 14-18 g CDW·L -1(preferably 24 g CDW·L -1 ), the concentration of glycerol is 130-140 mM (preferably 136 mM), the concentration of isopropylamine is 180-220 mM (preferably 200 mM), the temperature of the reaction is 28-35 °C (preferably 30 °C), the rotation speed is 200-240 rpm (preferably 220 rpm), and the time is at least 45 h.

[0028] In the present application, by controlling the addition amount of each reaction raw material within a specific range, 1,6-hexanediamine with high yield can be obtained in one pot in one step.

[0029] In a fourth aspect, the present application provides a method for synthesizing 1,6-hexanediamine in one pot in multiple steps by using the above biosynthetic system, comprising the following steps: taking 6-aminohexanoic acid as the substrate, adding cell module 4 with alcohol dehydrogenase ChnD gene integrated into the genome, glycerol and monosodium glutamate to carry out the first reaction; then replacing cell module 4 with alcohol dehydrogenase ChnD gene integrated into the genome with cell module 3, and adding isopropylamine to carry out the second reaction to obtain 1,6-hexanediamine; or taking caprolactam as the substrate, adding cell module 1 and glycerol to carry out the third reaction; then replacing cell module 1 with cell module 4 with alcohol dehydrogenase ChnD gene integrated into the genome, and adding glycerol and monosodium glutamate to carry out the fourth reaction; finally, replacing cell module 4 with alcohol dehydrogenase ChnD gene integrated into the genome with cell module 3, and adding isopropylamine to carry out the fifth reaction to obtain 1,6-hexanediamine.

[0030] In some embodiments, in the step of taking 6-aminohexanoic acid as the substrate, adding cell module 4 with alcohol dehydrogenase ChnD gene integrated into the genome, glycerol and monosodium glutamate to carry out the first reaction; then replacing cell module 4 with alcohol dehydrogenase ChnD gene integrated into the genome with cell module 3, and adding isopropylamine to carry out the second reaction to obtain 1,6-hexanediamine, the concentration of 6-aminohexanoic acid is 80-100 mM (preferably 90 mM), the total cell density is 14-18 g CDW·L -1 (preferably 16 g CDW·L -1 ), the concentration of glycerol is 160-170 mM (preferably 163 mM), the concentration of monosodium glutamate is 280-320 mM (preferably 300 mM), the temperature of the first reaction is 28-35 °C (preferably 30 °C), and the rotation speed is 200-240 rpm (preferably 220 rpm); after the first reaction is carried out for 27 h, the total cell density after replacing cell module 4 with cell module 3 is 14-18 g CDW·L -1 (preferably 16 g CDW·L -1), the concentration of isopropylamine is 130-170mM (preferably 150mM), the temperature of the second reaction is 28-35℃ (preferably 30℃), the rotation speed is 200-240rpm (preferably 220rpm), and the time is at least 31h.

[0031] In some embodiments, after the reaction of the third reaction is performed with caprolactam as the substrate, the addition of cell module 1 and glycerol; then the cell module 1 is replaced by the cell module 4 with the alcohol dehydrogenase ChnD gene integrated in the genome, and the fourth reaction is performed after the addition of glycerol and monosodium glutamate; finally, the cell module 4 with the alcohol dehydrogenase ChnD gene integrated in the genome is replaced by the cell module 3, and the fifth reaction is performed after the addition of isopropylamine, to obtain 1,6-hexanediamine. In the step of obtaining 1,6-hexanediamine, the concentration of caprolactam is 40-60mM (preferably 50mM), the total cell density of cell module 1 is 14-18g CDW·L -1 (preferably 16g CDW·L -1 ), the concentration of glycerol is 130-140mM (preferably 136mM), the temperature of the third reaction is 28-35℃ (preferably 30℃), and the rotation speed is 200-240rpm (preferably 220rpm); after the third reaction is performed for 24h, the total cell density after the replacement of cell module 1 is 14-18g CDW·L -1 (preferably 16g CDW·L -1 ), the concentration of glycerol is 130-140mM (preferably 136mM), the concentration of monosodium glutamate is 280-320mM (preferably 300mM), the temperature of the fourth reaction is 28-35℃ (preferably 30℃), and the rotation speed is 200-240rpm (preferably 220rpm); after the fourth reaction is performed for 50h, the total cell density after the replacement of cell module 3 is 14-18g CDW·L -1 (preferably 16g CDW·L -1 ), the concentration of isopropylamine is 130-170mM (preferably 150mM), the temperature of the fifth reaction is 28-35℃ (preferably 30℃), the rotation speed is 200-240rpm (preferably 220rpm), and the time is at least 42h.

