Transaminase mutants, recombinant plasmids, recombinant strains and their applications, and synthesis methods

Abstract

This application relates to the field of biotransformation technology, specifically to a transaminase mutant, recombinant plasmid, recombinant bacterial strain, its application, and synthesis method. The transaminase mutant is obtained by mutating a wild-type transaminase, which originates from heterotrophic Gram-negative bacteria and includes the amino acid sequence shown in SEQ ID NO. 5. The mutation sites of the transaminase mutant include any one or more of M439I, V419A, A239P, T276S, H310E, T36A, R43K, N48T, and A165S. By performing site-directed mutagenesis on the wild-type transaminase, a transaminase mutant with excellent overall enzyme activity is obtained and applied to the biocatalytic synthesis of 1,6-hexanediamine, significantly increasing the yield of 1,6-hexanediamine, reducing its industrial production cost, and improving production efficiency.

Description

Technical Field

[0001] This invention relates to the field of biotransformation technology, specifically to a transaminase mutant, recombinant plasmid, recombinant strain, its application, and synthesis method. Background Technology

[0002] 1,6-Hexanediamine, as an important chemical raw material, has a wide range of applications, and its synthesis technology has attracted much attention. Currently, the chemical synthesis methods for 1,6-hexanediamine suffer from problems such as high pollution, high energy consumption, and the use of highly toxic substances, placing significant pressure on the environment. In contrast, biosynthesis technology, with its green and environmentally friendly characteristics, has become a more promising research direction.

[0003] Current methods for biosynthesizing 1,6-hexanediamine share similar basic principles, typically involving the mixing of carboxylic acid reductase (CAR) with transaminase (TA) and using adipic acid or 6-aminohexanoic acid as substrates. For example, patent application CN202211085321.7 discloses the synthesis of 1,6-hexanediamine using adipic acid as a substrate by combining carboxylic acid reductase MAB CAR with one or two different transaminases. However, the synthesis efficiency of 1,6-hexanediamine using these methods needs improvement. To enhance the synthesis efficiency of 1,6-hexanediamine, existing technologies typically improve the efficiency by modifying carboxylic acid reductase, optimizing the synthesis pathway, or adjusting fermentation conditions, as illustrated by the technical solutions disclosed in patent applications CN202311855613.9, CN202511423758.0, and CN202311855614.3. Although these approaches have made some progress in the biosynthesis of 1,6-hexanediamine, there is still considerable room for improvement. Transaminases, as a key component of the 1,6-hexanediamine biosynthesis reaction system, have been the subject of relatively few research reports. This research bias has led to an imbalance or poor matching of transaminase and carboxylreductase activities within the synthesis system, limiting further improvements in the yield and efficiency of 1,6-hexanediamine biosynthesis. Summary of the Invention

[0004] The purpose of this invention is to provide a transaminase mutant that improves the catalytic efficiency of the transaminase mutant by performing single point mutations or multi-site combination mutations, thereby increasing the yield of 1,6-hexanediamine.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a transaminase mutant, wherein the transaminase mutant is obtained by mutating a wild-type transaminase, wherein the wild-type transaminase is derived from heterotrophic Gram-negative bacteria and its amino acid sequence is shown in SEQ ID NO.5, and the mutation sites of the transaminase mutant include any one or more of M439I, V419A, A239P, T276S, H310E, T36A, R43K, N48T and A165S.

[0006] Furthermore, the transaminase mutant is a multi-site mutant, and the mutation sites include A165S and / or T36A.

[0007] Further, the gene fragment encoding the wild-type transaminase is shown in SEQ ID NO.6; using the gene fragment encoding the wild-type transaminase as a template, the gene fragment encoding the transaminase mutant is obtained by PCR amplification.

[0008] This application provides a recombinant plasmid carrying a gene fragment encoding the above-mentioned transaminase mutant.

[0009] Furthermore, the construction process of the recombinant plasmid includes: digesting the pET28a plasmid and the gene fragment encoding the transaminase mutant obtained by amplification with restriction endonucleases NdeI and XhoI; and inserting the digested gene fragment between the NdeI and XhoI restriction sites of the pET28a plasmid.

[0010] This application provides recombinant strains expressing the above-mentioned transaminase mutants.

[0011] Furthermore, the recombinant strain was obtained by transforming the above-mentioned recombinant plasmid into Escherichia coli BL21(DE3) for expression.

