Carboxylate reductase mutants, recombinant cells and uses thereof for the production of 1,4-butanediamine

Abstract

This invention belongs to the fields of genetic engineering and enzyme engineering technology, specifically relating to a carboxylic acid reductase mutant, recombinant cells, and their application in the production of 1,4-butanediamine. The carboxylic acid reductase mutant of this invention has the amino acid sequence shown in SEQ ID NO:2, with valine at position 344 mutated to aspartic acid compared to the parent enzyme. The application of the mutant in the production of 1,4-butanediamine involves an enzyme system comprising: the carboxylic acid reductase mutant, or recombinant cells expressing the carboxylic acid reductase mutant, or a cell treatment thereof; a pantothenic acid thioethylaminotransferase modified with the carboxylic acid reductase; and a transaminase catalyzing the conversion of 4-aminobutyraldehyde to 1,4-butanediamine, or recombinant cells expressing the transaminase, or a cell treatment thereof. The carboxylic acid reductase mutant of this invention, when applied to the production of 1,4-butanediamine, significantly enhances the efficiency of 1,4-butanediamine synthesis.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and enzyme engineering technology, specifically relating to a carboxylic acid reductase mutant, recombinant cells, and their application in producing 1,4-butanediamine. Background Technology

[0002] Currently, the production of 1,4-butanediamine mainly relies on chemical synthesis. This involves reacting highly toxic styrene with hydrogen cyanide under expensive alkaline catalysts to produce flammable succinate, which is then converted to 1,4-butanediamine through a demanding hydrogenation reaction. This entire chemical synthesis route poses significant risks to human health and the environment, and the chemical raw materials used are derived from non-renewable petroleum resources. Given the shortage of petrochemical raw materials and increasing environmental pollution, finding a low-energy-consumption, environmentally friendly, sustainable, and efficient route for the production of 1,4-butanediamine is urgently needed. With the development of bioengineering, biosynthesis is gradually demonstrating its application value in numerous fields such as bio-based chemical product production, bioenergy, biomedicine, and pollution control.

[0003] The biosynthetic pathways of 1,4-butanediamine are mainly divided into two routes: direct decarboxylation of ornithine, two-step conversion of arginine, and γ-aminobutyric acid / glutamate derivatization. For example... Figure 1 As shown, the γ-aminobutyric acid pathway specifically includes two enzymatic reactions: (1) γ-aminobutyric acid is converted into 4-aminobutyraldehyde under the catalysis of carboxylic acid reductase, and the reaction process consumes ATP and NADPH; (2) 4-aminobutyraldehyde is converted into 1,4-butanediamine under the catalysis of transaminase, and the reaction process consumes L-alanine.

[0004] Compared to ornithine and arginine decarboxylation methods, γ-aminobutyric acid (GABA) has a wider range of sources, milder reaction conditions, a mature fermentation process, and lower costs. Furthermore, it exhibits weaker product feedback inhibition, and the carboxyl reductases and transaminases involved in GABA conversion are more tolerant of the product, reducing the impact of feedback inhibition on conversion efficiency. However, further improvements in actual conversion efficiency are still needed through optimizing enzyme combinations and modifying expression strains. Therefore, developing highly active carboxyl reductases and transaminases is crucial for achieving efficient biosynthesis of 1,4-butanediamine. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the purpose of this invention is to provide a carboxylic acid reductase mutant and its application in the production of 1,4-butanediamine.

[0006] The technical solution of this invention is summarized as follows: This invention provides a carboxylic acid reductase mutant, the amino acid sequence of which is shown in SEQ ID NO: 2.

[0007] The present invention also provides the encoding gene of the above-mentioned carboxylic acid reductase mutant.

[0008] The present invention also provides vectors, recombinant vectors or expression vectors containing the above-mentioned coding genes.

[0009] The present invention also provides a host cell containing the above-described coding gene or recombinant vector.

[0010] The present invention also provides a method for preparing the above-mentioned carboxylic acid reductase mutant, which involves gene recombination and expression using the coding gene of the carboxylic acid reductase mutant described in the present invention or an expression vector containing the coding gene.

[0011] The present invention also provides the application of the above-mentioned carboxylic acid reductase mutant in the production of 1,4-butanediamine.