[0032] In the present application, by controlling the addition amount of each reaction raw material within a specific range, 1,6-hexanediamine with high yield can be obtained from caprolactam as the substrate in one-pot multi-step, and the concentration of 1,6-hexanediamine is 43.5mM, which is the highest yield reported in the current in vivo biosynthesis.

[0033] The beneficial effects of the present application are: different from the prior art, the present application co-expresses the key genes optimized by mining in the biosynthesis pathway of 1,6-hexanediamine in a single cell module or a multi-cell module, and the different enzyme elements expressed by the above cell module can efficiently synthesize 1,6-hexanediamine by enzyme cascade reaction with caprolactam (preferably) or 6-aminocaproic acid as the substrate; further, after the carboxylate reductase MaCAR is mutated, the catalytic activity of the carboxylate reductase MaCAR can be significantly improved, thereby improving the yield of 1,6-hexanediamine; in addition, the biosynthesis system directly uses the cell module as a whole cell catalyst, avoiding the purification step of the enzyme, and therefore has the advantage of low cost. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 The biosynthesis system schematic diagram of different cell module whole cell catalysts for synthesizing HMD in the present application with CPL as the substrate; Figure 2A The cell module M CPL-ACA 2, M CPL-ACA 3 catalyzing the CPL reaction results; Figure 2B The cell module M CPL-ACA 3 catalyzing the CPL reaction results; Figure 2C The cell module M ACA-AH 1 catalyzing the ACA reaction results; Figure 2D The cell module M AH-HMD 1 catalyzing the ACA reaction results; Figure 2E The cell module M ACA-AH 1, M AH-HMD 1 one-pot one-step catalyzing the ACA reaction results; Figure 2F The cell module M CPL-ACA 3, M ACA-AH 1, M AH-HMD 1 one-pot one-step catalyzing the CPL reaction results; Figure 3 The cell module M ACA-HMD of the present application, wherein a) is the cell module M ACA-HMD The cascade reaction schematic diagram of combined catalysis, b) is the combination schematic diagram of the cell module M ACA-HMD The cascade reaction schematic diagram of combined catalysis, b) is the combination schematic diagram of the cell module M ACA-HMD The results of the cell module M ACA-HMD catalyzing the ACA reaction to obtain HMD; Figure 4 Results of screening MaCAR mutants with high ACA biosynthesis activity in Example 2 of the present application, wherein a) is the reaction pathway for screening MaCAR mutants; b) is the activity test results of double mutants based on MaCAR-L342E; c) is the activity test results of iterative mutations at L284 site based on MaCAR-L342E; d) is the activity test results of MaCAR mutants at S986 site based on L342E / L284T; e) is the activity test results of MaCAR mutants at D987 site based on L342E / L284T; f) is the activity test results of screened MaCAR mutants with enhanced activity; Figure 5 Cell module M ACA-HMD 17、 ACA-HMD 22Results of reacting ACA as substrate to obtain HMD (a) and cell module M ACA-HMD 19、 ACA-HMD 23Results of reacting ACA as substrate to obtain HMD (b); Figure 6 Cell module M ACA-HMD 22、 ACA-HMD 24Results of reacting ACA as substrate to obtain HMD (a) and cell module M ACA-HMD 23、 ACA-HMD 25Results of reacting ACA as substrate to obtain HMD (b); Figure 7 Results of preparing HMD from ACA as substrate by one-pot two-step method in Example 4 of the present application, wherein a) is a schematic diagram of a biosynthesis system composed of two cell modules and the types of enzymes expressed by each cell module, b) is cell module M ACA-HMD 24、 AH-HMD 1Results of catalyzing ACA to obtain HMD in two steps; Figure 8 Results of preparing HMD from CPL as substrate by one-pot three-step method in Example 4 of the present application, wherein a) is a schematic diagram of a biosynthesis system composed of three cell modules and the types of enzymes expressed by each cell module, b) is cell module M CPL-ACA 3、 ACA-HMD 24、 AH-HMD 1Results of catalyzing ACA to obtain HMD in three steps. DETAILED DESCRIPTION

[0035] The technical solutions in the embodiments of the present application will be clearly and completely described below with the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments of the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of the present application.

[0036] The experimental methods not specified in the embodiments are generally carried out according to the conventional experimental methods in the field of molecular biology, including but not limited to the experimental methods described in “Molecular Cloning: A Laboratory Manual” by M. R. Green, “Molecular Biology” by Robert·F·Weaver, or the experimental methods suggested by the manufacturers of reagent kits and instrument equipment. The reagents and biological materials used in the embodiments can be obtained from commercial channels if not otherwise specified.