[0012] This application provides the use of the above-mentioned transaminase mutant, the above-mentioned recombinant plasmid, or the above-mentioned recombinant strain in the preparation of 1,6-hexanediamine.

[0013] This application provides a whole-cell catalytic synthesis method for 1,6-hexanediamine, comprising:

[0014] The above recombinant strains and strains expressing carboxylic acid reductase MaCAR and phosphoubiotyltransferase BsSFP were obtained.

[0015] The recombinant strain and the strain were mixed and added to a reaction system including adipic acid, ammonia donor, glycerol and buffer solution, and stirred continuously to catalyze the synthesis of 1,6-hexanediamine.

[0016] Further, the pH range of the reaction system is 6.8-9.0; in the reaction system, the mass ratio of the bacterial strain to the recombinant bacterial strain is 1:(1.0-1.2), the concentration of adipic acid is any value between 8mM and 12mM, the concentration of glycerol is any value between 8mM and 12mM, and the concentration of the ammonia donor is 2-5 times that of the adipic acid.

[0017] The beneficial effects of this invention are as follows: This application focuses on transaminases in the biosynthetic pathway of 1,6-hexanediamine. By performing site-directed mutagenesis on wild-type transaminases, a transaminase mutant with excellent overall enzyme activity is obtained and applied to the biocatalytic synthesis of 1,6-hexanediamine, thereby significantly increasing the yield of 1,6-hexanediamine, effectively reducing the industrial production cost of 1,6-hexanediamine, and improving its production efficiency.

[0018] This application also optimizes the overall activity and substrate affinity of transaminase mutants by performing multi-site combined mutations on wild-type transaminases, thereby significantly improving substrate conversion rates and further promoting the yield of 1,6-hexanediamine, providing a more efficient and economical production method for related industries.

[0019] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Attached Figure Description

[0020] Figure 1 This is a comparison of the yields of wild-type transaminase and different transaminase mutants in the whole-cell catalytic synthesis of 1,6-hexanediamine, as shown in Example 1 of the present invention.

[0021] Figure 2 The image shows the HPLC detection of the synthesis of 1,6-hexanediamine catalyzed by the transaminase mutant RpTA-A165S as shown in Example 1 of this invention.

[0022] Figure 3 This is a comparison chart of the yields of different dual-site mutant transaminase mutants in the whole-cell catalytic synthesis of 1,6-hexanediamine, as shown in Example 2 of the present invention.

[0023] Figure 4 The yield of different multi-site transaminase mutants in the whole-cell catalytic synthesis of 1,6-hexanediamine, as shown in Example 3 of this invention. Detailed Implementation

[0024] The technical solutions of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Furthermore, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0025] Please see Figure 1 This application discloses a highly active transaminase mutant, obtained by mutating a wild-type transaminase. Transaminases are primarily responsible for catalyzing the aminotransfer reaction between the substrate and amino acids in the biocatalytic synthesis of 1,6-hexanediamine. Although the role of transaminases appears less significant compared to carboxylic acid reductases, their enzyme activity has a significant impact on the yield of 1,6-hexanediamine. Higher transaminase activity can improve substrate conversion efficiency, thereby increasing the overall catalytic efficiency of the reaction and significantly increasing the synthesis yield of the final product. This embodiment focuses on transaminases in the 1,6-hexanediamine biosynthesis pathway. By mutating a wild-type transaminase, a transaminase mutant with higher enzyme activity and catalytic efficiency was obtained and applied to the biosynthesis of 1,6-hexanediamine, thereby significantly improving the synthesis efficiency and yield of 1,6-hexanediamine. This wild-type transaminase is derived from the heterotrophic Gram-negative bacterium *Ruegeria pomeroyi*, and its amino acid sequence is shown in SEQ ID NO. 5 (NCBI GenBank: WP_011049154.1). The mutation sites of this transaminase mutant include, but are not limited to, any one or more of M439I, V419A, A239P, T276S, H310E, T36A, R43K, N48T, and A165S. These mutation sites are located in different structural regions of the transaminase, and the introduction of each mutation site aims to enhance the enzyme's structural stability, improve its activity and substrate affinity, thereby optimizing its catalytic efficiency. Specifically, mutations in amino acids at positions 439 and 419 are mainly used to improve the transaminase's affinity, making it easier to bind to the substrate and enhancing its binding affinity and stability, thus improving substrate conversion efficiency. Mutations at amino acids positions 165, 276, and 239 are primarily used to improve the structural stability of transaminases, optimize their spatial conformation, and thus enhance their overall activity. Mutations at positions 36 and 43 are mainly used to alter the amino acid environment of the active site, promoting electron transfer in catalytic reactions and thereby increasing enzyme activity.

