Carboxylic acid reductase mutant and application thereof

Abstract

The invention provides a carboxylate reductase mutant and application thereof. The mutant is characterized in that the 394th site of carboxylic acid reductase is subjected to site-specific mutagenesis, or the 419th site of carboxylic acid reductase is subjected to site-specific mutagenesis, or the 394th site and the 614th site of carboxylic acid reductase are subjected to site-specific mutagenesis at the same time. The amino acid sequence of the mutant G394F is as shown in SEQ ID NO.4, and the nucleotide sequence of the mutant G394F is as shown in SEQ ID NO.3; the amino acid sequence of the mutant G419H is as shown in SEQ ID NO.6, and the nucleotide sequence of the mutant G419H is as shown in SEQ ID NO.5; the amino acid sequence of the mutant MT1 is as shown in SEQ ID NO.8, and the nucleotide sequence of the mutant MT1 is as shown in SEQ ID NO.7. Compared with wild type carboxylic acid reductase, the carboxylic acid reductase mutant has the advantages that the enzyme activity is improved by 1.73-1.94 times, the yield of 1, 5-PDO is improved by 8.76%-32.67%, the production cycle of 1, 5-PDO is effectively shortened, and the production cost of 1, 5-PDO is reduced.

Description

Technical Field

[0001] The invention relates to a carboxylic acid reductase mutant and application thereof, belonging to the technical field of microbial enzymes. Background Art

[0002] 1,5-Pentanediol (1,5-PDO) is a key diol and intermediate in organic synthesis. It has received extensive attention and application in different fields in recent years. It is a green raw material for the production of new polyesters, coatings, adhesives, sealants and plasticizers. It can be used as an environmentally friendly green solvent for chemical synthesis such as antifreeze, inks, special detergents, etc. In the pharmaceutical field, it is used to produce penicillin intermediates, anti-tumor drugs and anesthetics. Compared with traditional plasticizers, 1,5-PDO can improve its poor temperature resistance and has high efficiency. With the expansion of demand for 1,5-PDO in many fields, the current production capacity cannot meet market demand. Therefore, the development prospects of efficient synthesis of 1,5-PDO are broad.

[0003] Industrial production of 1,5-PDO mainly relies on petroleum as raw material through redox reaction. However, the process route is complicated, there are many by-products of fusel alcohol, and a large amount of non-renewable petroleum energy is consumed, so the ability to produce 1,5-PDO is limited by limited C5 raw materials, making it difficult to achieve large-scale production. Renewable carbon source-biomass is not only widely available, low in raw material cost, but also has a short renewable cycle. Therefore, in recent years, chemical methods for converting biomass-derived furfural or tetrahydrofuran methanol into 1,5-PDO have received widespread attention. However, these processes usually include multiple chemical steps, including hydrogenation, dehydration, hydration and hydrogenation. There are disadvantages such as complex conversion process, increased production cost due to the use of precious metal catalysts, harsh reaction conditions for non-precious metal catalysts, unsatisfactory and unstable yield of target products, and unclear catalytic mechanism, which seriously restricts its industrial development.

[0004] Therefore, developing a green biosynthetic pathway for 1,5-PDO is an effective way to reduce people's dependence on fossil fuels. Carboxylic acid reductase (CAR) plays a vital role in this process. CAR can reduce the carboxyl group on 5-hydroxyvaleric acid (5-HV) to an aldehyde group to generate 5-hydroxyvaleraldehyde, which is a key step in the synthesis of 1,5-PDO. Compared with traditional chemical synthesis methods, the biosynthetic pathway catalyzed by CAR has the advantages of mild conditions, high selectivity, and environmental friendliness. In addition, using glucose as a raw material, low-cost and high-yield production of 1,5-PDO can be achieved through microbial fermentation and metabolic engineering. As a renewable and easily accessible carbon source, glucose can be converted into precursor substances such as 5-HV through metabolic pathways, providing sufficient substrates for the catalytic reaction of CAR. However, to achieve the efficient application of CAR in industrial production, it is necessary to further optimize its catalytic performance and stability, as well as develop fermentation processes and reaction systems that match it. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides a carboxylic acid reductase mutant and its application. The present invention uses site-directed mutagenesis technology to carry out directed evolution of wild-type carboxylic acid reductase, and obtains a mutant enzyme with higher catalytic efficiency of 5-HV (5-hydroxyvaleric acid) reduction, which is used for the biological fermentation production of 1,5-PDO, thereby replacing the chemical method with many by-products and difficult separation and purification to produce 1,5-pentanediol with broad application prospects.