[0037] In the present application, gene synthesis was completed by Wuhan Jin Kai Rui Biological Engineering Co., Ltd., and primer synthesis and DNA sequencing were completed by Shanghai Sunway Biotech Co., Ltd.

[0038] The sequences of phosphopantetheinyl transferase SFP gene, alcohol dehydrogenase ChnD gene and transaminase CV gene are described in the literature published by the inventors (Zhang Z, Fang L, Wang F, et al. Transforming Inert Cycloalkanes into α,ω-Diamines by Designed Enzymatic Cascade Catalysis. [J]. Angewandte Chemie, 2023:, e202215935. DOI:10.1002 / anie.202215935.).

[0039] Example 1 Construction and testing of cell modules in 1,6-hexanediamine biosynthesis system In order to realize the in vivo biosynthesis of 1,6-hexanediamine (HMD), the pathway enzymes were integrated into three different E. coli BL21 (DE3) cell modules: cell module 1 (M CPL-ACA ), cell module 2 (M ACA-AH ) and cell module 3 (M AH-HMD ) (named according to their catalytic reactions). These modules are combined to form E. coli consortium M CPL-ACA _M ACA-AH _M AH-HMD , which can realize the biosynthesis of HMD from caprolactam (CPL) (the reaction route is shown in Figure 1as shown).

[0040] 1.1 Cell module M CPL-ACA Construction and testing Firstly, cell module M CPL-ACA which can hydrolyze CPL to 6-aminocaproic acid (ACA). Through database gene mining, the cycloamidohydrolase 4270 gene / 4271 gene (the nucleotide sequences thereof are shown as SEQ ID NO. 1-2, respectively) was obtained, and after artificial synthesis of the cycloamidohydrolase 4270 gene / 4271 gene sequence, the cell module M Pseudomonas jessenii was constructed by the following method. CPL-ACA 2: The DNA fragments of 4270 gene, 4271 gene and linear plasmid backbone (pRSFDuet-1, pETDuet-1) were amplified by PCR using primers with 15-20 bp homologous arms, which can be used for subsequent recombination. The full length of 4270 gene and 4271 gene was assembled by overlap PCR and cloned into the linear vector in the presence of T5 exonuclease to generate 15 bp or 20 bp sticky ends to improve the recombination efficiency. The reaction mixture was 5 µL, containing linear vector, 4270 gene or 4271 gene, 4.0 buffer (New England Biolabs) and T5 exonuclease, incubated in ice water bath for 5 minutes, then 50 µL competent cells (E. coli DH5α) were quickly added for transformation, and spread on LB agar containing appropriate antibiotics. The obtained transformants were picked and DNA sequencing was performed for confirmation. The plasmid containing the target enzyme gene was transformed into E. coli BL21 (DE3) cells to obtain recombinant E. coli cell module M CPL-ACA 2 (containing pRSFDuet-4270 plasmid, pETDuet-4271 plasmid).

[0041] In order to improve the co-expression efficiency, 4270 gene and 4271 gene were cloned into pETDuet-1 plasmid according to the above method to obtain recombinant E. coli cell module M CPL-ACA 3 (containing pETDuet-4270-4271 plasmid), and the primer sequences used in the above construction are shown in Table 1.

[0042] Table 1 Primer sequences

[0043] Then the protein expression and preparation of whole cell catalyst of cell module M CPL-ACA were carried out, specifically as follows: the above constructed E. coli cell module M CPL-ACA 2, M CPL-ACA3Inoculate 2 mL of LB medium containing antibiotics (50 μg / mL kanamycin, 100 μg / mL ampicillin or chloramphenicol, corresponding to the selection marker of the vector) and incubate at 37 °C, 220 rpm for 6 h. Transfer the preculture (2 mL) to 100 mL of TB medium containing the appropriate antibiotic in a 250 mL flask and incubate at 37 °C, 220 rpm for 2-3 h until the OD 600 600.8 is reached, then IPTG is added to a final concentration of 0.2 mM. The temperature is adjusted to 25 °C and incubated for 14-16 h for protein expression. Cells are harvested by centrifugation at 5000 x g, 10 °C for 10 min, washed with 100 mM potassium phosphate buffer (pH 8.0) and the cell module M CPL-ACA is obtained.