[0026] In this embodiment or other embodiments, an initial gene fragment encoding the wild-type transaminase can be obtained through whole-genome synthesis based on the amino acid sequence of the wild-type transaminase. This initial gene fragment is then optimized according to the codon bias of the transformed strain, ultimately yielding the gene fragment encoding the wild-type transaminase as shown in SEQ ID NO. 6. Using this gene fragment encoding the wild-type transaminase as a template, amplification via polymerase chain reaction (PCR) yields the gene fragment encoding the transaminase mutant, thus providing the necessary gene resources for the expression and application of the transaminase mutant. In some embodiments, the gene fragment encoding the wild-type transaminase as shown in SEQ ID NO. 6 can be digested with enzymes and inserted into the similarly digested pET28a plasmid to obtain a recombinant plasmid; then, using the recombinant plasmid as a template, PCR amplification is performed to obtain the gene fragment encoding the transaminase mutant.

[0027] In one embodiment, the transaminase mutant is preferably a multi-site mutant including at least two mutation sites, and the mutation sites preferably include A165S and / or T36A. These transaminase mutants including multiple mutation sites have higher enzyme activity than transaminase mutants containing only a single mutation site.

[0028] One embodiment provides a recombinant plasmid carrying the gene encoding the aforementioned transaminase mutant. This plasmid can be pET28a or pET30a, or plasmids from the pGEX series (such as pGEX-4T-1 and pGEX-6P-1) and pMAL series, with pET28a being preferred. In this embodiment or other embodiments, the construction process of this recombinant plasmid includes: digesting the pET28a plasmid and the amplified gene fragment encoding the aforementioned transaminase mutant using restriction endonucleases NdeI and XhoI; and inserting the digested gene fragment between the NdeI and XhoI restriction sites of the pET28a plasmid.

[0029] One embodiment provides a recombinant strain expressing the above-mentioned transaminase mutant. This recombinant strain can efficiently express a highly active transaminase mutant. In this embodiment or other embodiments, the construction of the recombinant strain includes transforming the above-mentioned recombinant plasmid into *Escherichia coli* BL21(DE3) for expression to obtain the recombinant strain.

[0030] One embodiment provides the use of the above-described transaminase mutant, the above-described recombinant plasmid, or the above-described recombinant strain in the preparation of 1,6-hexanediamine.

[0031] One embodiment provides a method for the whole-cell catalytic synthesis of 1,6-hexanediamine, comprising:

[0032] The above recombinant strains and strains expressing carboxylic acid reductase MaCAR and phosphoubiotyltransferase BsSFP were obtained.

[0033] The recombinant strain and the strain were mixed and added to a reaction system containing adipic acid, isopropylamine, glycerol and buffer solution. The mixture was stirred continuously to catalyze the synthesis of 1,6-hexanediamine.

[0034] In some embodiments, the pH range of the reaction system is 6.8-9.0. Within this pH range, the enzymes maintain high activity, which is beneficial for the reaction. In the reaction system, the mass ratio of the strain to the recombinant strain is 1:(1.0-1.2) to achieve the best synergistic effect of the enzymes. Adipic acid is used as the substrate, and its concentration is preferably any value between 8 mM and 12 mM. Glycerol is used as a stabilizer to maintain enzyme or cell activity, and its concentration is preferably any value between 8 mM and 12 mM. The ammonia donor can be an organic amine such as isopropylamine, and its concentration is preferably two times or more than that of the substrate adipic acid. The buffer solution can be potassium phosphate buffer, etc., to maintain the pH stability of the reaction system. By reasonably limiting the concentrations of the substrate and components such as glycerol, the occurrence of side reactions can be effectively avoided, thereby achieving efficient and highly selective synthesis of 1,6-hexanediamine, and significantly improving the purity of the product while increasing the yield. During the catalytic synthesis process, it is preferable to continuously stir at a temperature of 20℃-37℃, and the stirring speed can be adjusted according to the scale of the reaction system to ensure that the reactants are fully mixed and improve the reaction efficiency.