[0012] The present invention also provides a composition for producing 1,4-butanediamine, comprising: the carboxylic acid reductase mutant of the present invention, or recombinant cells expressing the carboxylic acid reductase mutant, or a cell treatment thereof; A pantothenic acid thioethylamine transferase modified with the carboxylic acid reductase, or recombinant cells expressing the pantothenic acid thioethylamine transferase, or a cell treatment thereof; and A transaminase that catalyzes the conversion of 4-aminobutyraldehyde to 1,4-butanediamine, or a recombinant cell expressing said transaminase, or a cell treatment thereof; The amino acid sequence of the carboxylic acid reductase mutant is shown in SEQ ID NO: 2.

[0013] The beneficial effects of this invention are as follows: This invention uses materials derived from mycobacteria Mycobacteroides abscessus Based on the carboxylic acid reductase CAR, a highly active mutant was obtained by screening using an AI prediction platform. This mutant involves mutating valine at position 344 of the carboxylic acid reductase CAR to aspartic acid (i.e., CAR-V344D). The effect of the mutant enzyme on the synthesis efficiency of 1,4-butanediamine was evaluated. The CAR-V344D mutant enzyme (amino acid sequence shown in SEQ ID NO.2) provided by this invention, when used to replace the original enzyme in engineered bacteria producing 1,4-butanediamine, significantly enhances the synthesis efficiency of 1,4-butanediamine and promotes an increase in the yield of 1,4-butanediamine. Attached Figure Description

[0014] Figure 1 : The γ-aminobutyric acid pathway in the biosynthesis of 1,4-butanediamine.

[0015] Figure 2 Maps of pET28a plasmid, recombinant vectors pET28a-CAR-sfp and pET28a-SpuC. Detailed Implementation

[0016] The present invention will now be described through specific embodiments. Unless otherwise specified, all technical means used in this invention are methods well known to those skilled in the art. Furthermore, the embodiments should be understood as illustrative, not limiting, of the scope of the invention; the essence and scope of the invention are defined only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments without departing from the essence and scope of the invention also fall within the protection scope of this invention.

[0017] The following definitions are used in this invention: 1. Nomenclature of amino acids and DNA nucleic acid sequences Amino acid residues are named using the recognized IUPAC nomenclature, represented by three-letter abbreviations or single-letter symbols. DNA nucleic acid sequences are named using the recognized IUPAC nomenclature.

[0018] 2. Identification of mutants The term "amino acid replaced at the original amino acid position" is used to represent the mutated amino acid in the carboxylic acid reductase mutant. For example, V344D indicates that the 344th amino acid residue in the sequence from the N-terminus to the C-terminus is replaced by aspartic acid (D) instead of the parental valine (V). The numbering method of the carboxylic acid reductase in this invention is based on the sequence shown in SEQ ID NO:1.

[0019] 3. The amino acid sequence of the carboxylic acid reductase mutant is shown in SEQ ID NO:2: 4. The term "recombinant cell" refers to a cell obtained by introducing exogenous nucleic acid molecules (including but not limited to recombinant genes and recombinant vectors) into a host cell through genetic engineering. The cell thereby expresses genes or proteins that are not expressed in their natural state, and / or acquires new biological functions or characteristics. The "recombinant cell" includes "transformed organisms," "recombinant host cells," and their progeny.

[0020] 5. The term "cell-treated product" refers to a product in which the recombinant cells retain the corresponding enzyme catalytic activity after being treated by physical, chemical, or biological methods. This includes, but is not limited to: lysed cells, cell lysate, cell permeation products, crude enzyme extracts, crude enzyme solutions, immobilized cells, or immobilized enzymes.

[0021] The technical solution of this invention is described in detail below: This invention provides a carboxylic acid reductase mutant, the amino acid sequence of which is shown in SEQ ID NO:2.

[0022] The carboxylic acid reductase mutant described in this invention and the one derived from mycobacteria Mycobacteroides abscessus Compared to the parental carboxylic acid reductase CAR shown in SEQ ID NO:1, the amino acid sequence at position 344 is mutated to aspartic acid.

