Carboxylate reductase mutants and uses thereof

Abstract

The application provides a carboxylic acid reductase mutant and application thereof. The mutant is a carboxylic acid reductase mutant with site-directed mutation at position 394, or site-directed mutation at position 419, or site-directed mutation at position 394 and position 614. The amino acid sequence of the mutant G394F is shown as SEQ ID NO. 4, and the nucleotide sequence is shown as SEQ ID NO. 3; the amino acid sequence of the mutant G419H is shown as SEQ ID NO. 6, and the nucleotide sequence is shown as SEQ ID NO. 5; the amino acid sequence of the mutant MT1 is shown as SEQ ID NO. 8, and the nucleotide sequence is shown as SEQ ID NO. 7. Compared with the wild-type carboxylic acid reductase, the carboxylic acid reductase mutant provided by the application has an enzyme activity increased by 1.73-1.94 times, the 1,5-PDO yield is increased 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 present application relates to a carboxylate reductase mutant and its application, and belongs to the technical field of microbial enzymes. BACKGROUND

[0002] 1,5-pentanediol (1,5-PDO) is a key diol and intermediate in organic synthesis, which has attracted wide 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, etc. It can be used as an environmentally friendly solvent for chemical synthesis of antifreeze, ink, special detergent, etc. In the field of medicine, it is used to produce penicillin intermediates, antitumor drugs and anesthetic products. Compared with traditional plasticizers, 1,5-PDO can improve the poor temperature resistance and has high efficiency. With the expansion of demand for 1,5-PDO in multiple fields, the current production capacity cannot meet market demand. Therefore, the development prospect of high-efficiency synthesis of 1,5-PDO is broad.

[0003] The production of 1,5-PDO in industry mainly relies on petroleum as raw material through oxidation-reduction reaction. However, the process route is complicated, there are many by-products of fusel, a large amount of non-renewable petroleum energy is consumed, the production capacity of 1,5-PDO is limited by limited C5 raw materials, and it is difficult to realize large-scale production. Biomass, a renewable carbon source, not only has a wide source and is easy to obtain, but also has low raw material cost and short renewable cycle. Therefore, in recent years, the chemical method of converting biomass-derived furfural or tetrahydrofuran methanol to 1,5-PDO has attracted wide attention. However, these processes usually include multiple chemical steps, including hydrogenation, dehydration, hydration and hydrogenation. There are disadvantages such as complex conversion process, increase in production cost due to use of noble metal catalysts, harsh reaction conditions for non-noble metal catalysts, unsatisfactory yield and instability of target product, and unclear catalytic mechanism, which seriously limit the industrialization development.

[0004] Therefore, developing a green biosynthetic pathway for 1,5-PDO is an effective way to reduce our dependence on fossil fuels. Carboxylic acid reductase (CAR) plays a crucial role in this process. CAR can reduce the carboxyl group on 5-hydroxyvaleric acid (5-HV) to an aldehyde group, generating 5-hydroxyvaleraldehyde, a key step in the synthesis of 1,5-PDO. Compared with traditional chemical synthesis methods, the CAR-catalyzed biosynthetic pathway has the advantages of mild conditions, high selectivity, and environmental friendliness. Furthermore, using glucose as a feedstock, low-cost and high-yield 1,5-PDO production can be achieved through microbial fermentation and metabolic engineering. Glucose, as a renewable and readily available carbon source, can be converted into precursors such as 5-HV through metabolic pathways, providing sufficient substrate for the CAR-catalyzed reaction. However, to achieve the efficient application of CAR in industrial production, further optimization of its catalytic performance and stability, as well as the development of compatible fermentation processes and reaction systems, are required. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention provides a carboxylic acid reductase mutant and its application. Using site-directed mutagenesis, the present invention conducts directed evolution of wild-type carboxylic acid reductase, resulting in a mutant enzyme that catalyzes the reduction of 5-HV (5-hydroxyvaleric acid) with increased efficiency. This mutant enzyme can be used in the fermentation production of 1,5-PDO, thereby replacing chemical methods that produce numerous byproducts and are difficult to separate and purify, to produce 1,5-pentanediol, a promising method for broad application.

[0006] The technical solutions of the present invention are as follows:

[0007] A carboxylic acid reductase mutant, wherein the mutant is a carboxylic acid reductase (CAR) in which glycine at position 394 undergoes a site-directed mutation, or glycine at position 419 undergoes a site-directed mutation, or glycine at position 394 and 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] According to the present invention, the mutant is preferably 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] According to the present invention, the mutant is preferably 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 to histidine.

[0010] According to the application, preferably, the mutant is carboxylate reductase mutant MT1 (G419H-I614R), the amino acid sequence of which is shown as SEQ ID NO. 8, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 7; and the mutant is obtained by simultaneously mutating the 419th glycine in the carboxylate reductase amino acid sequence into histidine and mutating the 614th isoleucine into arginine.