[0035] Example 1

[0036] Obtain a wild-type transaminase derived from heterotrophic Gram-negative bacteria and containing the amino acid sequence shown in SEQ ID NO.5, and label it as RpTA.

[0037] A complete gene sequence encoding the wild-type transaminase was synthesized based on its amino acid sequence, yielding an initial gene fragment. Subsequently, this initial gene fragment was optimized according to the codon bias of *E. coli*, resulting in a gene fragment with the nucleotide sequence shown in SEQ ID NO. 6. This fragment was then inserted between the NdeI and XhoI restriction sites of the pET28a plasmid, yielding a recombinant plasmid carrying the gene fragment encoding the wild-type transaminase, labeled pET28a-RpTA. Finally, a portion of the recombinant plasmid pET28a-RpTA was transformed into *E. coli* BL21(DE3) to obtain a recombinant strain expressing the wild-type transaminase, labeled BL21 / pET28a-RpTA.

[0038] Based on the gene fragment encoding the wild-type transaminase obtained above, primers RpTA-1 (SEQ ID NO.7) and RpTA-2 (SEQ ID NO.8) were designed and synthesized. Next, using the recombinant plasmid pET28a-RpTA as a template and RpTA-1 and RpTA-2 as primers, PCR amplification was performed using the GeneMorphII random mutation PCR kit purchased from Agilent Technologies (China) Co., Ltd. The amplified PCR product was purified by agarose gel electrophoresis to obtain the purified PCR product. Then, the PCR product and the recombinant plasmid pET28a-RpTA were digested with restriction endonucleases NdeI and XhoI, respectively. The digested PCR product fragment was ligated with the digested recombinant plasmid pET28a-RpTA fragment to obtain a recombinant plasmid carrying the mutant gene. In this process, the purpose of digesting the recombinant plasmid pET28a-RpTA with NdeI and XhoI is to remove the original gene fragment encoding the wild-type transaminase between its NdeI and XhoI restriction sites, so that the digested PCR product fragment can be inserted between its NdeI and XhoI restriction sites to complete the construction of the recombinant plasmid.

[0039] The recombinant plasmid carrying the mutant gene was transformed into *E. coli* BL21(DE3) and plated on LBK kanamycin-resistant plates, incubated upside down at 37°C. After transformants appeared, single colonies were picked and transferred to 96-well deep-well plates, and 1 mL of LBK (Luria-Bertany kanamycin-containing medium) containing 0.1 mM IPTG (isopropyl-β-D-thiogalactopyranoside) was added to each well. Simultaneously, a single colony of BL21 / pET28a-RpTA was streaked onto an LBK-resistant plate and inoculated into 96-well deep-well plates under the same conditions. The 96-well deep-well plates were incubated at 37°C and 250 rpm for approximately 8 hours, and then 200 μL of the culture was collected for preservation. The remaining bacterial culture was centrifuged to remove the supernatant, then resuspended in buffer, and subjected to repeated freeze-thaw cycles to break up the cell walls, in order to obtain E. coli cell lysates containing wild-type transaminase RpTA and transaminase mutants.

[0040] Alanine aminotransferase assay kit (C009-1-1) purchased from Nanjing Jiancheng Biotechnology Institute was used, and the enzyme activity of the cell lysates was measured according to the instructions. The results showed that the enzyme activity of most cell lysates containing mutants was different from that of cell lysates containing wild-type transaminases. Through multiple rounds of ordered mutagenesis and screening, 14 highly active transaminase mutants with a single mutation site were finally identified and labeled as: RpTA-D72S, RpTA-M439I, RpTA-V419A, RpTA-S176G, RpTA-A239P, RpTA-T276S, RpTA-V304I, RpTA-H310E, RpTA-T36A, RpTA-R43K, RpTA-N48T, RpTA-K93Q, RpTA-S164A, and RpTA-A165S. The specific enzyme activity data of these transaminase mutants are shown in Table 1.