[0023] The present invention also provides a coding gene for the above-mentioned carboxylate reductase mutant. According to the present invention, the coding gene is not limited to the nucleotide sequence shown in SEQ ID NO:7 in the specific embodiments, and may also include a mutant nucleotide sequence as the sequence shown in SEQ ID NO:7 or a gene homologous to the sequence shown in SEQ ID NO:7 that encodes the carboxylate reductase (SEQ ID NO:2). The coding gene may be a variant nucleotide sequence due to the degeneracy of the genetic code.

[0024] The present invention also provides vectors, recombinant vectors or expression vectors containing the above-mentioned coding genes, such as a recombinant vector composed of vector plasmid pET28a and the coding gene described in the present invention.

[0025] The present invention also provides a host cell comprising the above-described coding gene or recombinant vector. The host cell can be any host suitable for producing the carboxylate reductase mutant of the present invention from the gene or vector of the present invention, such as microbial expression systems including *Escherichia coli*, Bacillus (e.g., *Bacillus subtilis*, *Bacillus amyloliquefaciens*), and yeasts (e.g., *Pichia pastoris*, *Saccharomyces cerevisiae*). *Escherichia coli* is preferred.

[0026] The present invention also provides a method for preparing the above-mentioned carboxylate reductase mutant, which involves gene recombination and expression using the coding gene of the carboxylate reductase mutant described in this invention or an expression vector containing the coding gene. Gene recombination methods and expression hosts known to those skilled in the art can be used, and suitable culture media and culture conditions for host expression can be selected. The method may further include a step of recovering the carboxylate reductase mutant, which may involve isolating or purifying the carboxylate reductase mutant from the host culture or expression product, and can be performed using any method known to those skilled in the art.

[0027] This invention also provides the application of the above-mentioned carboxylic acid reductase mutant in the production of 1,4-butanediamine, particularly in the catalytic conversion of γ-aminobutyric acid to 4-aminobutyraldehyde. The carboxylic acid reductase mutant of this invention exhibits improved enzymatic catalytic activity compared to the parental carboxylic acid reductase.

[0028] The present invention also provides a composition for producing 1,4-butanediamine, comprising: the carboxylic acid reductase mutant of the present invention, or recombinant cells expressing the carboxylic acid reductase mutant, or a cell treatment thereof; A pantothenic acid thioethylamine transferase modified with the carboxylic acid reductase, or recombinant cells expressing the pantothenic acid thioethylamine transferase, or a cell treatment thereof; and A transaminase that catalyzes the conversion of 4-aminobutyraldehyde to 1,4-butanediamine, or a recombinant cell expressing said transaminase, or a cell treatment thereof; The amino acid sequence of the carboxylic acid reductase mutant is shown in SEQ ID NO: 2.

[0029] The present invention also provides a method for producing 1,4-butanediamine, comprising the following steps: Under suitable reaction conditions, an enzyme system is used to catalyze the production of 1,4-butanediamine from the substrate γ-aminobutyric acid. The enzyme system comprises: the carboxylic acid reductase mutant of the present invention, or recombinant cells expressing the carboxylic acid reductase mutant, or a cell treatment thereof; A pantothenic acid thioethylamine transferase modified with the carboxylic acid reductase, or recombinant cells expressing the pantothenic acid thioethylamine transferase, or a cell treatment thereof; and A transaminase that catalyzes the conversion of 4-aminobutyraldehyde to 1,4-butanediamine, or a recombinant cell expressing said transaminase, or a cell treatment thereof; The method includes the step of mixing and contacting the substrate with the three enzymes described above, or their recombinant cells, or their cell treatments.

[0030] In some embodiments, the transaminase is preferably a highly active mutant SpuC-M298R, whose amino acid sequence is shown in SEQ ID NO:5.

[0031] The present invention will be described in more detail below through specific embodiments. Unless otherwise defined, the technical and scientific terms used in the following embodiments have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0032] Example 1: This example illustrates mutation site prediction and mutant library construction.

[0033] (1) Mutation site prediction Using an AI prediction platform to obtain carboxylic acid reductase CAR (from cart Based on the kinetic parameters of the gene encoding (NCBI-ProteinID: WP_145044229.1), and according to the screening criteria of high affinity and high catalytic constant, 10 potential high-activity mutation sites were selected for mutant expression and catalytic verification.

