Carboxylate reductase mutants and uses thereof

Abstract

This invention discloses a carboxylic acid reductase mutant and its applications, belonging to the field of bioengineering technology. This invention relates to marine mycobacteria (…). Mycobacterium marinum Carboxylic acid reductase Mm The A domain of CAR and Mycobacterium avium ( Mycobacterium avium Carboxylic acid reductase Mav Directed evolution of the PR domain of CAR yielded single-site and multi-site mutants with significantly enhanced catalytic activity. Mm CAR's advantageous mutation A domain and Mav By combining the PR domain with the CAR mutant, a chimeric carboxylic acid reductase with further enhanced catalytic activity was obtained. This invention also utilizes recombinant engineered bacteria containing this mutant to catalyze the reduction of 3-methoxy-4-hydroxybenzoic acid to aldehydes. Results show that the recombinant engineered bacteria exhibit significantly superior catalytic activity to the wild-type in the 3-methoxy-4-hydroxybenzoic acid reduction reaction, demonstrating promising industrial application prospects.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to a carboxylic acid reductase mutant and its applications. Background Technology

[0002] Carboxylic acid reductase (CAR) is a class of multifunctional enzymes that depend on ATP and NAD(P)H to catalyze the reduction of carboxylic acids to the corresponding aldehydes. Structurally, CAR typically comprises three functional domains: an adenylation domain (A domain, responsible for substrate activation and the formation of adenosine intermediates), a phosphoproteopancreatoyl thioethylamine transferase recognition domain (P domain), and a reductase domain (R domain, responsible for the reduction of acyl-AMP to aldehydes). Due to their broad substrate spectrum and excellent chemoselectivity, CARs have shown great potential for application in the synthesis of fragrances, pharmaceutical intermediates, and biofuels.

[0003] Mycobacterium marinum Carboxylic acid reductase from Mm CAR (UniProtKB / Swiss-Prot: B2HN69.1) and Mycobacterium avium Carboxylic acid reductase from Mav CAR (NCBI Reference Sequence: WP_011725490.1) are two types of carboxylic acid reductases with different catalytic properties. However, the catalytic activity, thermal stability, and substrate specificity of wild-type CARs often fail to meet the requirements of industrial applications. Therefore, modifying CAR enzymes through protein engineering to improve their catalytic activity has significant research and application value.

[0004] Currently, the modification of CAR enzymes mainly focuses on two aspects: domain-level recombination and amino acid-level site-directed mutagenesis. Domain recombination, by combining the advantageous domains of CAR enzymes from different sources, holds promise for obtaining hybrid enzymes with excellent catalytic performance. Site-directed mutagenesis, on the other hand, involves screening for saturated mutations at key amino acid sites to obtain mutants with enhanced activity. However, research on the organic combination of these two strategies to systematically modify different domains of CAR enzymes and ultimately obtain high-performance hybrid enzymes through domain recombination has not yet been reported. Mycobacterium marinum CAR source ( Mm CAR) and Mycobacterium avium CAR source ( Mav CARs are two types of carboxylic acid reductases with different catalytic properties. The question is how to... Mm Favorable mutations in the CAR A domain and MavEffective structural recombination through favorable mutations in the CAR PR domain to construct chimeric CARs with both high activity and high stability is of great significance for improving the defects of wild-type CARs. Summary of the Invention