[0006] The technical solution of the present invention is as follows:

[0007] A carboxylic acid reductase mutant, wherein the mutant is a carboxylic acid reductase (CAR) in which the glycine at position 394 undergoes a site-directed mutation, or a glycine at position 419 undergoes a site-directed mutation, or a glycine at position 394 and an isoleucine at position 614 undergo a site-directed mutation simultaneously; the nucleotide sequence of the carboxylic acid reductase (CAR) is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0008] Preferably according to the present invention, the mutant is the carboxylic acid reductase mutant G394F, whose amino acid sequence is shown in SEQ ID NO.4, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.3; the glycine at position 394 of the carboxylic acid reductase amino acid sequence is mutated into phenylalanine.

[0009] Preferably according to the present invention, the mutant is the carboxylic acid reductase mutant G419H, whose amino acid sequence is shown in SEQ ID NO.6, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.5; the glycine at position 419 of the carboxylic acid reductase amino acid sequence is mutated into histidine.

[0010] Preferably according to the present invention, the mutant is a carboxylic acid reductase mutant MT1 (G419H-I614R), whose amino acid sequence is shown in SEQ ID NO.8, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.7; the glycine at position 419 of the carboxylic acid reductase amino acid sequence is mutated into histidine, and the isoleucine at position 614 is mutated into arginine.

[0011] A recombinant vector is a plasmid vector in which the coding gene of the carboxylic acid reductase mutant is inserted.

[0012] Preferably according to the present invention, the plasmid vector is pET-28a.

[0013] A recombinant strain is obtained by transforming the above recombinant vector into a host cell.

[0014] Preferably, according to the present invention, the method for constructing the recombinant strain is specifically as follows: the above-mentioned carboxylic acid reductase mutant encoding gene or a recombinant vector containing the above-mentioned carboxylic acid reductase mutant encoding gene is transferred into the genetically engineered Escherichia coli LE02G to obtain a recombinant strain for producing 1,5-pentanediol.

[0015] Among them, the Escherichia coli genetically engineered bacteria LE02G is an existing strain, and its construction method has been disclosed in Chinese patent document CN118726222A, a genetically engineered bacteria for efficiently producing 1,5-pentanediol and its construction method and application. For details, please refer to paragraphs

[0060] to

[0168] of its specification.

[0016] Application of the carboxylic acid reductase mutant, recombinant vector or recombinant strain in the preparation of 1,5-pentanediol.

[0017] Preferably, according to the present invention, the specific process for preparing 1,5-pentanediol using the above-mentioned recombinant strain is as follows:

[0018] The recombinant strain was activated on LB solid medium, and a single colony after activation was picked and inoculated into LB liquid medium, and cultured at 37°C and 200 r / min for 12 hours; then, the inoculation amount was 1.5-2.5% and inoculated into fermentation medium, and kanamycin with a concentration of 50 μg / mL was added to maintain the plasmid; after growing at 37°C for 4 hours, an inducer IPTG (final concentration 1 mM) was added, and fermentation production was continued for 5-7 days to obtain 1,5-pentanediol;

[0019] Among them, the fermentation medium composition ( / L): glucose 20g / L, peptone 10g / L, yeast extract powder 5g / L, NaCl 10g / L.