[0011] A recombinant vector is obtained by inserting the encoding gene of the carboxylate reductase mutant into a plasmid vector.

[0012] According to the application, preferably, the plasmid vector is pET-28a.

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

[0014] According to the application, preferably, the construction method of the recombinant strain is as follows: the carboxylate reductase mutant encoding gene or the recombinant vector containing the carboxylate reductase mutant encoding gene is transformed into Escherichia coli genetically engineered bacteria LE02G to obtain a recombinant strain for producing 1,5-pentanediol.

[0015] The Escherichia coli genetically engineered bacteria LE02G is an existing strain, and the construction method thereof is disclosed in the Chinese patent document CN118726222A, and the specific construction method is described in the specification

[0060] to

[0168] .

[0016] The carboxylate reductase mutant, the recombinant vector or the recombinant strain is used in the preparation of 1,5-pentanediol.

[0017] According to the application, preferably, the specific process for preparing 1,5-pentanediol by using the recombinant strain is as follows:

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

[0019] The fermentation medium is composed of (per liter) 20g / L of glucose, 10g / L of proteose peptone, 5g / L of yeast extract powder and 10g / L of NaCl.

[0020] The application has the following beneficial effects:

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

[0022] Figure 1 The electrophoresis results of the carboxylate reductase mutant are shown in the following table:

[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 enzyme activity comparison results of the carboxylate reductase mutant and the wild-type carboxylate reductase are shown in the following table:

[0025] In the figure, (a) is the relative enzyme activity comparison results of the partial carboxylate reductase single mutant and the wild-type carboxylate reductase; (b) is the relative enzyme activity comparison results of the partial carboxylate reductase double mutant and the wild-type carboxylate reductase.

[0026] Figure 3 The effects of temperature on the enzyme activity and stability of the carboxylate reductase mutant are shown in the following table:

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

[0028] Figure 4 The effects of pH on the enzyme activity and stability of the carboxylate reductase mutant are shown in the following table:

[0029] In the figure, (a) is the optimum 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 use of 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) is LE G394F LE G419H and LE MT1 (d) is the yield of 5-hydroxyvaleraldehyde; G394F LE G419H and LE MT1 of 1,5-PDO production. DETAILED DESCRIPTION

[0032] The technical solutions of the present invention are further described below in conjunction with the embodiments and the accompanying drawings, but the scope of protection 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, 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: Detection was performed by ultraviolet high performance liquid chromatography under the following conditions: mobile phase: 5 mM H2SO4; volume flow rate: 0.6 mL / min; column temperature: 65°C; detection wavelength: 210 nm.

[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 identify 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: using public databases such as RCSB PDB, UniProt, etc., to retrieve the protein model of wild-type carboxylate reductase (CAR) from Nocardia iowensis. Autodock Vina software is used for virtual screening to predict the binding mode of substrate and cofactor with CAR. The three-dimensional structure model of CAR is analyzed using Pymol software to identify the amino acid residues in the active center. The CAR is subjected to alanine scanning using Discover studio software to evaluate the effect of each amino acid residue on enzyme activity. The CAVER software is used to predict the channel leading to the active site of CAR and analyze the amino acid composition of the channel. The predicted key amino acid residues are subjected to virtual mutation to simulate the effect of mutation on enzyme activity and substrate channel.

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

[0041] Among them, the amino acid sequence of carboxylate 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 carboxylate 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 carboxylate 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 carboxylate reductase single mutants (such as Y418P, Y418A, Y418F, Y418H, I614A, K616A, S395H, S395F, G613T, etc.) and other carboxylate reductase double mutants (such as MT2 and MT3, etc.) is not described in detail here.

[0043] 2. Heterologous expression of carboxylate reductase

[0044] (1) Taking the carboxylic acid reductase mutant G394F as an example, single-point mutation primers G394-F / R were designed; then, using the nucleotide sequence of wild-type carboxylic acid reductase (CAR) as a template, primers G394F-F / R were used to perform site-directed mutagenesis by inverse PCR amplification. After amplification, the unmutated original template was removed using Dpn I 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 GeneWeiZ Company according to the sequence information;

[0049] (2) The mutant G394F gene was ligated to pET28a to obtain the recombinant plasmid pET28a-CAR-G394F; the recombinant plasmid pET28a-CAR-G394F was then 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 cells were collected by centrifugation at 4000g and 4°C for 20 min using a high-speed refrigerated centrifuge. The cells obtained by centrifugation were resuspended in 50 mL of 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 incubate on ice for 30 minutes, stirring every 5 to 6 minutes to allow it to fully function; then use an ultrasonic cell disruptor for disruption, which needs to be run under low temperature conditions and takes 25 minutes; after disruption, set a high-speed refrigerated centrifuge: 4°C, 13,000g centrifugation conditions 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 pre-packed Ni-NTA column, the carboxylate reductase mutant G394F crude enzyme solution was combined with the Ni-NTA column for 1 hour (4°C) after the resin was equilibrated with ddH2O and Lysis Buffer, the impurities were washed away with Lysis Buffer and Wash Buffer, and finally the target protein was eluted with Elution buffer, collected in centrifuge tubes, and dialyzed to remove imidazole, to obtain the heterologously expressed carboxylate reductase mutant G394F.