[0005] The purpose of this invention is to provide a carboxylic acid reductase mutant and its applications, thereby addressing the problems existing in the prior art. This invention, through... Mm CAR's A domain and Mav By rationally designing and site-directed mutagenesis of the PR domain of CAR, mutants with significantly enhanced activity were obtained. Then, through domain recombination, a chimeric carboxylate reductase with both high activity and high stability was constructed and applied to the biocatalytic preparation of 3-methoxy-4-hydroxybenzaldehyde compounds, overcoming the limitations of the wild-type enzyme. Mm CAR and Mav CAR catalysts suffer from low catalytic activity, poor thermal stability, and unsatisfactory substrate specificity, making it difficult to meet the needs of industrial production.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides a carboxylic acid reductase mutant comprising any one of the following mutants (1)-(3): (1) For marine mycobacteria ( Mycobacterium marinum Carboxylic acid reductase from ) Mm The CAR is obtained by substituting one or more of the following substitution mutations into the amino acid sites I303, T390, Y415, I425, R277, E281, M299, Q302, E336, and T338 in the A domain: R277A, R277C, R277S, R277L, R277G, R277V, E281F, E281T, E281L, E281D, E281G, E281V, E2 81S, M299A, M299G, M299C, M299L, M299D, M299I, M299V, M299N, M299T, Q302A, Q302G, Q302S , Q302C, Q302L, Q302T, Q302P, I303A, I303D, I303E, I303I, I303K, I303L, I303M, I303N, I3 03P, I303R, I303Y, I303G, I303H, E336A, E336P, E336G, E336I, E336V, E336C, T338S, T338N , T338L, T338V, T390A, T390D, T390H, T390C, T390I, T390H, T390L, T390M, T390N, T390P, T 390E, T390Q, T390R, T390W, Y415C, Y415H, Y415P, Y415W, Y415E, Y415F, Y415G, Y415I, Y415 M, Y415N, Y415H, Y415P, Y415Q, Y415S, Y415T, Y415V, I425A, I425C, I425F, I425N, I425Q, I425E, I425F, I425G, I425K, I425N, I425K, I425N, I425R, I425S, I425W, and I425Y; wherein, the carboxyl reductase Mm The amino acid sequence of CAR is shown in SEQ ID NO:1; (2) For Mycobacterium avium ( Mycobacterium avium Carboxylic acid reductase from ) Mav The CAR is obtained by substituting the amino acid sites P995 and / or D996 in the PR domain with the following substitution mutations: P995L, P995E, P995G, P995H, P995M, P995Q, P995R, D996E, D996G, D996L, D996M, D996P, D996Q, D996S, and D996T; wherein, the carboxylic acid reductase... Mav The amino acid sequence of CAR is shown in SEQ ID NO:15; (3) Carboxyl reductase derived from marine mycobacteria Mm CAR A domain mutants and Mycobacterium avium carboxylate reductase Mav The CAR PR domain mutant was fused to obtain the carboxylic acid reductase. MmThe A domain mutant of CAR is at least one of the mutants shown in item (1), the carboxylic acid reductase Mav The PR domain mutant of CAR is at least one of the mutants shown in item (2).

[0007] Preferably, in the mutant of item (1), the carboxylic acid reductase Mm The A domain of CAR contains the following combinations of mutations: T390A / Q302C, T390A / Q302C / R277L, T390A / T338S / R277G, T390A / T338I / R277A, T390A / T338N / R277L, T390A / T338S / R277C, T390A / T338V / R277C, T390A / T338G / R277V, and T390A / T338N / R277L. / Q302C, T390A / T338S / R277G / E336P, T390A / T338N / R277L / E336P, T390A / T338N / R277 A / E336G / M299C, T390A / T338S / R277G / Q302A / E336P, T390A / T338N / R277L / M299T, T39 0A / T338N / R277L / M299A, T390A / T338N / R277L / M299G, T390A / T338N / R277L / M299C, T3 90A / T338N / R277L / M299L, T390A / T338N / R277L / M299D, T390A / T338N / R277L / M299N, T3 90A / T338N / R277L / M299I, T390A / T338N / R277L / M299V, T390A / T338N / R277L / Q302C / R 301L, T390A / T338N / R277L / Q302C / E281G, T390A / T338N / R277L / Q302C / E281L, T390A / T 338N / R277L / Q302C / E281D, T390A / T338N / R277L / Q302C / E281C, T390A / T338N / R277L / Q302C / E281V, T390A / T338N / R277L / Q302C / E281T, T390A / T338N / R277L / Q302C / E281S.

[0008] Preferably, in the mutant of item (2), the carboxylic acid reductase Mav The PR domain of CAR includes the following combination of mutations: P995E / D996Q, P995H / D996E, P995G / D996Q, P995R / D996Q, P995H / D996Q, P995Q / D996E, P995G / D996E, P995M / D996E, P995G / D996L, P995H / D996L.