[0020] The beneficial effects of the present invention are:

[0021] The present invention is based on carboxylic acid reductase (CAR), and selects the amino acid at position 394, 419 or 614 for site-directed mutagenesis, and mutates the glycine at position 394 to phenylalanine (G394F), or mutates the glycine at position 419 to histidine (G419H), or mutates the glycine at position 419 to histidine and the isoleucine at position 614 to arginine (G419H-I614R). The carboxylic acid reductase mutant provided by the present invention has higher enzyme activity and higher efficiency in catalyzing the synthesis of 1,5-PDO. Compared with the wild-type carboxylic acid reductase, the enzymatic activity of the carboxylic acid reductase mutant was increased by 1.73 to 1.94 times, and the 1,5-PDO yield was increased by 8.76% to 32.67%, which effectively shortened the production cycle of 1,5-PDO and reduced the production cost of 1,5-PDO, thereby solving the problems of low carboxylic acid reductase activity, low expression level, limited application, and low synthesis yield of 1,5-PDO in the prior art, and provided technical support for industrial applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The electrophoresis results of carboxylic acid reductase mutants are shown;

[0023] In the figure, lane M represents protein marker, lane 1 represents G394F, lane 2 represents G419H, and lane 3 represents MTI (G419H-I614R).

[0024] Figure 2 The relative enzymatic activity comparison results of carboxylic acid reductase mutant and wild-type carboxylic acid reductase;

[0025] In the figure, (a) is the relative enzyme activity comparison result of some carboxylic acid reductase single mutants and wild-type carboxylic acid reductase; (b) is the relative enzyme activity comparison result of some carboxylic acid reductase double mutants and wild-type carboxylic acid reductase.

[0026] Figure 3 The effect of temperature on the activity and stability of carboxylic acid reductase mutants;

[0027] In the figure, (a) is the optimal reaction temperature of G394F, G419H and MT1; (b) is the temperature stability of G394F; (c) is the temperature stability of G419H; and (d) is the temperature stability of MT1.

[0028] Figure 4 The effect of pH on the activity and stability of carboxylic acid reductase mutants;

[0029] In the figure, (a) is the optimal reaction pH of G394F, G419H, and MT1; (b) is the pH stability of G394F; (c) is the pH stability of G419H; (d) is the pH stability of MT1

[0030] Figure 5 To study the carboxylic acid reductase mutants G394F, G419H and MT1 (G419H-I614R) for the synthesis of 1,5-PDO;

[0031] In the figure, (a) is the synthesis pathway of 1,5-PDO; (b) is the synthesis pathway of LE G394F LE G419H and LE MT1 5-HV production; (c) LE G394F LE G419H and LE MT1 The yield of 5-hydroxyvaleraldehyde; (d) is LE G394F LE G419H and LE MT1 of 1,5-PDO production. DETAILED DESCRIPTION

[0032] The technical solution of the present invention is further described below in conjunction with the embodiments and the accompanying drawings, but the protection scope of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the present invention are methods well known to those skilled in the art.

[0033] In the embodiment, the YahK enzyme is alcohol dehydrogenase, and the GenBank number of its gene is EG13595.

[0034] In the embodiment, the genetically engineered Escherichia coli LE02G is an existing strain, and its construction method has been disclosed in Chinese patent document CN118726222A, a genetically engineered bacterium for efficiently producing 1,5-pentanediol, and its construction method and application. For details, please see paragraphs

[0060] to

[0168] of its specification.

[0035] The determination method of 5-hydroxyvaleric acid (5-HV) adopts ultraviolet high performance liquid chromatography.

[0036] 5-HV detection method: Detected by ultraviolet high performance liquid chromatography, the detection conditions are as follows: mobile phase: 5mM H2SO4; volume flow rate: 0.6mL / min; column temperature: 65℃; detection wavelength: 210nm.

[0037] Example 1. Preparation of carboxylic acid reductase mutants

[0038] 1. The inventors conducted bioinformatics analysis on the wild-type carboxylic acid reductase (CAR) from Nocardia iowensis to determine the key amino acid sites. The nucleotide sequence of the wild-type carboxylic acid reductase (CAR) is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0039] Specifically, the protein model of the wild-type carboxylic acid reductase (CAR) from Nocardia iowensis was retrieved using public databases such as RCSB PDB and UniProt. Autodock Vina software was used for virtual screening to predict the binding mode of substrates and cofactors with CAR. The three-dimensional structural model of CAR was analyzed using Pymol software to identify the amino acid residues in the active center. Alanine scanning of CAR was performed using Discover studio software to evaluate the effect of each amino acid residue on enzyme activity. CAVER software was used to predict the channel leading to the active site of CAR and analyze the amino acid composition of the channel. Virtual mutations were performed on the predicted key amino acid residues to simulate the effects of mutations on enzyme activity and substrate channels.