[0052] Other carboxylate reductase mutants were heterologously expressed and purified according to the same method as above, wherein the single-point mutation primers used for carboxylate reductase mutants G419H and MT1 (G419H-I614R) were G419H-F / R, MT1-F1 / R1 and MT1-F2 / R2, and the specific sequences were as follows:

[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'-TTTCGCgcgACCAGACAGCAGGCCATTCGCAA-3'.

[0059] The PCR amplification system was prepared according to the kit instructions; 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); final extension, 72°C for 10 min; and finally 4°C for incubation.

[0060] The expression of the purified recombinant proteins G394F, G419H and MT1 (G419H-I614R) was identified by polyacrylamide gel electrophoresis (SDS-PAGE), and the results are shown in Figure 1 .

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

[0062] Example 2, enzyme activity determination of carboxylate reductase mutants

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

[0064] 1. Enzymatic reaction

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

[0066] Among them, each component of the reaction system is: 5-hydroxyvaleric acid 10g / L, carboxylate reductase mutant 0.3mg / mL, YahK enzyme 0.3mg / mL, NADPH 2mM, ATP 2.5mM, and the total volume is 10mL;

[0067] The reaction was immediately transferred to boiling water for 10min to terminate the reaction. The reaction solution was centrifuged, and the reaction supernatant obtained was filtered with a 0.22μm filter membrane. Three sets of parallel experiments were set up, and HPLC was used for detection, and the conversion rate was also determined.

[0068] 2. Detection method

[0069] First, the standard sample was prepared. The standard samples of 5-HV and 1,5-pentanediol were prepared. A 20g / L 5-HV standard solution was prepared as a mother liquor, which was diluted to 15g / L, 10g / L, 5g / L and 2.5g / L in turn, and 700μL of the standard sample solution was filtered with a 0.22μm filter membrane (organic membrane) and injected into a sample bottle. Two other standard samples were prepared with the same concentration. The 5-aminovaleric acid in the sample was detected by ultraviolet high performance liquid chromatograph. The sample was centrifuged at 10000r / min for 10min, and the supernatant was subjected to DEEMM derivatization treatment, and then filtered with a 0.22μm organic filter membrane and placed in a liquid phase vial for HPLC detection.

[0070] 5-Hydroxyvaleric acid (5-HVA) was detected in the samples using ultraviolet high-performance liquid chromatography (HPLC) under the following conditions: mobile phase: 5 mm H₂SO₄; flow rate: 0.6 mL / min; column temperature: 65°C; detection wavelength: 210 nm. A standard curve was generated based on area and concentration, and the consumption of the substrate 5-HVA in the samples was calculated.

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

[0072] 3. The 86 carboxylic acid reductase single mutants and 10 carboxylic acid reductase double mutants designed in Example 1 were tested in sequence according to the above methods 1 and 2. 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 were as follows: 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, respectively. 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] Determine the optimal reaction temperature for carboxylic acid reductase mutants: Perform the assay in 100 mmol / L PBS buffer (pH 7.0). Set the reaction temperature at 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, and 50°C. Incubate the sample at each temperature for 8 hours. At the end of the reaction time, immediately transfer the sample to boiling water to ensure that the high temperature inactivates the enzyme. Boiling for approximately 10 minutes is generally sufficient to terminate the enzymatic reaction. After processing the sample, the relative activity is calculated, with the highest enzyme activity defined as 100%.