[0009] Preferably, in the mutant of item (3), the fusion mutant includes any combination of the following: Mm (A)_WT- Mav (PR)_WT、 Mm (A)_M1- Mav (PR)_WT、 Mm (A)_M2- Mav (PR)_WT、 Mm (A)_M3- Mav (PR)_WT、 Mm (A)_M4- Mav (PR)_WT、 Mm (A)_M5- Mav (PR)_WT、 Mm (A)_M1- Mav (PR)_M2、 Mm (A)_M2- Mav (PR)_M2、 Mm (A)_M3- Mav (PR)_M2、 Mm (A)_M4- Mav (PR)_M2、 Mm (A)_M5- Mav (PR)_M2; where the Mm (A)_WT- Mav (PR)_WT is a carboxylic acid reductase. Mm The A domain of CAR and carboxylic acid reductase Mav Chimeric carboxylate reductase obtained by fusing the PR domain of CAR; Mm (A)_M1- Mav (PR)_WT is a carboxylic acid reductase. Mm T390A mutant of CAR A domain and carboxyl reductase Mav Chimeric carboxylate reductase obtained by fusing the PR domain of CAR; Mm (A)_M2- Mav (PR)_WT is a carboxylic acid reductase. Mm The T390A / Q302C mutant of the A domain of CAR and carboxyl reductase MavChimeric carboxylate reductase obtained by fusing the PR domain of CAR; Mm (A)_M3- Mav (PR)_WT is a carboxylic acid reductase. Mm The T390A / Q302C / R277L mutant of the A domain of CAR and carboxyl reductase Mav Chimeric carboxylate reductase obtained by fusing the PR domain of CAR; Mm (A)_M4- Mav (PR)_WT is a carboxylic acid reductase. Mm The T390A / T338N / R277L / Q302C mutant of the A domain of CAR and carboxyl reductase Mav Chimeric carboxylate reductase obtained by fusing the PR domain of CAR; Mm (A)_M5- Mav (PR)_WT is a carboxylic acid reductase. Mm The T390A / T338N / R277L / Q302C / E281S mutant of the A domain of CAR and carboxyl reductase Mav Chimeric carboxylate reductase obtained by fusing the PR domain of CAR; Mm (A)_M1- Mav (PR)_M2、 Mm (A)_M2- Mav (PR)_M2、 Mm (A)_M3- Mav (PR)_M2、 Mm (A)_M4- Mav (PR)_M2、 Mm (A)_M5- Mav (PR)_M2 is a carboxylic acid reductase. Mm T390A mutant of CAR A domain, carboxyl reductase Mm T390A / Q302C mutant of CAR A domain, carboxyl reductase Mm T390A / Q302C / R277L mutant of CAR A domain, carboxyl reductase Mm The T390A / T338N / R277L / Q302C mutant of the A domain of CAR and carboxyl reductase Mm The T390A / T338N / R277L / Q302C / E281S mutants of the A domain of CAR, respectively, are associated with carboxyl reductase. Mav Chimeric carboxylic acid reductase obtained by fusing the P995E / D996Q mutant of the PR domain of CAR.

[0010] The present invention also provides a nucleic acid molecule that encodes the carboxylic acid reductase mutant.

[0011] The present invention also provides a biomaterial comprising the aforementioned nucleic acid molecules, wherein the biomaterial includes a recombinant vector or recombinant engineered bacteria.

[0012] The present invention also provides a whole-cell catalyst, wherein the whole-cell catalyst comprises the recombinant engineered bacteria described above.

[0013] The present invention also provides a method for preparing 3-methoxy-4-hydroxybenzaldehyde, using 3-methoxy-4-hydroxybenzoic acid as a substrate and the recombinant engineered bacteria or the whole-cell catalyst described herein to carry out a catalytic reaction to obtain 3-methoxy-4-hydroxybenzaldehyde.

[0014] The present invention also provides the application of the carboxylic acid reductase mutant, the nucleic acid molecule, the biomaterial, or the whole-cell catalyst in catalyzing the reduction of carboxylic acid substrates to corresponding aldehydes or alcohols.

[0015] Preferably, the application is in the preparation of 3-methoxy-4-hydroxybenzaldehyde.

[0016] The present invention discloses the following technical effects: (1) This invention has obtained multiple enzymes through enzyme engineering modification. Mm CAR enzyme A domain single mutants and combined mutants exhibited significantly higher carboxyl reductase activity than the wild type. Mm CAR. The relative activity of the combined mutant T390A / T338N / R277L / Q302C / E281S was 6.22 times that of the wild type, indicating that synergistic modification of the substrate binding pocket and key sites of the A domain can effectively improve catalytic efficiency.