[0034] (2) Construction of mutant libraries Since carboxylic acid reductase (CAR) requires pantothenic thiol-ethylamine modification, this embodiment selected Bacillus subtilis as the precursor. Bacillus subtilis The pantothenic thioethylamine transferase Sfp (by sfp Gene encoding, NCBI-ProteinID: ACG68433.1).

[0035] First, pET28a plasmid ( Figure 2 Using pET28a-F / R as a template, a linearized vector was obtained by reverse PCR, and then obtained using primer CAR-F / R. cart Gene fragment (encoding amino acid sequence as shown in SEQ ID NO:1, nucleotide sequence as shown in SEQ ID NO:6), pET28a linearized vector and cart The gene fragment was ligated using the ClonExpress® II One StepCloning Kit series of homologous recombinases (purchased from Nanjing Novizan Biotechnology Co., Ltd.) to obtain the recombinant vector pET28a-CAR.

[0036] The PCR reaction system consisted of: a total volume of 50 μL, 19 μL of sterile water, 2 μL of upstream primer, 2 μL of downstream primer, 2 μL of template, and 25 μL of Primestar DNA Polymerase. The PCR reaction program was: 98℃ for 30 s, 98℃ for 10 s, 57℃ for 30 s, 72℃ for 5 s / kb, 72℃ for 10 min, 16℃ ± ∞, with 30 cycles. The homologous recombination reaction conditions were: 37℃ for 10 min. The reaction system consisted of: a total volume of 10 μL, 2 μL of 5×CE II Buffer, 1 μL of linearized cloning vector, 1 μL of insert fragment, 1 μL of Exnase® II, and 5 μL of ddH2O.

[0037] The recombinant vector pET28a-CAR linker system was transformed into Escherichia coli via CaCl2 chemical transformation. E. coli After DH5α, the cells were plated on LB agar plates containing 50 mg / L kanamycin sulfate and incubated overnight at 37°C. Once single colonies appeared on the plates, colony PCR was performed using primers JD-pET28a-F / R to identify the recombinant strain. E. coli DH5α-pET28a-CAR. DNA was extracted from the recombinant strain using the OMEGA DNA Extraction Kit (Omega, catalog number D3350-01). E. coli plasmid pET28a-CAR was extracted from DH5α-pET28a-CAR.

[0038] pET28a-CAR recombinant plasmid ( Figure 2 Using pET28a-CAR-F / R as a template, a linearized vector was obtained by reverse PCR. Then, using primers sfp-F / R, the vector was further obtained... sfp Gene fragment (encoding amino acid sequence as shown in SEQ ID NO:3, nucleotide sequence as shown in SEQ ID NO:8), pET28a-CAR linearized vector and sfp Gene fragment ligation products were chemically transformed into E. coli E. coli DH5α was plated on LB agar plates containing 50 mg / L kanamycin sulfate and incubated overnight at 37°C. After single colonies grew on the plates, colony PCR was performed using primers JD-pET28a-F / R to identify the recombinant strain. E. coli DH5α-pET28a-CAR-sfp.

[0039] DNA was extracted from recombinant strains using the OMEGA DNA Extraction Kit (brand: Omega, catalog number: D3350-01). E. coli The plasmid pET28a-CAR-sfp was extracted from DH5α-pET28a-CAR-sfp. The recombinant plasmid was chemically transformed into... E. coliAfter single colonies of BL21(DE3) competent cells grew on plates, colony PCR was performed using primers JD-pET28a-F / R to identify the recombinant expression strain. E. coli BL21-pET28a-CAR-sfp.

[0040] Using recombinant plasmid pET28a-CAR-sfp ( Figure 2 Using a template, circular mutant plasmids were obtained by full plasmid PCR using mutant primers (SEQ ID NO: 20~SEQ ID NO: 39 shown in Table 1). The PCR reaction system was as follows: total volume 50 μL, sterile water 19 μL, upstream primer 2 μL, downstream primer 2 μL, template 2 μL, Primestar DNA Polymerase 25 μL; the PCR reaction program was as follows: 98℃ for 30 s, 98℃ for 10 s, 57℃ for 30 s, 72℃ for 5 s / kb, 72℃ for 10 min, 16℃ ± ∞, 30 cycles.