[0040] It was found that the amino acids at positions 394, 395, 418, 419, 394, 613, 614 and 616 of the amino acid sequence of wild-type carboxylic acid reductase (CAR) were located near the active center and highly conserved regions, and site-directed mutagenesis of them was very likely to improve the enzymatic activity and catalytic efficiency of carboxylic acid reductase. Therefore, saturated single-point mutations and double-point mutations were performed on amino acids at positions 394, 395, 418, 419, 394, 613, 614 and 616 of the amino acid sequence of wild-type carboxylic acid reductase (CAR), respectively, and a total of 86 carboxylic acid reductase single mutants and 10 carboxylic acid reductase double mutants were obtained.

[0041] Among them, the amino acid sequence of the carboxylic acid reductase mutant G394F is shown in SEQ ID NO.4, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.3. The amino acid sequence of the carboxylic acid reductase mutant G419H is shown in SEQ ID NO.6, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.5. The amino acid sequence of the carboxylic acid reductase mutant MT1 (G419H-I614R) is shown in SEQ ID NO.8, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.7.

[0042] The specific information of other carboxylic acid reductase single mutants (such as Y418P, Y418A, Y418F, Y418H, I614A, K616A, S395H, S395F, G613T, etc.) and other carboxylic acid reductase double mutants (such as MT2 and MT3, etc.) will not be described in detail here.

[0043] 2. Heterologous expression of carboxylic acid reductase

[0044] (1) Taking the carboxylic acid reductase mutant G394F as an example, single point mutation primers G394-F / R were designed; then, the nucleotide sequence of the wild-type carboxylic acid reductase (CAR) was used as a template, and primers G394F-F / R were used to perform site-directed mutagenesis by inverse PCR amplification. After the amplification was completed, the unmutated original template was removed using DpnⅠ enzyme to obtain the mutant G394F gene as shown in SEQ ID NO.3;

[0045] The primer sequences are:

[0046] G394F-F: 5'-AGTTtttTCTGCTCCGCTGGCGGCGGAGATGA-3',

[0047] G394F-R: 5'-GCGGAGCAGAaaaAACTACAGCCACCAGGAAACGA-3';

[0048] Among them, the nucleotide sequence of wild-type carboxylic acid reductase (CAR) was artificially synthesized by Genewise Company according to the sequence information;

[0049] (2) The mutant G394F gene was connected to pET28a to obtain the recombinant plasmid pET28a-CAR-G394F; then the recombinant plasmid pET28a-CAR-G394F was transformed into Escherichia coli BL21star (DE3) competent cells to construct the recombinant strain CAR-G394F;

[0050] (3) The recombinant strain CAR-G394F was cultured at 37°C and 200 rpm with shaking until the OD was 0.6-0.8, IPTG (final concentration 0.05 mmol / L) was added, and induced at 25°C for 12 h; the induced expression bacteria were collected by centrifugation at 4000g and 4°C for 20 min using a high-speed refrigerated centrifuge. The centrifuged bacteria were resuspended in 50 mL Lysis Buffer (0.05 mol / L Tris-HCl pH 8.0, 0.4 mol / L NaCl, 10% (V / V) glycerol). Add 80 μL of 50 mg / mL lysozyme, 50 μL of 1 mol / L protease inhibitor-phenylmethylsulfonyl fluoride (PMSF), and 10 μL of β-thiol reducing agent, and place in an ice bath for 30 minutes, stirring every 5 to 6 minutes to allow it to fully function; then use an ultrasonic cell disruptor to disrupt the cells, and the process needs to be run under low temperature conditions for 25 minutes; after the disruption is completed, set a high-speed refrigerated centrifuge: 4°C, 13000g centrifugation for 1 hour, cool to 4°C and centrifuge to remove insoluble matter such as cell debris, and the supernatant is the crude enzyme solution of the carboxylic acid reductase mutant G394F;

[0051] (4) Using a pre-packed Ni-NTA column, the resin was equilibrated with ddH2O and Lysis Buffer, the crude enzyme solution of the carboxylic acid reductase mutant G394F was combined with the Ni-NTA column for 1 hour (4°C), and impurities were washed away with Lysis Buffer and Wash Buffer. Finally, the target protein was eluted with Elution buffer and collected in a centrifuge tube. The imidazole was removed by dialysis to obtain the heterologously expressed carboxylic acid reductase mutant G394F.