[0017] (2) This invention has obtained multiple enzymes through enzyme engineering modification. Mav CAR enzyme PR domain single mutants and combined mutants showed significantly higher carboxyl reductase activity than wild-type. Mav CAR, among which the double mutant P995E / D996Q has a relative activity 1.90 times that of the wild type.

[0018] (3) This invention obtained through enzyme engineering modification. Mm CAR A domain mutants and those derived from Mav The chimeric enzyme of the PR domain mutant of CAR has higher activity than the parental enzyme. Mm (A)- Mav (PR) wild-type) further improved, optimal chimeric enzyme Mm (A)_M5- Mav (PR)_M2 has the relative activity of wild typeMm 4.94 times that of CAR (4) The mutants and chimeric enzymes provided by this invention have broad application prospects in the biocatalytic preparation of 3-methoxy-4-hydroxybenzaldehyde compounds. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 for Mm CAR and Mav Schematic diagram of CAR domains, domain boundary delineation, and annotation of key mutation sites; Figure 2 for Mm Comparison of relative activities of CAR A domain single mutants; Figure 3 for Mm Comparison of relative activities of CAR A-domain iterative combinatorial mutants; Figure 4 for Mav Comparison of relative activities of CAR PR domain single mutants; Figure 5 for Mav Comparison of relative activities of CAR PR domain iterative combinatorial mutants; Figure 6 This is a schematic diagram illustrating the construction of a chimeric enzyme mutant; Figure 7 Comparison of catalytic activities of different chimeric enzyme mutants; Figure 8 SDS-PAGE electrophoresis image of the recombinant strain expressing the optimal mutant; S represents the supernatant, P represents the precipitate; Figure 9 The time-product yield curves for 3-methoxy-4-hydroxybenzoic acid catalyzed by the optimal chimeric mutant, wild-type enzyme, and wild-type enzyme chimera are shown. Detailed Implementation

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0026] Experimental methods not specified in the following examples are generally performed under standard conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual, or as recommended by the manufacturer.

[0027] The strains, plasmids, and reagents used in the following examples are all commercially available. E. coli The K-12MG1655 RARE strain was purchased from Addgene. Primer synthesis and sequencing were performed by Beijing Qingke Biotechnology Co., Ltd. and Shanghai Sangon Biotech Co., Ltd.

[0028] This invention utilizes techniques such as protein molecule docking, alanine scanning, and Caver web channel prediction to... Mycobacterium marinum The carboxylic acid reductase was modified to obtain a mutant with a higher yield of 3-methoxy-4-hydroxybenzaldehyde.

[0029] Example 1: Targeting carboxyl reductase Mm Construction and screening of site-directed mutants of CAR domain A 1. Carboxylic acid reductaseMm CAR plasmid construction For origin Mycobacterium marinum CAR enzyme ( Mm CAR) and derived from Bacillus subtilis Phosphopantyl thioethylamine transferase (PPT) Bs The SFP gene was codon-optimized based on E. coli host preference to obtain the optimized target gene. Both were then co-cloned into the multiple cloning site of the pETDuet-1 vector using homologous recombination. After sequencing verification, the recombinant plasmid was transformed into the E. coli host strain. E. coli Recombinant engineered bacteria were obtained from K-12 MG1655 RARE.

[0030] PCR amplification reaction system (50 μL system): 2×ToroBlue Flash KOD Dye Mix 25 μL; template (pET30b synthesis gene or pETDuet-1 plasmid vector) 2 μL; forward primer (10 μM) 2 μL; reverse primer (10 μM) 2 μL; ddH2O to 50 μL. Primers are shown in Table 1.

[0031] Table 1 Primers for constructing the carboxylic acid reductase MmCAR plasmid PCR amplification was performed using a falling PCR program. The reaction conditions were: pre-denaturation at 98℃ for 2 min; followed by decreasing annealing temperature cycles, each cycle consisting of denaturation at 98℃ for 10 s, decreasing annealing at 63℃ to 58℃ for 55 s, and extension at 68℃ (the extension time was determined according to the principle of 5 s / kb, and the specific duration was based on the length of the amplified fragment). The annealing temperature for the first cycle was 63℃, and the annealing temperature was decreased by 0.5℃ for each subsequent cycle until the annealing temperature reached 58℃. Then, 30 cycles of amplification reaction were performed at 58℃. After the cycle, a final extension was performed at 72℃ for 4 min.