[0041] PCR products were digested with the fast digester Q.Cut Mr. Digest at 37℃ for 1 hour. The digestion system consisted of: 43 μL of PCR product, 5 μL of buffer, and Q. Cut. Mr. I 2μL, take 10μL of the digestion system and chemically convert it to E. coli DH5α competent cells were used to obtain recombinant mutant strains after verification with primers JD-pET28a-S / A.

[0042] Mutant plasmids were extracted using the OMEGA DNA Extraction Kit (brand: Omega, catalog number: D3350-01), and then chemically transformed into... E. coli After single colonies of BL21(DE3) competent cells grew on plates, colony PCR was performed using primers JD-pET28a-S / A to identify the mutant strain.

[0043] (3) Transaminase SpuC and its mutant SpuC-M298 Since the synthesis of 1,4-butanediamine requires a two-step enzymatic reaction, and the key enzyme in the second step is a transaminase, a transaminase derived from *Pseudomonas* was selected for this pathway. Pseudomonas transaminase (by say The gene encoding (NCBI-ProteinID: WP_016502144.1) was used. A linearized vector was obtained via reverse PCR using primer pET28a-F / R with pET28a plasmid as a template. The vector was then used with primer SpuC-F / R to obtain... say Gene fragments (encoding amino acid sequences as shown in SEQ ID NO:4 and nucleotide sequences as shown in SEQ ID NO:9), the pET28a linearized vector and sayThe gene fragment was ligated into the recombinant vector pET28a-SpuC using the ClonExpress® II One Step Cloning Kit series of homologous recombinases (purchased from Nanjing Novizan Biotechnology Co., Ltd.). The PCR reaction system and procedure were the same as above, and then the vector was transformed into E. coli. E. coli DH5α was plated on LB agar plates containing 50 mg / L kanamycin sulfate and incubated overnight at 37°C. After single colonies grew on the plates, colony PCR was performed using primers JD-pET28a-F / R to identify the recombinant strain. E. coli DH5α-pET28a-SpuC.

[0044] Using the OMEGA DNA Extraction Kit (brand: Omega, catalog number: D3350-01), DNA was extracted from recombinant strains. E. coli Plasmid pET28a-SpuC was extracted from DH5α-pET28a-SpuC. The recombinant plasmid was chemically transformed into... E. coli After single colonies of BL21(DE3) competent cells grew on plates, colony PCR was performed using primers JD-pET28a-F / R to identify the recombinant expression strain. E. coli BL21-pET28a-SpuC.

[0045] Using recombinant plasmid pET28a-SpuC as a template, a circular mutant plasmid was obtained by full-plasmid PCR using the mutant primers SpuC-M298R-F / R. The PCR reaction system consisted of: 50 μL total volume, 19 μL sterile water, 2 μL upstream primer, 2 μL downstream primer, 2 μL template, and 25 μL Primestar DNA Polymerase. The PCR program was as follows: 98℃ for 30 s, 98℃ for 10 s, 57℃ for 30 s, 72℃ for 5 s / kb, 72℃ for 10 min, 16℃ ± ∞, for a total of 30 cycles.

[0046] PCR products were digested with the fast digester Q.Cut Mr. Digest at 37℃ for 1 hour. The digestion system consisted of: 43 μL of PCR product, 5 μL of buffer, and Q. Cut. Mr. I 2μL, take 10μL of the digestion system and chemically convert it to E. coli DH5α competent cells were used to obtain recombinant mutant strains after verification with primers JD-pET28a-S / A.

[0047] Mutant plasmids were extracted using the OMEGA DNA Extraction Kit (brand: Omega, catalog number: D3350-01), and then chemically transformed into... E. coliAfter single colonies of BL21(DE3) competent cells grew on plates, colony PCR was performed using primers JD-pET28a-S / A to identify the mutant strain.

[0048] Table 1

[0049] Example 2: This example illustrates the activity screening and verification of mutant enzymes and their application in the production of 1,4-butanediamine.

[0050] Screening method for mutant strains: The mutant strains obtained in Example 1 were cultured and fermented as follows.