[0041] Table 1. Enzyme activity of transaminase mutants with single point mutations

[0042]

[0043] As shown in Table 1, the enzyme activities of these transaminase mutants are significantly higher than those of wild-type transaminase RpTA, especially RpTA-A165S, RpTA-T36A, RpTA-D72S, and RpTA-N48T, whose enzyme activities reach over 6.5 U / mL, significantly higher than wild-type transaminase RpTA, demonstrating good application potential. This also indicates that these 14 mutation sites are effective mutation sites. Simultaneously, recombinant strains expressing these transaminase mutants were obtained through preservation and labeled as follows: BL21 / pET28a-RpTA-D72S, BL21 / pET28a-RpTA-M439I, BL21 / pET28a-RpTA-V419A, BL21 / pET28a-RpTA-S176G, BL21 / pET28a-RpTA-A239P, BL21 / pET28a-RpTA-T276S, BL21 / pET28a-RpTA-V304I, BL21 / pET28a-RpTA-H310E, BL21 / pET28a-RpTA-T36A, BL21 / pET28a-RpTA-R43K, BL 21 / pET28a-RpTA-N48T, BL21 / pET28a-RpTA-K93Q, BL21 / pET28a-RpTA-S164A, BL21 / pET28a-RpTA-A165S.

[0044] To verify the effectiveness of the above-mentioned transaminase mutants in the biosynthesis of 1,6-hexanediamine, the 14 recombinant strains expressing transaminase mutants and the recombinant strain expressing wild-type transaminase were respectively co-blended with the recombinant strain BL21 / pACYCDuet-MaCAR-BsSFP, and 1,6-hexanediamine was synthesized via whole-cell catalysis. The construction process of the recombinant strain BL21 / pACYCDuet-MaCAR-BsSFP included:

[0045] First, carboxylic acid reductase (MaCAR) and phosphoproteoylthiotransferase (BsSFP) were obtained. The carboxylic acid reductase was derived from *Mycobacteroides abscessus*, and its amino acid sequence is shown in SEQ ID NO.1 (NCBI GenBank: WP_206481270.1). The phosphoproteoylthiotransferase was derived from *Bacillus subtilis*, and its amino acid sequence is shown in SEQ ID NO.3 (UnProt ID: P39135). Next, based on the amino acid sequences of MaCAR and BsSFP, whole-genome synthesis was performed to obtain initial gene fragments encoding MaCAR and BsSFP, respectively. Subsequently, according to the codon bias of *E. coli*, the initial gene fragments encoding MaCAR and BsSFP were optimized, ultimately yielding the gene fragment encoding MaCAR with the nucleotide sequence shown in SEQ ID NO.2 and the gene fragment encoding BsSFP with the nucleotide sequence shown in SEQ ID NO.4, respectively. Next, the gene fragment encoding MaCAR was inserted between the BamHI and EcoRI restriction sites of the pACYCDuet-1 plasmid. Simultaneously, to prevent frameshift mutations, a "G" base was added to the front of the MaCAR gene fragment. Then, the gene fragment encoding BsSFP was inserted between the NdeI and KpnI restriction sites of the pACYCDuet-1 plasmid, resulting in the plasmid vector pACYCDuet-MaCAR-BsSFP, which simultaneously carries both the MaCAR and BsSFP genes. Finally, this plasmid vector pACYCDuet-MaCAR-BsSFP was transformed into *E. coli* BL21(DE3) to obtain the recombinant strain BL21 / pACYCDuet-MaCAR-BsSFP, which simultaneously expresses the carboxylic acid reductase MaCAR and the phosphoubilityltransferase BsSFP.

[0046] The specific experimental procedure for synthesizing 1,6-hexanediamine using a whole-cell catalytic approach is as follows:

[0047] The recombinant strain BL21 / pACYCDuet-MaCAR-BsSFP was inoculated into 10 mL of LB broth containing chloramphenicol (final concentration 34 μg / mL); the recombinant strain BL21 / pET28a-RpTA and 14 recombinant strains expressing transaminase mutants were inoculated into 10 mL of LB broth containing kanamycin (final concentration 50 μg / mL); all media were cultured at 37℃ and 220 rpm for 16 hours to obtain the corresponding seed cultures. Then, appropriate amounts of these seed cultures were inoculated into 100 mL of LB broth containing the corresponding antibiotics, and the initial absorbance (OD600) was adjusted to 0.1; after culturing at 37℃ and 220 rpm until the OD600 reached 0.6-0.8, IPTG (isopropyl-β-D-thiogalactoside) was added to a final concentration of 0.2 mM, and the culture was transferred to 16℃ and 180 rpm for 20 hours to induce the expression of the target protein. After induction of expression, the supernatant was removed by centrifugation, and the recombinant strains were collected. The collected recombinant strains were washed with 100mM potassium phosphate buffer (pH 8.0), and centrifugation was repeated to remove the supernatant. Finally, the recombinant strains BL21 / pACYCDuet-MaCAR-BsSFP, BL21 / pET28a-RpTA, and 14 recombinant strains expressing transaminase mutants were obtained, which can be used as whole-cell biocatalysts.