[0044] Finally, the cell module M CPL-ACA is used as a whole-cell biocatalyst to react with CPL. The cell module M CPL-ACA 2, M CPL-ACA 3The cell module M -1 3is resuspended in 3 mL of phosphate buffer (pH 8.0, 100 mM) to a total cell density of 8 g CDW L CPL-ACA -1. The reaction is carried out at 25 °C, 220 rpm with 50 mM CPL and E. coli cells providing coenzyme NAD(P)H / ATP from 68 mM glycerol. The results are shown in Figure 2A .

[0045] As can be seen from Figure 2A , co-expression of 4270 and 4271 genes in a single plasmid has higher activity in catalyzing the hydrolysis of CPL.

[0046] Further, the cell module M CPL-ACA 3is used as a whole-cell biocatalyst to react with CPL. The reaction conditions are as above, except that 50 mM CPL is added first, and then additional CPL is added in stages (20 mM CPL is added at 5 h, and 30 mM CPL is added at 12 h). The results are shown in Figure 2B .

[0047] As can be seen from Figure 2B , the cell module M CPL-ACA 3reacts with CPL to obtain 92.2 mM ACA within 23 h.

[0048] 1.2 Construction and testing of the cell module M ACA-AH First, the cell module M ACA-AH is constructed.which can convert ACA to 6-amino-1-hexanol (AH). Through database gene mining, we obtained Mycobacterium abscessus carboxylate reductase MaCAR gene (nucleotide sequence as shown in SEQ ID NO. 3, amino acid sequence as shown in SEQ ID NO. 4) from ATCC 19977 and Bacillus subtilis phosphopantetheinyl transferase SFP gene from ATCC 19977. After artificial synthesis of MaCAR gene and SFP gene sequences, MaCAR gene and SFP gene were cloned into pRSFDuet-1 plasmid according to the construction method in step 1.1, and mutation was performed on L342 site to obtain MaCAR(L342E) mutant, and then obtain recombinant Escherichia coli cell module M ACA-AH 1 (containing pRSFDuet-MaCAR(L342E)-SFP plasmid), wherein the co-expressed carboxylate reductase MaCAR(L342E) and phosphopantetheinyl transferase SFP catalyze the reduction of ACA to the corresponding aldehyde; then the aldehyde ketone reductase endogenous to Escherichia coli reduces the aldehyde to the corresponding alcohol (AH); the primer sequences used in the above construction are shown in Table 2.

[0049] Table 2 Primer sequences

[0050] Then the method in step 1.1 was used to obtain cell module M ACA-AH 1 whole cell biocatalyst.

[0051] Finally, cell module M ACA-AH 1 was used, and ACA was used as the substrate for the reaction, and the reaction conditions were as follows: total cell density was 10 g CDW·L -1 , 50mM ACA, 37℃, 220rpm reaction. The results are shown in Figure 2C .

[0052] As can be seen from Figure 2C , cell module M ACA-AH 1 can obtain 6.4mM AH within 23h using ACA as the substrate.

[0053] 1.3 Construction and testing of cell module M AH-HMD First, cell module M AH-HMD was constructed, which can convert ACA to 6-amino-1-hexanol (AH). Through database gene mining, we obtained Acinetobacter sp. alcohol dehydrogenase ChnD gene from NCIMB9871 and Chromobacterium violaceum ​The transaminase CV gene was obtained from the source. After artificially synthesizing the ChnD gene and CV gene sequences, the ChnD gene and CV gene were cloned into the pETDuet-1 plasmid according to the construction method in step 1.1 to obtain the recombinant E. coli cell module M. AH-HMD 1 (containing pETDuet-ChnD-CV plasmid), the primer sequences used in the above construction are shown in Table 3 below.

[0054] Table 3 Primer Sequences

[0055] Then, using the method in step 1.1, cell module M is obtained. AH-HMD 1. Whole-cell biocatalyst.

[0056] Finally, use cell module M. AH-HMD 1. The reaction was carried out using AH as a substrate under the following conditions: 10 mM AH and 100 mM isopropylamine were used as amine donors in 3 mL of phosphate buffer (pH 8.0, 100 mM), and the reaction was carried out at 25 °C and 220 rpm. The results are as follows: Figure 2D As shown.

[0057] from Figure 2D As can be seen from this, cell module M AH-HMD 1. Using AH as a substrate, 8.8 mM HMD was obtained within 23 h.