[0032] PCR products were detected by 1% agarose gel electrophoresis, using a DNA marker as a molecular weight control to verify the correctness of the target band size. After confirmation, 2 µL of DpnI restriction endonuclease was added to the PCR reaction solution, and the mixture was incubated at 37°C for 30 min to eliminate methylated template plasmids. After digestion, the mixture was heated in an 80°C metal bath for 15 min. Dpn I was inactivated, and the PCR products were then recovered by agarose gel electrophoresis to obtain... Mm CAR gene fragments, Bs SFP gene fragment and linearized pETDuet-1 vector fragment.

[0033] Recombination ligation and transformation: The recovered target gene fragment and the linearized vector were mixed at a volume ratio of 1:1, and 2×SoSoo recombinase was added. Homologous recombination was performed by incubating at 50℃ for 30 min. The ligation product was transformed into DH5α chemically competent cells and plated on LB agar plates containing ampicillin (Amp). The cells were incubated at 37℃ for 12 h until single colonies appeared. Single colonies were picked for DNA sequencing verification. The recombinant strain with the correct sequence was the successfully constructed carboxylic acid reductase. Mm CAR engineered strains (containing pETDuet- Mm CAR-BsSFP recombinant plasmid.

[0034] 2. Mm Construction of CAR single-point mutants Using alphafold3 pairs Mm The A domain of CAR (see) Figure 1 A separate model was constructed to create its three-dimensional structure. The substrate 3-methoxy-4-hydroxybenzoic acid was docked to the crystal structure, and rational analysis was performed. Based on the interaction between the substrate binding pocket and the substrate molecule, [the appropriate model was selected]. Mm Site-directed mutations were performed at four sites (I303, T390, Y415, I425) of the amino acid sequence of the A domain of the CAR enzyme (SEQ ID NO: 1).

[0035] Furthermore, protein channels can affect enzyme catalytic activity by influencing the efficiency of substrate transport to the catalytic center. Based on this, analysis of potential substrate transport channels using CAVER Web software revealed that the bottleneck of the substrate channel consists of E281, R277, M299, Q302, E336, and T338. The large side chains of these amino acid residues create steric hindrance, hindering substrate diffusion and exacerbating inhibition at high substrate concentrations. Therefore, it was decided to mutate six amino acid residues—R277, E281, M299, Q302, E336, and T338—with smaller and different amino acids (such as glycine, alanine, serine, valine, threonine, cysteine, leucine, isoleucine, and aspartic acid). The PCR amplification, DpnI digestion, and transformation involved in this scheme are as described in Part 1 of this embodiment. The primers used are shown in Table 2.

[0036] Table 2 Mm CAR single-point mutant construction primers Note: In the nucleotide sequence, M represents A or C, K represents G or T, and N represents A, G, C, or T.

[0037] 3. Mm CAR site-directed mutation screening Transform the mutant plasmid that was verified to be correct by sequencing into E. coli K-12 MG1655 RARE competent cells were selected, and single colonies were inoculated into 5 mL of LB liquid medium (containing 50 mg / L ampicillin) and cultured at 37°C and 220 rpm for 12 h to obtain seed culture. This seed culture was then transferred at a 1% inoculation rate to 50 mL of LB liquid medium (containing 50 mg / L ampicillin) and cultured at 37°C and 220 rpm until the bacterial OD reached the target value. 600 Approximately 0.6–0.8, with a final concentration of 0.5. Mm Isopropyl-β-D-thiogalactoside (IPTG) was used to induce the expression of the target protein by inducing culture at 18°C ​​for 20 h. After induction, the culture medium was centrifuged and the bacterial cells were collected.