[0077] Determining the temperature stability of carboxylic acid reductase mutants: The enzyme was incubated at reaction temperatures of 30°C, 35°C, 40°C, and 45°C, with the initial enzyme activity determined. Samples were taken after incubation for 30, 60, 90, and 120 minutes, respectively, and reacted with the substrate 5-HV at 35°C for 60 minutes. After the reaction, the reaction mixture was immediately boiled for 10 minutes or transferred to a 100°C metal bath to completely terminate the reaction and deactivate the enzyme. The reaction mixture was then processed and analyzed by HPLC using the same method as in Example 2. The highest enzyme activity was defined as 100%, and the relative activity was 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 optimal temperature of the carboxylic acid reductase mutant G394F is 35°C, the optimal temperature of the carboxylic acid reductase mutant G419H is 40°C, and the optimal temperature of the carboxylic acid reductase mutant MT1 (G419H-I614R) is also 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 reaction pH for the carboxylic acid reductase mutants: Buffers with a pH of 4.0 to 9.0 (all at 100 mmol / L) were selected. Sodium citrate buffer (pH 4.0 to 6.0), PBS buffer (pH 5.5 to 8.0), and Tris-HCl buffer (pH 6.5 to 9.0) were reacted at 35°C for 60 min. After completion, the enzymatic reaction was terminated by boiling in boiling water for 10 min. Samples were then analyzed by HPLC using the same assay as in Example 2, with the highest enzyme activity considered 100%.

[0082] To determine the pH stability of carboxylic acid reductase mutants, the enzyme solution was placed in sodium citrate buffer (pH 4.0) or PBS buffer (pH 5.0-8.0) at 4°C for 30, 60, 90, and 120 minutes, respectively. The reaction was continued at 35°C for 60 minutes. After completion of the reaction, the enzyme was processed as for the temperature determination and the residual enzyme activity was determined. The highest enzyme activity was considered 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 optimal pH of the carboxylic acid reductase mutant G394F is 6, the optimal pH of the carboxylic acid reductase mutant G419H is 7, and the optimal 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 = 6-7°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 carboxylic acid reductase mutant G394F gene sequence 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 LE 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 The cells were activated on LB solid medium, and single colonies were picked and inoculated into LB liquid medium and cultured at 37°C and 200 rpm for 12 hours. A 2% inoculum was then inoculated into a fermentation medium, and 50 μg / mL of kanamycin was added to maintain the plasmid. After growing at 37°C for 4 hours, the inducer IPTG (final concentration of 1 mM) was added, and fermentation production was continued for 6 days to produce 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 determined on days 1, 2, 3, 4, 5 and 6 of the fermentation process. The results were as follows: Figure 5 As shown in b, c and d.

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

[0094] Depend on Figure 5 b As can be seen from the figure, as the fermentation time gets longer, 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 the 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 can be seen from the figure, as the fermentation time gets longer, 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 the 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 becomes longer, 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 WTAll are obviously increased. In particular, at the 7th day of fermentation, it is increased by 8.76%, 12.43% and 32.67% respectively, which shows that the carboxylic acid reductase mutant G394F, the carboxylic acid reductase mutant G419H and the carboxylic acid reductase mutant MT1 (G419H-I614R) provided in the application 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 glycine at position 394 of the carboxylic acid reductase is mutated to phenylalanine, resulting in the carboxylic acid reductase mutant G394F; or the glycine at position 419 of the carboxylic acid reductase is mutated to histidine, resulting in the carboxylic acid reductase mutant G419H; or the glycine at position 419 of the carboxylic acid reductase is mutated to histidine and the isoleucine at position 614 of the carboxylic acid reductase is mutated to arginine, resulting in the carboxylic acid reductase mutant MT1 (G419H-I614R). 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.

2. A recombinant vector, characterized in that The gene encoding the carboxylic acid reductase mutant according to claim 1 is inserted into a plasmid vector.

3. The recombinant vector according to claim 2, wherein The plasmid vector is pET-28a.

4. A recombinant strain, characterized in that The method is obtained by transforming the encoding gene of the carboxylic acid reductase mutant according to claim 1 or the recombinant vector according to claim 2 into a host cell.

5. The recombinant strain according to claim 4, characterized in that The specific construction method is: the coding gene of the carboxylic acid reductase mutant according to claim 1 or the recombinant vector according to claim 2 is transferred into a genetically engineered Escherichia coli to obtain a recombinant strain for producing 1,5-pentanediol.

6. Use of the carboxylic acid reductase mutant according to claim 1, the recombinant vector according to claim 2, or the recombinant strain according to claim 4 in the preparation of 1,5-pentanediol.

7. The use according to claim 6, characterized in that The specific process for preparing 1,5-pentanediol using the recombinant strain according to claim 4 is as follows: The recombinant strain according to claim 4 is activated on LB solid medium, and a single colony after activation is picked and inoculated into LB liquid medium, and cultured at 37°C and 200 rpm for 12 hours; then, an inoculum size of 1.5-2.5% is inoculated into a fermentation medium, and kanamycin at a concentration of 50 μg / mL is added to maintain the plasmid; after growing at 37°C for 4 hours, an inducer IPTG is added, and fermentation production is continued for 5-7 days to obtain 1,5-pentanediol; The fermentation medium consists of 20 g / L glucose, 10 g / L peptone, 5 g / L yeast extract powder, and 10 g / L NaCl.

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