[0052] Other carboxylic acid reductase mutants were obtained by heterologous expression and purification according to the same method as above, wherein the single point mutation primers used for the carboxylic acid reductase mutants G419H and MT1 (G419H-I614R) were G419H-F / R, MT1-F1 / R1 and MT1-F2 / R2, and the specific sequences were:

[0053] G419H-F: 5'-TGGTTACcatTCTACGGAAGCCGGTGCTTCGG-3',

[0054] G419H-R: 5'-CCGTAGAatgGTAACCATCGTGCAACGGCAGG-3',

[0055] MT1-F1: 5'-TGGTTACcatTCTACGGAAGCCGGTGCTTCGG-3',

[0056] MT1-R1: 5'-CCGTAGAatgGTAACCATCGTGCAACGGCAGG-3',

[0057] MT1-F2: 5'-TGTCTGGTcgcGCGAAACTGTTGCGTCCGAAT-3',

[0058] MT1-R2: 5'-TTTCGCgcgACCAGACAGCAGGCATTCGCAA-3'.

[0059] The PCR amplification system was prepared according to the instructions of the kit; the PCR amplification program was as follows: pre-denaturation, 94°C for 2 min; denaturation, 98°C for 10 sec; annealing, 60°C for 30 sec; extension, 72°C for 3 min (30 cycles); termination of extension, 72°C for 10 min; and finally incubation at 4°C.

[0060] The expression of purified recombinant proteins G394F, G419H and MT1 (G419H-I614R) was identified by SDS-PAGE. Figure 1 shown.

[0061] Depend on Figure 1 It can be seen that the band sizes of carboxylic acid reductase mutants G394F, G419H and MT1 (G419H-I614R) are all around 128 kDa, which is consistent with the predicted value, indicating that the carboxylic acid reductase mutants G394F, G419H and MT1 (G419H-I614R) are successfully heterologously expressed.

[0062] Example 2: Determination of the enzymatic activity of carboxylic acid reductase mutants

[0063] The nucleotide sequence of the wild-type carboxylic acid reductase was artificially synthesized by Genewise Company, and the wild-type carboxylic acid reductase was obtained according to the heterologous expression method described in Example 1.

[0064] 1. Enzyme-catalyzed reaction

[0065] 5-Hydroxyvaleric acid (5-HV), the carboxylic acid reductase mutant enzyme solution prepared in Example 1, YahK enzyme solution, NADPH solution and ATP solution were added to PBS buffer to prepare a reaction system; the reaction was carried out at 35° C. for 8 h to obtain a reaction solution containing 1,5-pentanediol;

[0066] The components of the reaction system are: 5-hydroxyvaleric acid 10 g / L, carboxylic acid reductase mutant 0.3 mg / mL, YahK enzyme 0.3 mg / mL, NADPH 2 mM, ATP 2.5 mM, and the total volume is 10 mL;

[0067] After 8 hours of reaction, the mixture was immediately transferred to boiling water for inactivation for 10 minutes to terminate the reaction. The reaction solution was centrifuged and the reaction supernatant was filtered through a 0.22 μm filter membrane. Three parallel experiments were set up and HPLC was used for detection, and the conversion rate was determined at the same time.

[0068] 2. Detection method

[0069] First, prepare the standard products. Prepare the standards of 5-HV and 1,5-pentanediol. Prepare 20g / L of 5-HV standard solution as the mother solution, dilute it to the concentrations of 15g / L, 10g / L, 5g / L and 2.5g / L in sequence, use a disposable syringe to draw 700μL of the standard solution, filter it with a 0.22μm filter membrane (organic membrane), and inject it into the sample bottle. The other two standards are configured with the same concentration. Use ultraviolet high performance liquid chromatography to detect 5-aminovaleric acid in the sample. Centrifuge the sample at 10000r / min for 10min, and the supernatant after centrifugation is treated with DEEMM derivatization, then filtered through a 0.22μm organic filter membrane and placed in a liquid phase vial for HPLC detection.