[0058] 1.4 Preparation of HMD using CPL or ACA as substrates via a one-pot, one-step method The steps for preparing HMD using ACA as a substrate in a one-pot, one-step process include: ... (The text abruptly ends here, likely due to an incomplete sentence or a formatting error.) ACA-AH 1 and the cell module M prepared in step 1.3 AH-HMD 1 cell was resuspended in 3 mL of phosphate buffer (pH 8.0, 100 mM) at a ratio of 1:1, with a total cell density of 16 g CDW·L⁻¹. -1 The reaction was performed at 60 mM ACA, 30 °C, and 220 rpm. E. coli cells utilized 68 mM glycerol and 200 mM isopropylamine (amine donor) to provide the coenzyme NAD(P)H / ATP. The results are as follows: Figure 2E As shown.

[0059] from Figure 2E As can be seen, HMD gradually accumulates as the reaction proceeds, reaching 9.7 mM at 30 h.

[0060] The steps for preparing HMD using CPL as a substrate in a one-pot, one-step process include: ... (The text abruptly ends here, likely due to an incomplete sentence or a formatting error.) CPL-ACA 3. Cell module M prepared in step 1.2 ACA-AH1 and the cell module M prepared in step 1.3 AH-HMD 1 was resuspended in 3 mL phosphate buffer (pH 8.0, 100 mM) at a ratio of 1:1:1, with a total cell density of 24 g CDW·L -1 , 60 mM CPL, 30°C, 220 rpm reaction, E. coli cells provided coenzyme NAD(P)H / ATP using 136 mM glycerol and 200 mM isopropylamine (amine donor), and the results are shown in Figure 2F

[0061] As can be seen from Figure 2F , HMD gradually accumulated as the reaction proceeded, reaching 4.7 mM at 45 h.

[0062] 1.5 Integration and regulation of multiple cell modules In the cell module M ACA-AH 1, the endogenous aldehyde-ketone reductase in E. coli reduces the intermediate product 6-aminohexanal to AH, and AH needs another cell module M AH-HMD to be reoxidized, which greatly reduces the efficiency. Based on this, a cell module 4 (M ACA-HMD ) co-expressing carboxylate reductase MaCAR(L342E) mutant gene, phosphopantetheinyl transferase SFP gene, and transaminase PatA gene was constructed, which can biosynthesize HMD from ACA. Through database gene mining, the transaminase PatA gene (nucleotide sequence as shown in SEQ ID NO. 5) from Escherichia coli After artificially synthesizing the PatA gene sequence, the MaCAR(L342E) mutant gene and SFP gene were cloned into the pRSFDuet-1 plasmid according to the construction method in step 1.1, and the PatA gene was cloned into the pACYCDuet-1 and pETDuet-1 plasmids, respectively, to obtain recombinant E. coli cell module M ACA-HMD 17 (containing pRSFDuet-MaCAR(L342E)-SFP plasmid and pACYCDuet-PatA plasmid) and recombinant E. coli cell module M ACA-HMD 19 (containing pRSFDuet-MaCAR(L342E)-SFP plasmid and pETDuet-PatA plasmid), and the primer sequences used in the above construction are shown in Table 4.

[0063] Table 4 Primer sequences

[0064] Then, using the method in step 1.1, cell module M ACA-HMD 17, M ACA-HMD 19 whole-cell biocatalysts were obtained, respectively.​

[0065] Finally, the cell modules M ACA-HMD 17、M ACA-HMD 19 were used respectively as substrate ACA, and the reaction conditions were as follows: the cell modules M ACA-HMD 17、M ACA-HMD 19 were respectively resuspended in 3 mL of phosphate buffer (pH 8.0, 100 mM), and the total cell density was 12 g CDW·L -1 , 50 mM ACA, 30℃, 220 rpm for 25 h, and the E. coli cells used 68 mM glycerol as an energy source and 200 mM monosodium glutamate to provide coenzyme NAD(P)H / ATP, and the results are shown in Figure 3 .

[0066] As can be seen from Figure 3 , the cell modules M ACA-HMD 17、M ACA-HMD 19 used ACA as substrate, and 7.5-11.4 mM HMD was obtained within 25 h, and the relative content of the byproduct AH was low.

[0067] Example 2 Combination mutation of MaCAR(L342E) mutant in cell module M ACA-AH 1 Since the activity of the MaCAR(L342E) (M1) mutant is low, the structure of L342E is modeled using AlphaFold3, and through molecular docking, 9 amino acids (W283, L284, H301, L302, S394, G395, Y419, S421, G426) within the 5Å range of ACA are determined, and the above-mentioned sites are mutated to alanine using the mutant M1 as a template to obtain MaCAR double mutants, and the activity of the above-mentioned MaCAR double mutants is tested (the screening test reaction pathway is shown in Figure 4 a), specifically, the mutant M1, MaCAR double mutant cell module was resuspended in 3 mL of phosphate buffer (pH 8.0, 100 mM), and the total cell density was 10 g CDW·L -1 , 20 mM ACA, 37℃, 220 rpm for 27 h, and the E. coli cells used 68 mM glycerol as an energy source to provide coenzyme NAD(P)H / ATP, and the results showed that the activity of the MaCAR(L342E / L284A) double mutant was improved by 9% higher than that of the mutant M1 (as shown in Figure 4 b).