[0038] The collected bacterial cells were washed twice with Tris-HCl buffer (50 mM, pH 7.5). Whole-cell biocatalysis was performed in 1 mL Tris-HCl buffer (50 mM, pH 7.5) with the following composition (all reagent concentrations are final): glucose 2% (w / v), MgCl2 6 mM, ATP 3 mM, NADPH 3 mM, and bacterial cell concentration 0.5 g·CDW / L. The substrate 3-methoxy-4-hydroxybenzoic acid (VA) was added to the reaction system at a concentration of 1 g / L, and the reaction was carried out at 30 °C and 800 rpm for 6 h with shaking. After the reaction, the product 3-methoxy-4-hydroxybenzaldehyde (VAN) was diluted using the same pretreatment method and the peak area was detected by high-performance liquid chromatography (HPLC).

[0039] Liquid chromatography (HPLC) detection: Take 100 μL of reaction solution, add 900 μL of ethanol, sonicate for 2 min, centrifuge at 12000 rpm for 3 min, take the supernatant and filter it through a 0.22 μm microporous membrane for HPLC analysis.

[0040] HPLC detection conditions: AQ-C18 column (4.6 mm × 250 mm, 5 μm); detection wavelength 270 nm; column temperature 40℃; mobile phase flow rate 1.0 mL / min; injection volume 10 μL; single detection time 20 min. Mobile phase A was ammonium formate solution (0.1 M, pH 3.0), and mobile phase B was acetonitrile. Gradient elution was used, and the elution program is shown in Table 3.

[0041] Table 3 HPLC gradient elution program Data processing was performed on the HPLC detection results to... Mm Wild-type CAR enzyme (Mm The liquid phase peak area corresponding to CAR_WT was used as the denominator, and the liquid phase peak area corresponding to the mutant enzyme was used as the numerator. The calculated result was recorded as the relative activity. The results are shown in Table 4 and 5. Figure 2 .

[0042] Table 4 Mm Changes in activity caused by a single point mutation in the A domain of CAR enzyme From Table 4 and Figure 2 It can be seen that, Mm The CAR enzyme T390A mutant exhibits the highest activity and is named [name missing]. Mm CAR_M1.

[0043] Example 2: Targeting carboxyl reductase Mm Construction and screening of iterative combinatorial mutants of CAR domain A 1. Construction of MmCAR iterative combinatorial mutants As shown in the results of Example 1, the mutant with the highest relative activity was T390A, which is located near the substrate binding pocket. In this example, it was combined with amino acid residues within the substrate transport channel to construct two-, three-, four-, and five-mutant hybrid mutants. The PCR amplification, DpnI digestion, and transformation involved in this scheme are as described in Example 1. The primers used are shown in Table 5.

[0044] Table 5 Primers for constructing MmCAR iterative combinatorial mutants Note: In the nucleotide sequence, M represents A or C, K represents G or T, and N represents A, G, C, or T.

[0045] 2. Mm Screening of CAR iterative combinatorial mutants Transform the combined mutant plasmid, which was verified to be correct by sequencing, into... E. coli K-12 MG1655 RARE competent cells were used, and single colonies were selected for induced expression and whole-cell catalysis, as described in Example 1. The concentration of the substrate 3-methoxy-4-hydroxybenzoic acid was 1 g / L. After the catalytic reaction, the peak area of ​​3-methoxy-4-hydroxybenzaldehyde corresponding to different mutants was detected by HPLC. The HPLC results were processed to... MmThe liquid phase peak area corresponding to CAR_WT is used as the denominator, and the liquid phase peak area corresponding to the combined mutant enzyme is used as the numerator. The calculated result is taken as the relative activity. The results are shown in Table 6 and 7. Figure 3 .

[0046] Table 6 Mm Changes in activity caused by combination mutations at the A domain site of CAR enzyme According to Table 6 and Figure 3 Based on the data, the mutant with the best relative activity, T390A / Q302C, was named Mm CAR_M2, the triple mutant T390A / Q302C / R277L was named Mm CAR_M3, the four mutants T390A / T338N / R277L / Q302C are named Mm CAR_M4, the five mutants T390A / T338N / R277L / Q302C / E281S are named Mm CAR_M5.

[0047] Example 3: Targeting carboxyl reductase Mav Construction and screening of site-directed mutants of CAR domain PR 1. Carboxylic acid reductase Mav CAR plasmid construction For origin Mycobacterium avium CAR enzyme ( Mav CAR) and derived from Bacillus subtilis ( Bacillus subtilis ) phosphopantoylthioethylamine transferase ( Bs The SFP gene was codon-optimized based on E. coli host preference to obtain the optimized target gene. Both were then co-cloned into the multiple cloning site of the pETDuet-1 vector using homologous recombination. After sequencing verification, the recombinant plasmid was transformed into the E. coli host strain. E. coli Recombinant engineered bacteria were obtained from K-12 MG1655 RARE. This protocol involves PCR amplification, Dpn The digestion and transformation screening were the same as in Example 1. Primers are shown in Table 7.