[0070] The 5-hydroxyvaleric acid in the sample was detected by ultraviolet high performance liquid chromatography, and the detection conditions were as follows: mobile phase 5mmH2SO4; volume flow rate 0.6mL / min; column temperature 65℃; detection wavelength 210nm. A standard curve was made based on the area and concentration, and then the consumption of the substrate 5-hydroxyvaleric acid in the sample was calculated.

[0071] The 1,5-pentanediol in the sample was detected by differential high performance liquid chromatography. The detection conditions were as follows: mobile phase 5 mmH2SO4; volume flow rate 0.6 mL / min; column temperature 60°C. A standard curve was made based on the area and concentration, and the generation of the product 5-hydroxyvaleraldehyde in the sample was calculated.

[0072] 3. According to the above methods 1 and 2, the 86 carboxylic acid reductase single mutants and 10 carboxylic acid reductase double mutants designed in Example 1 were measured in turn. Among them, the activities of 5 single mutants and 2 double mutants were significantly improved, the activities of 3 single mutants and 1 double mutant were significantly decreased, and the activities of 76 single mutants and 7 double mutants were not significantly changed. The enzyme activities of wild-type carboxylic acid reductase (WT), some carboxylic acid reductase single mutants and some carboxylic acid reductase double mutants are shown in Figure 2. Figure 2 shown.

[0073] Depend on Figure 2 It can be seen that the enzyme activities of the carboxylic acid reductase mutants G394F, G419H and MT1 (G419H-I614R) are 1.94 times, 1.91 times and 1.73 times that of the wild-type carboxylic acid reductase, and the enzyme activities are significantly improved, and can be used for the preparation of 1,5-PDO.

[0074] Example 3: Effects of different temperatures and pH on the activity of carboxylic acid reductase mutants

[0075] 1. Effect of temperature on the activity and stability of carboxylic acid reductase mutants

[0076] Determination of the optimal reaction temperature of carboxylic acid reductase mutants: Determination was performed under 100mmol / L PBS buffer (pH7.0). The reaction temperatures were set at 20℃, 25℃, 30℃, 35℃, 40℃, 45℃, and 50℃, and the samples were placed at the corresponding temperatures for 8h. When the reaction time was up, they were quickly transferred to boiling water to ensure that the high temperature inactivated the enzyme. Generally, the enzymatic reaction can be terminated by boiling for about 10min. The samples to be tested were processed, and the sample with the highest enzyme activity was taken as 100% and the relative enzyme activity was calculated.

[0077] Determination of temperature stability of carboxylic acid reductase mutants: set the reaction temperature to 30°C, 35°C, 40°C and 45°C, respectively put the enzyme in it for insulation, take the enzyme activity before insulation as the original enzyme activity, and take samples after insulation for 30min, 60min, 90min and 120min respectively, and react with substrate 5-HV at 35°C for 60min. After the reaction, immediately boil for 10min or transfer to a 100°C metal bath to completely terminate the enzyme activity, and treat the reaction liquid. Detect the sample by HPLC, the detection method is as shown in Example 2, and the highest enzyme activity is 100% and the relative enzyme activity is calculated.

[0078] The results of the effect of temperature on the activity and stability of carboxylic acid reductase mutants are as follows Figure 3 shown.

[0079] Depend on Figure 3 It can be seen that the optimum temperature of the carboxylic acid reductase mutant G394F is 35°C, the optimum temperature of the carboxylic acid reductase mutant G419H is 40°C, and the optimum temperature of the carboxylic acid reductase mutant MT1 (G419H-I614R) is 40°C. In addition, the carboxylic acid reductase mutants G394F, G419H and MT1 (G419H-I614R) all have good stability at 30-35°C.

[0080] 2. Effect of pH on the activity and stability of carboxylic acid reductase mutants

[0081] Determination of the optimal buffer and optimal reaction pH of carboxylic acid reductase mutants: Select a buffer of pH 4.0-9.0 (all 100 mmol / L). Sodium citrate buffer (pH 4.0-6.0), PBS buffer (pH 5.5-8.0), Tris-HCl buffer (pH 6.5-9.0), react at 35°C for 60 min. After the reaction is completed, boil in boiling water for 10 min to terminate the enzymatic reaction, and treat the sample for HPLC detection. The detection method is as shown in Example 2, and the highest enzyme activity is 100%.