[0068] Further, based on the mutant M1, the L284 site was subjected to NNK saturation mutation, and the mutant L342E / L284T (M2) was screened, which had a 41% increase in catalytic activity compared with the single mutant L342E (M1) Figure 4 c). The activity screening test method was as follows: the MaCAR mutant cell module was resuspended in 3 mL phosphate buffer (pH 8.0, 100 mM), and the total cell density was 12 g CDW·L -1 , 40 mM ACA, 37°C, 220 rpm for 8 h, and the E. coli cells used 68 mM glycerol as an energy source to provide coenzyme NAD(P)H / ATP.

[0069] Further, based on the mutant M2, NNK-based site-directed saturation mutation was performed to obtain the triple mutants L342E / L284T / D987C (M3a) and L342E / L284T / S986A (M3b), and the activities were improved Figure 4 d, 4e). The activity screening test method was as follows: the MaCAR mutant was screened by a 96-well plate, the mutant cell module was resuspended in 3 mL phosphate buffer (pH 8.0, 100 mM), and the total cell density was 10 g CDW·L -1 , 80 mM ACA, 37°C, 220 rpm for 24 h, and the E. coli cells used 68 mM glycerol as an energy source to provide coenzyme NAD(P)H / ATP.

[0070] Further, based on M3b, iterative saturation mutation was performed, and finally the quadruple mutant L342E / L284T / S986A / D987N (M4) was obtained. Under the standard reaction conditions, the catalytic activities of the wild-type MaCAR (WT) and the representative mutants were compared, and the MaCAR (M4) achieved a 10.6 mM HMD yield, which was 5.3 times that of the MaCAR (WT) Figure 4 f). The activity screening test method was as follows: the MaCAR and its mutant cell module were resuspended in 3 mL phosphate buffer (pH 8.0, 100 mM), and the total cell density was 8 g CDW·L -1 , 80 mM ACA, 37°C, 220 rpm for 8 h, and the E. coli cells used 68 mM glycerol as an energy source to provide coenzyme NAD(P)H / ATP.

[0071] In the above mutation, the primer sequences used are shown in Table 5.

[0072] Table 5 Primer sequences

[0073] Example 3 Cell module MACA-HMD Redesign The mutant genes L342E / L284T / S986A / D987N (M4) obtained in Example 2 were used to replace cell module M. ACA-HMD 17 and M ACA-HMD The (L342E) gene in 19 was used to obtain recombinant E. coli cell module M. ACA-HMD 22 and M ACA-HMD 23. Then, an activity test was performed using ACA as a substrate. The test method is as follows: E. coli cell module M ACA-HMD Resuspended in 3 mL phosphate buffer (pH 8.0, 100 mM), total cell density 16 g CDW·L -1 90 mM ACA, reacted at 30℃ and 220 rpm for 6 h, E. coli cells utilized 136 mM glycerol and 300 mM monosodium glutamate to provide the coenzyme NAD(P)H / ATP. The results showed that cell module M ACA-HMD 22 and M ACA-HMD 23. The catalytic activity was significantly enhanced, the concentration of the product HMD was higher, and the content of the byproduct AH was lower. Figure 5 ).