[0051] Plate culture: The recombinant carboxylic acid reductase CAR strain and the recombinant transaminase SpuC strain stored at -80℃ were inoculated onto activation plates and incubated overnight at 37℃ to obtain slant seed culture; the solid culture medium consisted of: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, 20 g / L agar, and the remainder was water, with natural pH.

[0052] In vitro seed culture: After scraping the bacterial cells with a toothpick, they were inoculated into test tubes containing 5 mL of LB medium and cultured overnight with shaking at 37℃ and 220 rpm to obtain the primary seed culture. The seed culture medium consisted of: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, and the remainder was water, with natural pH.

[0053] 24-well plate fermentation: The primary seed culture was inoculated at a rate of 10% (v / v) into 24-well plates containing 4.5 mL of fermentation medium (total system 5 mL). The plates were cultured at 37℃ and 220 rpm with shaking until OD600 = 0.6-0.8. IPTG was then added at a final concentration of 0.1 mM, and expression was induced at 20℃ and 200 rpm for 20 h. The fermentation medium consisted of 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, and the remainder was water, with the pH set to natural.

[0054] Shake-flask fermentation: The primary seed culture was inoculated at a rate of 2% (v / v) into shake flasks containing 50 mL of fermentation medium. The culture was shaken at 37℃ and 220 rpm until OD600 = 0.6-0.8. IPTG was then added at a final concentration of 0.1 mM, and expression was induced at 20℃ and 200 rpm for 20 h. The fermentation medium consisted of 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, and the remainder was water, with the pH set to natural.

[0055] After fermentation, the fermentation broth of the two strains was centrifuged at 4℃ and 8000 rpm for 10 min, and then resuspended in 0.05 M, pH 7.0 PBS buffer solution. This process was repeated three times to remove interference from the fermentation supernatant.

[0056] Ultrasonic disruption: The bacterial cells were resuspended in 0.05M, pH 7.0 PBS buffer solution, and then disrupted using an ultrasonic disruptor. The ultrasonic conditions were: voltage 150 W, ultrasonic on for 2 s and off for 4 s, ultrasonic time 10-20 min, to obtain crude enzyme solution of recombinant strain.

[0057] The catalytic reaction system consisted of a final volume of 5 mL of 0.05 M PBS buffer solution (pH 7.0), to which 1 mL each of the crude enzyme solution (OD600=30) of the recombinant carboxylic acid reductase strain and the crude enzyme solution (OD600=30) of the recombinant transaminase strain were added. The system also contained 0.1 M GABA and 10 mM Mg. 2+ 0.1 mM PLP, 2.5 g / L glucose, and 20 mM alanine were mixed thoroughly and reacted in a 30°C water bath for 24 h. After the reaction, recombinant cells were removed by centrifugation at 4°C and 8000 rpm for 10 min. The control group consisted of Escherichia coli. E. coli BL21-pET28a-CAR-sfp and E. coli BL21-pET28a-SpuC.

[0058] The liquid chromatography method for detecting 1,4-butanediamine in the reaction system was as follows: reversed-phase column (Shim-pack GIST-HP-C18 column, 2.1 x 100 mm, particle size 3 μm), detection wavelength 284 nm. The mobile phase system consisted of: 100% acetonitrile (A); 25 mM sodium acetate aqueous buffer (pH 4.8) (B). The flow rate was 0.5 mL / min. The gradient elution program was as follows: 0–2 min: 80%–75% B; 2–27 min: 75%–37.5% B; 27.01–37 min: 80% B. The flow rate was 0.5 mL / min, column temperature was 30 °C, UV detection wavelength was 284 nm, and retention time was 40 min.

[0059] The mutant strain from Example 1 was subjected to 24-well plate fermentation and catalytic reaction according to the above method. The enzyme catalytic system for producing 1,4-butanediamine included: a carboxylic acid reductase CAR mutant, a pantothenic thioethylamine transferase Sfp wild-type, and a transaminase SpuC wild-type. The catalytic reaction results are shown in Table 2. (The text abruptly ends here, likely due to an incomplete sentence or missing information.) E. coli BL21-pET28a-CAR-V344D-sfp and E. coliThe yield of 1,4-butanediamine in the catalytic reaction system of crude enzyme solution of BL21-pET28a-SpuC was 2.015 g / L, compared with the control group ( E. coli BL21-pET28a-CAR-sfp and E. coli Compared to BL21-pET28a-SpuC, the carboxyl reductase mutant CAR-V344D increased the yield of 1,4-butanediamine by 18.2%.