[0048] The recombinant strain BL21 / pACYCDuet-MaCAR-BsSFP, which can serve as a whole-cell biocatalyst, was mixed with the recombinant strain BL21 / pET28a-RpTA or a recombinant strain expressing a transaminase mutant. This mixture was then added to a reaction system comprising 10 mM adipic acid, 50 mM potassium phosphate buffer (pH 8.0), 10 mM glycerol, and 20 mM isopropylamine. The concentrations of the recombinant strain BL21 / pACYCDuet-MaCAR-BsSFP and the recombinant strain BL21 / pET28a-RpTA or the recombinant strain expressing a transaminase mutant were ensured to be 8 g CDW / L. The reaction system was continuously stirred at 25°C and 200 rpm for 24 hours to catalyze the synthesis of 1,6-hexanediamine. After the reaction was complete, the yield of 1,6-hexanediamine was determined by HPLC. The results are shown in Table 2. Figure 1 , Figure 2 As shown. To ensure the accuracy and reliability of the experimental results and minimize the interference of random errors, all experiments in this application consist of five parallel experiments, and the yield increase is evaluated by comparing the means. The following tables list the yields of two random parallel experiments.

[0049] Table 2. Yields of wild-type transaminases and different transaminase mutants in the whole-cell catalytic synthesis of 1,6-hexanediamine

[0050]

[0051] Table 2 shows that, compared to wild-type transaminases, the aforementioned transaminase mutants significantly increased the yield of 1,6-hexanediamine during whole-cell catalytic synthesis, with yield increases ranging from 4% to 24%. This indicates that single-point mutations in wild-type transaminases can significantly improve their catalytic efficiency, leading to a substantial increase in the final yield of synthesized 1,6-hexanediamine. Among them, the transaminase mutant RpTA-A165S showed the largest yield increase, reaching 23.7%.

[0052] Example 2

[0053] To further increase the yield of 1,6-hexanediamine, several dual-site mutant transaminase mutants were constructed by superimposing other mutation sites that can enhance yield, based on the transaminase mutant RpTA-A165S. Whole-cell catalytic synthesis of 1,6-hexanediamine was then tested using these mutants. The specific experimental steps are as follows:

[0054] The above-mentioned recombinant strain BL21 / pET28a-RpTA-A165S was inoculated, and the recombinant plasmid pET28a-RpTA-A165S was extracted. Using recombinant plasmid pET28a-RpTA-A165S as a template, a high-fidelity DNA polymerase (KOD Fx Neo) kit (model KFX-201) purchased from Toyobo Shanghai Biotechnology Co., Ltd. was used. Primer pairs were employed as follows: T36A-1 (SEQ ID NO. 9) and T36A-2 (SEQ ID NO. 10), R43K-1 (SEQ ID NO. 11) and R43K-2 (SEQ ID NO. 12), N48T-1 (SEQ ID NO. 13) and N48T-2 (SEQ ID NO. 14), A239P-1 (SEQ ID NO. 15) and A239P-2 (SEQ ID NO. 16), T276S-1 (SEQ ID NO. 17) and T276S-2 (SEQ ID NO. 18), and H310E-1 (SEQ ID NO. 19) and H310E-2 (SEQ ID NO. 10). PCR amplification was performed on NO.20, V419A-1 (SEQ ID NO.21) and V419A-2 (SEQ ID NO.22), M439I-1 (SEQ ID NO.23) and M439I-2 (SEQ ID NO.24) to obtain PCR amplification products.

[0055] PCR amplification products were digested with the restriction endonuclease DpnI, and 10 μL of the digested product was then transformed into E. coli Top10 competent cells purchased from Beijing Zhuangmeng International Biotechnology Co., Ltd. The transformed cells were plated on LBK kanamycin-resistant plates for positive transformant selection. Subsequently, the correct transformants were identified by colony PCR, and plasmids were extracted from LB medium and sequenced for verification. Finally, eight correct recombinant plasmids carrying the gene encoding the two-site mutant transaminase were obtained, namely: pET28a-RpTA-A165S-T36A, pET28a-RpTA-A165S-R43K, pET28a-RpTA-A165S-N48T, pET28a-RpTA-A165S-A239P, pET28a-RpTA-A165S-T276S, pET28a-RpTA-A165S-H310E, pET28a-RpTA-A165S-V419A, and pET28a-RpTA-A165S-M439I.