[0074] Furthermore, due to cell module M ACA-HMD The endogenous aldehyde-ketone reductase in the *E. coli* still reduces the intermediate aldehyde to the corresponding alcohol (AH), limiting HMD production. Therefore, integrating the alcohol dehydrogenase ChnD gene into the *E. coli* genome... ldhA The method for site-specific gene integration is as follows: A CRISPR-Cas9-mediated gene editing system is used to integrate gene editing sites on the *E. coli* BL21(DE3) genome. ldhA The gene was replaced with the ChnD gene. In short, the system consists of two plasmids (pCas and pTarget) and a donor DNA (assembled via overlap PCR, carrying...) ldhA The pCas plasmid was composed of the ChnD gene with 500 bp homologous arms upstream and downstream. First, cells containing the pCas plasmid were cultured at 30°C for 6 hours in a mixture of kanamycin (50 mg / mL) and L-arabinose (2 mM) to induce λ-Red expression. The resulting cells were then used to prepare competent cells. Furthermore, pTarget and donor DNA were co-transformed into pCas-containing *E. coli* BL21(DE3) cells via electroporation. After a 3-hour recovery period at 30°C, the cells were plated on LB agar (50 mg / mL kanamycin and 100 mg / mL spectinomycin) and cultured overnight at 30°C. Colony PCR and DNA sequencing were used to confirm successful integration of the ChnD gene into the host genome. Finally, after two rounds of plasmid elimination, engineered *E. coli* cells with ChnD inserted into the genome, i.e., cell module M, were obtained. ACA-HMD 24 and MACA-HMD 25. Then the activity test was carried out with ACA as the substrate, and the test method was as follows: E. coli cell module M ACA-HMD was resuspended in 3 mL phosphate buffer (pH 8.0, 100 mM) with a total cell density of 16 g CDW·L -1 , 90 mM ACA, 30°C, 220 rpm for 20 h, and E. coli cell module M ACA-HMD 24 utilized 136 mM glycerol and 300 mM monosodium glutamate to provide coenzyme NAD(P)H / ATP, and the results showed that E. coli cell module M ACA-HMD 24 and M ACA-HMD 25 had significantly enhanced catalytic activity, higher concentration of product HMD, and less content of by-product AH Figure 6 .

[0075] The primer sequences required for gene integration are shown in Table 6.

[0076] Table 6 Primer sequences

[0077] Since the content of by-product AH in E. coli cell module M ACA-HMD 24 was lower than that in E. coli cell module M ACA-HMD 24 was selected for subsequent experiments. Although the performance of E. coli cell module M ACA-HMD 24 was significantly improved, a large amount of AH by-product was still accumulated. Therefore, it was combined with E. coli cell module M AH-HMD 1 to construct E. coli combination M ACA-HMD 24_M AH-HMD 1, which can catalyze the biosynthesis of HMD from ACA. Further integration with E. coli cell module M CPL-ACA 3 to construct E. coli combination M CPL-ACA 3_M ACA-HMD 24_M AH-HMD 1, which can catalyze the biosynthesis of HMD from CPL.

[0078] Example 4 Preparation of HMD from CPL or ACA as the substrate using one-pot multi-step method The steps of preparing HMD from ACA as the substrate using one-pot two-step method include: resuspending E. coli cell module M ACA-HMD 24 prepared in Example 3 in 3 mL phosphate buffer (pH 8.0, 100 mM) with a total cell density of 16 g CDW·L -1 , 90 mM ACA, 30°C, 220 rpm, and E. coli cell module M ACA-HMD24 is replaced with cell module M AH-HMD 1. Total cell density was 16 g CDW·L -1 150 mM isopropylamine was added, and the reaction was continued at 30 °C and 220 rpm. The results were as follows. Figure 7 As shown.

[0079] from Figure 7 As can be seen, HMD gradually accumulates as the reaction proceeds, reaching 59.4 mM at 58 h, which is 4 times that of the one-pot, one-step process.

[0080] The steps for preparing HMD using CPL as a substrate and a one-pot three-step method include: preparing cell module M CPL-ACA The cells were resuspended in 3 mL of phosphate buffer (pH 8.0, 100 mM) at a total cell density of 16 g CDW·L⁻¹. -1 The reaction was carried out at 50 mM CPL (20 mM added after 6 h, and 30 mM added after 10 h) at 30 °C and 220 rpm. E. coli cells utilized 136 mM glycerol as an energy source to provide the coenzyme NAD(P)H / ATP. After 24 h, the cell module M in the reaction solution was... CPL-ACA 3 is replaced with cell module M ACA-HMD 24, total cell density was 16 g CDW·L -1 300 mM sodium glutamate and 136 mM glycerol were added, and the reaction was continued at 30°C and 220 rpm. After 50 h, the cell module M in the reaction solution was removed. ACA-HMD 24 is replaced with cell module M AH-HMD 1. Total cell density was 16 g CDW·L -1 150 mM isopropylamine was added, and the reaction was continued at 30 °C and 220 rpm. The results were as follows. Figure 8 As shown.

[0081] from Figure 8 As can be seen, HMD gradually accumulates as the reaction proceeds, reaching 43.5 mM at 92 h, which is 7.5 times that of the one-pot, one-step process.

[0082] It should be noted that all the above embodiments belong to the same inventive concept, and the descriptions of each embodiment have different focuses. Where the description in a particular embodiment is not detailed, please refer to the description in other embodiments.