[0082] Determination of pH stability of carboxylic acid reductase mutants: The enzyme solution was placed in a pH 4.0 sodium citrate buffer and a pH 5.0-8.0 PBS buffer at 4°C for 30 min, 60 min, 90 min and 120 min respectively. The reaction was carried out at 35°C for 60 min. After the reaction was completed, the residual enzyme activity of the enzyme was determined in the same manner as the temperature determination. The highest enzyme activity was 100%.

[0083] The results of the effect of pH on the activity and stability of carboxylic acid reductase mutants are as follows Figure 4 shown.

[0084] Depend on Figure 4It can be seen that the optimum pH of the carboxylic acid reductase mutant G394F is 6, the optimum pH of the carboxylic acid reductase mutant G419H is 7, and the optimum pH of the carboxylic acid reductase mutant MT1 (G419H-I614R) is 7. In addition, the carboxylic acid reductase mutants G394F, G419H and MT1 (G419H-I614R) all have good stability at pH = 6 to 7°C.

[0085] Example 4: Application of carboxylic acid reductase mutants in the production of 1,5-PDO

[0086] A method for preparing 1,5-pentanediol comprises the following steps:

[0087] (1) The gene sequence of the carboxylic acid reductase mutant G394F was connected to the plasmid pET-28a to obtain the plasmid vector pET-28a-G394F; then the plasmid vector pET-28a-G394F was transferred into the genetically engineered Escherichia coli LE02G to obtain the mutant recombinant strain LE02G for producing 1,5-pentanediol. G394F ;

[0088] The recombinant strain LE was obtained by the same method. WT , mutant recombinant strain LE G419H and mutant recombinant strain LE MT1 ;

[0089] (2) The recombinant strain LE WT , mutant recombinant strain LE G394F , mutant recombinant strain LE G419H and mutant recombinant strain LE MT1 Activate on LB solid culture medium respectively, pick out the activated single colony and inoculate into LB liquid culture medium, and culture at 37°C and 200r / min for 12h; then inoculate into fermentation medium at a 2% inoculation amount, and add kanamycin with a concentration of 50μg / mL to maintain the plasmid; after growing at 37°C for 4h, add inducer IPTG (final concentration 1mM), and continue fermentation production for 6 days to obtain 1,5-pentanediol;

[0090] Among them, the fermentation medium composition ( / L): glucose 20g / L, peptone 10g / L, yeast extract powder 5g / L, NaCl 10g / L.

[0091] The principle process of preparing 1,5-pentanediol in this embodiment is as follows Figure 5 As shown in a.

[0092] The contents of 5-hydroxyvaleric acid (5-HV), 5-hydroxyvaleraldehyde (5-Hydroxytryptophanal) and 1,5-pentanediol (1,5-PDO) in the fermentation broth were measured on the 1st, 2nd, 3rd, 4th, 5th and 6th days of the fermentation process. The results were as follows: Figure 5 As shown in b, c and d.

[0093] Depend on Figure 5 a It can be seen that glucose first generates 5-HV under the action of Escherichia coli genetically engineered bacteria LE02G, and then 5-HV generates 1,5-pentanediol under the action of carboxylic acid reductase mutant and YahK enzyme in Escherichia coli.

[0094] Depend on Figure 5 b As can be seen from the figure, as the fermentation time increases, the mutant recombinant strain LE G394F , mutant recombinant strain LE G419H and mutant recombinant strain LE MT1 The content of 5-HV in the fermentation broth was higher than that of wild-type recombinant strain LE WT Especially on the 6th day of fermentation, the decreases were 28.57%, 20.57%, and 40.68%, respectively, indicating that the substrate 5-HV was significantly reduced in the mutant recombinant strain LE G394F , mutant recombinant strain LE G419H and mutant recombinant strain LE MT1 Fully consumed.