[0056] The above eight recombinant plasmids were transformed into Escherichia coli BL21(DE3) to obtain recombinant E. coli strains expressing dual-site mutant transaminase mutants, which were labeled as BL21 / pET28a-RpTA-A165S-T36A, BL21 / pET28a-RpTA-A165S-R43K, and BL21 / pET28a-RpTA-A165S-N48T, respectively. , BL21 / pET28a-RpTA-A165S-A239P, BL21 / pET28a-RpTA-A165S-T276S, BL21 / pET28a-Rp TA-A165S-H310E, BL21 / pET28a-RpTA-A165S-V419A and BL21 / pET28a-RpTA-A165S-M439I.

[0057] To verify the effect of the aforementioned dual-site mutant transaminase in the biosynthesis of 1,6-hexanediamine, it was applied to the whole-cell catalytic synthesis of 1,6-hexanediamine, and the same whole-cell catalytic synthesis experimental procedure as in Example 1 was used. The results are shown in Table 3 and... Figure 3 As shown.

[0058] Table 3. Yields of different transaminase mutants in the whole-cell catalytic synthesis of 1,6-hexanediamine

[0059]

[0060] Table 3 shows that, compared with the single-point mutation transaminase mutant RpTA-A165S, most of the double-site mutation transaminase mutants significantly improved the whole-cell catalytic synthesis of 1,6-hexanediamine, with increases ranging from 4% to 32%. Among them, the three double-site mutation transaminase mutants RpTA-A165S-T36A, RpTA-A165S-A239P, and RpTA-A165S-V419A showed particularly outstanding performance, with increases in catalytic 1,6-hexanediamine synthesis of 31.8%, 10.9%, and 9.8%, respectively. This result indicates that by rationally combining the above mutation sites and utilizing the synergistic effect between them, the structural stability and activity of transaminases can be further improved, as well as their binding ability to substrates enhanced, thereby further improving the synthesis efficiency of 1,6-hexanediamine.

[0061] Example 3

[0062] The 1,6-hexanediamine yield of the multi-site transaminase mutant was 10-46% higher than that of the two-site mutant A165S-T36A.

[0063] To further increase the yield of 1,6-hexanediamine, based on the transaminase mutant RpTA-A165S-T36A, other mutation sites that can enhance yield were added to construct three-site mutation transaminase mutants and four-site mutation transaminase mutants. Whole-cell catalytic synthesis of 1,6-hexanediamine was then tested. The specific experimental steps are as follows:

[0064] Using the recombinant plasmid pET28a-RpTA-A165S-T36A as a template, PCR amplification was performed using primer pairs A239P-1 and A239P-2 and V419A-1 and V419A-2, respectively, yielding amplification products. The amplification products were then digested, transformed, screened, and sequenced using the same methods as in Example 2, resulting in two correct recombinant plasmids carrying the gene encoding a three-point mutation transaminase mutant: pET28a-RpTA-A165S-T36A-A239P and pET28a-RpTA-A165S-T36A-V419A. The two recombinant plasmids were transformed into Escherichia coli BL21(DE3) to obtain recombinant strains BL21 / pET28a-RpTA-A165S-T36A-A239P and BL21 / pET28a-RpTA-A165S-T36A-V419A expressing three-point mutant transaminase mutants.

[0065] Simultaneously, using the recombinant plasmid pET28a-RpTA-A165S-T36A-A239P as a template, PCR amplification was performed on V419A-1 and V419A-2 using the aforementioned primers to obtain amplification products. The amplification products were digested, transformed, screened, and sequenced using the same methods as in Example 2 to obtain the correct recombinant plasmid pET28a-RpTA-A165S-T36A-A239P-V419A carrying the gene encoding the four-site mutant transaminase. This plasmid was then transformed into *E. coli* BL21(DE3) to obtain the corresponding recombinant strain BL21 / pET28a-RpTA-A165S-T36A-A239P-V419A expressing the four-site mutant transaminase.

[0066] The three-site and four-site transaminase mutants were applied to the whole-cell catalytic synthesis of 1,6-hexanediamine, and the same whole-cell catalytic synthesis procedure as in Example 1 was used to verify their effectiveness in the biosynthesis of 1,6-hexanediamine. The results are shown in Table 4. Figure 4 As shown.