[0048] Table 7 Carboxylic acid reductases Mav CAR plasmid construction primers 2. Mav Construction of CAR site-directed mutants Using alphafold3 pairs Mav PR domain of CAR (see) Figure 1A separate model was constructed to create its three-dimensional structure. Thioester analogs of 3-methoxy-4-hydroxybenzoic acid were docked to the crystal structure for rational analysis. Based on the substrate binding pocket and substrate molecule interactions, the source was selected. Mav Site-directed mutagenesis was performed at two sites (P995 and D996) in the amino acid sequence of the PR domain of the CAR enzyme (SEQ ID NO: 15) (SEQ ID NO: 17). This protocol involves PCR amplification, Dpn The digestion and transformation screening were the same as in Example 1. The primers used are shown in Table 8.

[0049] Table 8 Mav CAR single-point mutant construction primers Note: In the nucleotide sequence, M represents A or C, K represents G or T, and N represents A, G, C, or T.

[0050] 3. Mav Screening of CAR site-directed mutants Transform the mutant plasmid that was verified to be correct by sequencing into E. coli K-12 MG1655 RARE competent cells were used, and single colonies were selected for induced expression and whole-cell catalysis, following the same procedures as in Example 1. The concentration of the substrate 3-methoxy-4-hydroxybenzoic acid was 1 g / L. After the catalytic reaction, the peak area of ​​3-methoxy-4-hydroxybenzaldehyde corresponding to different mutants was detected by HPLC. The HPLC results were processed to... Mav CAR enzyme wild type ( Mav The liquid phase peak area corresponding to CAR_WT was used as the denominator, and the liquid phase peak area corresponding to the combined mutant enzyme was used as the numerator. The calculated result was taken as the relative activity. The results are shown in Table 9. Figure 4 .

[0051] Table 9. Activity changes caused by single point mutations in the PR domain of MavCAR enzymes From Table 9 and Figure 4 It can be seen that, except for the alanine mutation (P995A) which causes a sharp drop in activity, the other seven substitutions at the P995 site all showed varying degrees of improvement. Among them, charged / polar side chains (E, R, H) and small side chain flexible amino acids (G) showed the best effects, with P995E being the optimal choice for this site. Similarly, at the D996 site, except for the alanine mutation (D996A) which significantly decreased activity, all other substitutions showed improvement. The elongation of acidic amino acids (D996E) and amidation (D996Q) showed the best effects, with D996E being the optimal choice for this site.

[0052] Example 4: Iterative combinatorial mutations targeting the PR domain of the carboxylic acid reductase MavCAR 1. Mav Construction of CAR iterative combinatorial mutants This embodiment involves combined mutations at the P995 and D996 sites, preferably constructing 10 double-combination mutants: P995E / D996Q, P995H / D996E, P995G / D996Q, P995R / D996Q, P995H / D996Q, P995Q / D996E, P995G / D996E, P995M / D996E, P995G / D996L, and P995H / D996L. The PCR amplification, DpnI digestion, and transformation involved in this scheme are the same as in Example 1. The primers used are shown in Table 10.

[0053] Table 10 Mav Primers for constructing CAR iterative combinatorial mutants 2. Screening of MavCAR iterative combinatorial mutants Transform the combined mutant plasmid, which was verified to be correct by sequencing, into... E. coli K-12 MG1655 RARE competent cells were used, and single colonies were selected for induced expression and whole-cell catalysis, following the same procedures as in Example 1. The concentration of the substrate 3-methoxy-4-hydroxybenzoic acid was 1 g / L. After the catalytic reaction, the peak area of ​​3-methoxy-4-hydroxybenzaldehyde corresponding to different mutants was detected by HPLC. The HPLC results were processed to... Mav The liquid phase peak area corresponding to CAR_WT is used as the denominator, and the liquid phase peak area corresponding to the combined mutant enzyme is used as the numerator. The calculated result is taken as the relative activity. The results are shown in Table 11 and 12. Figure 5 .