[0060] Table 2

[0061] Example 3: This embodiment illustrates a method for producing 1,4-butanediamine using the highly active mutant CAR-V344D and its enzyme catalytic system.

[0062] Based on the preliminary results of 24-well plate fermentation and catalytic reaction screening, the scale-up reaction system was set to 50 mL. The enzyme catalytic system included: carboxylic acid reductase mutant CAR-V344D, pantothenic acid thioethylamine transferase Sfp wild-type, transaminase SpuC wild-type, or its highly active mutant SpuC-M298R. The 50 mL shake-flask catalytic reaction system consisted of: a final volume of 50 mL, 0.05 M PBS buffer (pH 7.0), 10 mL each of crude enzyme solution (OD600=30) of the recombinant carboxylic acid reductase strain and the recombinant transaminase strain, and the system contained 0.3 M GABA and 30 mM Mg. 2+ 0.1 mM PLP, 6 g / L glucose, and 60 mM alanine were mixed thoroughly and reacted in a 30°C water bath for 24 h. After the reaction, recombinant cells were removed by centrifugation at 4°C and 8000 rpm for 10 min. The control group consisted of Escherichia coli. E. coli BL21-pET28a-CAR-sfp and E. coli BL21-pET28a-SpuC.

[0063] The results are shown in Table 3, in the presence of recombinant strains E. coli BL21-pET28a-CAR-V344D-sfp and E. coli In the catalytic reaction system of BL21-pET28a-SpuC, the yield of 1,4-butanediamine was 14.4 g / L, which was similar to that of the control group ( E. coli BL21-pET28a-CAR-sfp and E. coliCompared to BL21-pET28a-SpuC, the carboxylic acid reductase mutant CAR-V344D increased the yield of 1,4-butanediamine by 16%. Even with the introduction of the highly active transaminase mutant SpuC-M298R into the catalytic system, the addition of the carboxylic acid reductase mutant CAR-V344D further increased the yield of 1,4-butanediamine from 15.4 g / L to 16.5 g / L.

[0064] Table 3

[0065] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various changes, modifications, substitutions and variations in form and detail to these embodiments without departing from the spirit and principles of the present invention. The scope of the present invention is defined by the claims and their equivalents.

Claims

1. A carboxylic acid reductase mutant, the amino acid sequence of which is shown in SEQ ID NO:

2.

2. The encoding gene of the carboxylic acid reductase mutant according to claim 1.

3. The encoding gene as described in claim 2, wherein the nucleotide sequence is shown in SEQ ID NO:

7.

4. A recombinant vector, characterized in that, It includes the encoding gene as described in claim 2.

5. A host cell, characterized in that, It contains the coding gene of claim 2 or the recombinant vector of claim 4.

6. The application of the carboxylic acid reductase mutant according to claim 1, characterized in that, The carboxylic acid reductase mutant is used to catalyze the conversion of γ-aminobutyric acid to 4-aminobutyraldehyde.

7. A method for preparing the carboxylic acid reductase mutant of claim 1, characterized in that, This method involves gene recombination and expression using the coding gene of the carboxylic acid reductase mutant or an expression vector containing the coding gene.

8. A composition for producing 1,4-butanediamine, comprising: the carboxylic acid reductase mutant of claim 1, or recombinant cells expressing the carboxylic acid reductase mutant, or a cell treatment thereof; A pantothenic acid thioethylamine transferase modified with the carboxylic acid reductase, or recombinant cells expressing the pantothenic acid thioethylamine transferase, or a cell treatment thereof; and A transaminase that catalyzes the conversion of 4-aminobutyraldehyde to 1,4-butanediamine, or a recombinant cell expressing said transaminase, or a cell treatment thereof.

9. The composition according to claim 8, characterized in that, The amino acid sequence of the transaminase is shown in SEQ ID NO:5.

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