[0067] Table 4. Yields of different transaminase mutants in the whole-cell catalytic synthesis of 1,6-hexanediamine

[0068]

[0069] Table 4 shows that, compared with the two-site mutant transaminase RpTA-A165S-T36A, the three-site or four-site mutant transaminases were more effective in increasing the yield of 1,6-hexanediamine. This further confirms that by rationally combining the mutant sites selected above, the interactions between the mutant sites can significantly optimize the enzyme activity and substrate affinity of transaminases, thereby significantly improving their binding performance and catalytic efficiency with substrates, increasing substrate conversion efficiency, and ultimately achieving a substantial increase in product yield. Among them, the three transaminase mutants RpTA-A165S-T36A-A239P, RpTA-A165S-T36A-V419A, and RpTA-A165S-T36A-A239P-V419A increased the yield of 1,6-hexanediamine by 25.1%, 13.6%, and 46.6%, respectively. Furthermore, the transaminase mutant RpTA-A165S-T36A-A239P-V419A catalyzed the synthesis of 1,6-hexanediamine with a yield as high as 146.95 mg / L, representing a 2.41-fold increase compared to the wild-type transaminase. This fully demonstrates the enormous potential of multi-site mutation strategies in optimizing transaminase performance and increasing 1,6-hexanediamine yield, providing a new reference and direction for further modifying transaminases through protein engineering to achieve more efficient biosynthesis.

[0070] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0071] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively 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 transaminase mutant, characterized in that, The transaminase mutant was obtained by mutating a wild-type transaminase derived from heterotrophic Gram-negative bacteria, and its amino acid sequence is shown in SEQ ID NO.

5. The mutation site of the transaminase mutant is any one of the following: A165S; Any combination of T36A, R43K, A239P and V419A with A165S; A combination of A165S, T36A and A239P; A combination of A165S, T36A, and V419A; or, A combination of A165S, T36A, A239P and V419A.

2. The transaminase mutant as described in claim 1, characterized in that, The gene fragment encoding the wild-type transaminase is shown in SEQ ID NO.6; using the gene fragment encoding the wild-type transaminase as a template, the gene fragment encoding the transaminase mutant was obtained by PCR amplification.

3. A recombinant plasmid carrying a gene fragment encoding the transaminase mutant of claim 1 or 2.

4. The recombinant plasmid as described in claim 3, characterized in that, The construction process of the recombinant plasmid includes: digesting the pET28a plasmid and the gene fragment encoding the transaminase mutant obtained by amplification with restriction endonucleases NdeI and XhoI; and inserting the digested gene fragment between the NdeI and XhoI restriction sites of the pET28a plasmid.

5. A recombinant strain expressing the transaminase mutant of claim 1 or 2.

6. The recombinant strain according to claim 5, characterized in that, The recombinant strain is obtained by expressing the recombinant plasmid as described in claim 3 or 4 into Escherichia coli BL21(DE3).

7. The use of the transaminase mutant of claim 1 or 2, the recombinant plasmid of claim 3 or 4, or the recombinant strain of claim 5 or 6 in the preparation of 1,6-hexanediamine.

8. A whole-cell catalytic synthesis method for 1,6-hexanediamine, characterized in that, include: Obtain the recombinant strain according to claim 5 or 6 and the strain expressing carboxylic acid reductase MaCAR and phosphoproteotransferase BsSFP; wherein the carboxylic acid reductase MaCAR is derived from Mycobacterium abscessus, and the amino acid sequence is shown in SEQ ID NO.1; wherein the phosphoproteotransferase BsSFP is derived from Bacillus subtilis, and the amino acid sequence is shown in SEQ ID NO.3; The recombinant strain and the strain were mixed and added to a reaction system including adipic acid, ammonia donor, glycerol and buffer solution, and stirred continuously to catalyze the synthesis of 1,6-hexanediamine.

9. The synthesis method as described in claim 8, characterized in that, The pH range of the reaction system is 6.8-9.0; in the reaction system, the mass ratio of the bacterial strain to the recombinant bacterial strain is 1:(1.0-1.2), the concentration of adipic acid is any value between 8mM and 12mM, the concentration of glycerol is any value between 8mM and 12mM, and the concentration of the ammonia donor is 2-5 times the concentration of adipic acid.

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