[0095] Depend on Figure 5 c As the fermentation time increases, the mutant recombinant strain LE G394F , mutant recombinant strain LE G419H and mutant recombinant strain LE MT1 The content of 5-hydroxyvaleraldehyde in the fermentation broth was higher than that of wild-type recombinant strain LE WT All of them increased significantly, indicating that the intermediate product 5-hydroxyvaleraldehyde was in the mutant recombinant strain LE G394F , mutant recombinant strain LE G419H and mutant recombinant strain LE MT1 Generate in large quantities.

[0096] Depend on Figure 5 d It can be seen that as the fermentation time increases, the mutant recombinant strain LE G394F , mutant recombinant strain LE G419H and mutant recombinant strain LE MT1 The content of 1,5-PDO in the fermentation broth was higher than that of the wild-type recombinant strain LE WTEspecially on the 7th day of fermentation, the percentages increased by 8.76%, 12.43% and 32.67%, respectively, indicating that the carboxylic acid reductase mutant G394F, carboxylic acid reductase mutant G419H and carboxylic acid reductase mutant MT1 (G419H-I614R) provided by the present invention can effectively increase the yield of 1,5-PDO, shorten the production cycle of 1,5-PDO and reduce the production cost of 1,5-PDO.

Claims

1. A carboxylic acid reductase mutant, characterized in that: The mutant is a carboxylic acid reductase in which the glycine at position 394 undergoes a site-directed mutation, or a glycine at position 419 undergoes a site-directed mutation, or a glycine at position 394 and an isoleucine at position 614 undergo a site-directed mutation simultaneously; the nucleotide sequence of the carboxylic acid reductase is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.

2.

2. The carboxylic acid reductase mutant according to claim 1, characterized in that The mutant is a carboxylic acid reductase mutant G394F, whose amino acid sequence is shown in SEQ ID NO.4 and whose nucleotide sequence of the encoding gene is shown in SEQ ID NO.3; the glycine at position 394 of the carboxylic acid reductase amino acid sequence is mutated into phenylalanine.

3. The carboxylic acid reductase mutant according to claim 1, characterized in that The mutant is a carboxylic acid reductase mutant G419H, whose amino acid sequence is shown in SEQ ID NO.6 and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.5; the glycine at position 419 of the carboxylic acid reductase amino acid sequence is mutated into histidine.

4. The carboxylic acid reductase mutant according to claim 1, characterized in that The mutant is a carboxylic acid reductase mutant MT1 (G419H-I614R), whose amino acid sequence is shown in SEQ ID NO.8, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.7; the glycine at position 419 of the carboxylic acid reductase amino acid sequence is mutated into histidine, and the isoleucine at position 614 is mutated into arginine.

5. A recombinant vector, characterized in that: The gene encoding the carboxylic acid reductase mutant according to any one of claims 1 to 4 is inserted into a plasmid vector; preferably, the plasmid vector is pET-28a.

6. A recombinant strain, characterized in that: The method is obtained by transforming the recombinant vector according to claim 5 into a host cell.

7. The recombinant strain according to claim 6, characterized in that The construction method is specifically as follows: the coding gene of the carboxylic acid reductase mutant described in any one of claims 1 to 4 or the recombinant vector described in claim 5 is transferred into the genetically engineered Escherichia coli LE02G to obtain a recombinant strain for producing 1,5-pentanediol.

8. Use of the carboxylic acid reductase mutant according to any one of claims 1 to 4, the recombinant vector according to claim 5 or the recombinant strain according to claim 6 in the preparation of 1,5-pentanediol.

9. The use according to claim 8, characterized in that The specific process for preparing 1,5-pentanediol using the recombinant strain according to claim 6 is as follows: Activate the recombinant strain of claim 6 on LB solid culture medium, pick up the activated single colony and inoculate it into LB liquid culture medium, and culture it at 37°C and 200r / min for 12h; then inoculate it into fermentation medium at an inoculum of 1.5-2.5%, and add kanamycin at a concentration of 50μg / mL to maintain the plasmid; after growing at 37°C for 4h, add inducer IPTG (final concentration 1mM), and continue fermentation production for 5-7 days to obtain 1,5-pentanediol; Among them, the fermentation medium composition ( / L): glucose 20g / L, peptone 10g / L, yeast extract powder 5g / L, NaCl 10g / L.

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