[0083] The embodiments described above are merely illustrative of implementation methods of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A biosynthetic system for high yield production of 1,6-hexanediamine, characterized in that, a cell module 2 comprising co-expressing a carboxylate reductase MaCAR or a mutant gene thereof and a phosphopantetheinyl transferase SFP gene, a cell module 3 co-expressing an alcohol dehydrogenase ChnD gene and a transaminase CV gene, or a cell module 4 comprising co-expressing a carboxylate reductase MaCAR or a mutant gene thereof, a phosphopantetheinyl transferase SFP gene and a transaminase PatA gene; The nucleotide sequence of the carboxylic acid reductase MaCAR gene is shown in SEQ ID NO.3, and the phosphate pantothenic acid thiotransferase SFP gene is derived from... Bacillus subtilis The alcohol dehydrogenase ChnD gene is derived from Acinetobacter sp. NCIMB9871, the transaminase CV gene is derived from Chromobacterium violaceum The nucleotide sequence of the transaminase PatA gene is shown in SEQ ID NO.

5.

2. The biosynthetic system of claim 1, wherein, When caprolactam is used as the substrate, the biosynthetic system further comprises a cell module 1 co-expressing a cycloamidohydrolase 4270 gene and a cycloamidohydrolase 4271 gene; wherein the nucleotide sequence of the cycloamidohydrolase 4270 gene is shown as SEQ ID NO. 1, and the nucleotide sequence of the cycloamidohydrolase 4271 gene is shown as SEQ ID NO.

2.

3. The biosynthetic system of claim 1, wherein, The carboxylate reductase MaCAR mutant is a mutation at at least one of the following positions of the amino acid sequence shown as SEQ ID NO. 4: A1) L342E, A2) L284T, A3) S986A, A4) D987N, A5) D987C.

4. The biosynthetic system of claim 3, wherein, The carboxylate reductase MaCAR mutant is at least one selected from the group consisting of L342E, L342E / L284T, L342E / L284T / D987C, L342E / L284T / S986A, L342E / L284T / S986A / D987N.

5. The biosynthetic system according to any one of claims 1 to 4, wherein, The biosynthetic system comprises a cell module 4 co-expressing a carboxylic acid reductase MaCAR or mutant gene thereof, a phosphopantetheinyl transferase SFP gene, a transaminase PatA gene, the genome of the cell module 4 having ldhA Site integration of the alcohol dehydrogenase ChnD gene.

6. The biosynthetic system of claim 5, wherein, The biosynthetic system further comprises a cell module 3 co-expressing an alcohol dehydrogenase ChnD gene and a transaminase CV gene.

7. The biosynthetic system according to any one of claims 1 to 4, wherein, The cells in the cell module 1, the cell module 2, the cell module 3 and the cell module 4 comprise Escherichia coli.

8. The biosynthetic system of any one of claims 1-7 for use in high-yield production of 1,6-hexanediamine.

9. A method for one-pot one-step synthesis of 1,6-hexanediamine using the biosynthetic system of any one of claims 1-4, characterized in that, comprising the following steps: reacting 6-aminohexanoic acid as the substrate with the mixture of the cell module 2 and the cell module 3, glycerol and isopropylamine, or with the cell module 4, glycerol and monosodium glutamate to obtain 1,6-hexanediamine; or reacting caprolactam as the substrate with the mixture of the cell module 1, the cell module 2 and the cell module 3, glycerol and isopropylamine, or with the mixture of the cell module 1 and the cell module 4, glycerol and monosodium glutamate to obtain 1,6-hexanediamine.

10. A method for one-pot multi-step synthesis of 1,6-hexanediamine using the biosynthetic system of claim 5 or 6, characterized in that, comprising the following steps: reacting 6-aminohexanoic acid as the substrate with the cell module 4 having the alcohol dehydrogenase ChnD gene integrated in the genome, glycerol and monosodium glutamate to perform a first reaction; then replacing the cell module 4 having the alcohol dehydrogenase ChnD gene integrated in the genome with the cell module 3, and adding isopropylamine to perform a second reaction to obtain 1,6-hexanediamine; Or taking caprolactam as the substrate, adding cell module 1 and glycerol to carry out the third reaction; then replacing cell module 1 with cell module 4 in which the alcohol dehydrogenase ChnD gene is integrated in the genome, and adding glycerol and monosodium glutamate to carry out the fourth reaction; finally, replacing cell module 4 in which the alcohol dehydrogenase ChnD gene is integrated in the genome with cell module 3, and adding isopropylamine to carry out the fifth reaction, to obtain 1,6-hexanediamine.

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