[0054] Table 11 Activity changes caused by combined mutations in the PR domain sites of MavCAR enzymes From Table 11 and Figure 5 It can be seen that the two mutants P995E / D996Q have the best relative activity, and they are named P995E / D996Q. Mav CAR_M2.

[0055] Example 5: Construction and screening of chimeric carboxylic acid reductase 1. Construction of chimeric carboxylic acid reductase This embodiment uses the above-mentioned carboxylic acid reductase. Mm CAR mutant domain A and carboxylic acid reductase Mav The CAR mutant domain PR was fused to construct 11 domain recombinant chimeras. Figure 6(and Table 13). The protocol involves PCR amplification, Dpn The digestion and transformation screening were the same as in Example 1. The primers used are shown in Table 12.

[0056] Table 12 Primers for constructing chimeric carboxylate reductase 2. Screening of chimeric carboxylic acid reductases Transform the combined mutant plasmid, which was verified to be correct by sequencing, into... E. coli K-12 MG1655 RARE competent cells were used, and single colonies were selected for induced expression and whole-cell catalysis, following the same procedures as in Example 1. The concentration of the substrate 3-methoxy-4-hydroxybenzoic acid was 15 g / L. After the catalytic reaction, the peak area of ​​3-methoxy-4-hydroxybenzaldehyde corresponding to different mutants was detected by HPLC. The HPLC results were processed to... Mm The liquid phase peak area corresponding to CAR_WT was used as the denominator, and the liquid phase peak area corresponding to the chimeric carboxyl reductase was used as the numerator. The calculated result was taken as the relative activity. The results are shown in Table 13 and... Figure 7 .

[0057] Table 13 Comparison of activities of recombinant chimeras containing carboxylate reductase domains As shown in Table 13, the data is obtained from carboxyl reductase. Mm CAR mutant domain A and carboxylic acid reductase Mav Chimeric carboxylate reductases formed by the fusion of the CAR mutant domain and the PR domain exhibit higher activity than the wild-type. Mm CAR is also higher than that produced by carboxyl reductase. Mm CAR wild-type domain A and carboxylic acid reductase Mav Chimeric carboxylate reductase formed by the fusion of the CAR wild-type domain and PR. Mm (A)_WT- Mav (PR)_WT.

[0058] Example 6: Enzymatic characterization of chimeric carboxylic acid reductase 1. Protein expression and purification This embodiment selects wild-type carboxylic acid reductase. Mm CAR_WT, wild-type chimeric carboxyl reductase Mm (A)_WT- Mav (PR)_WT, chimeric carboxylate reductase mutant Mm (A)_M4- Mav (PR)_WT and chimeric carboxylate reductase mutation Mm (A)_M5- Mav(PR)_M2 was characterized for enzymatic properties. The protein expression method is described in Example 1.

[0059] Cell cultures induced by protein expression were collected by centrifugation at 4°C (8000 rpm, 10 min) and washed with PBS buffer. Cells were then placed at 0°C and sonicated for 20 min using an ultrasonic cell disruptor (JY92-IIDN, Ningbo Xinzhi Biotechnology Co., Ltd.) (3 s operation time, 5 s interval), followed by centrifugation at 8000 rpm for 10 min to remove cell debris. The supernatant and precipitate after centrifugation of the cell disruption buffer were identified by SDS-PAGE analysis. The results are shown below. Figure 8 The supernatant was filtered through a membrane and then purified and eluted by Ni-NTA affinity chromatography under the following elution conditions: Solution A (20 mM PBS, pH 7.4, 0.5 M sodium chloride, 10 mM imidazole); Solution B (20 mM PBS, pH 7.4, 0.5 M sodium chloride, 500 mM imidazole). Wash once with 10 column volumes of solution A, then wash with solutions A and B sequentially at volume ratios of 5%, 10%, 20%, 30%, 40%, 50%, 80%, and 100% for approximately 5 column volumes, collecting the eluted fractions at each ratio. The highest concentration and purity of the target protein in the elution buffer were observed at ratios of 30% and 40%.

[0060] The target protein fraction with the highest concentration and purity was concentrated by ultrafiltration. The concentrated protein was flash-frozen in liquid nitrogen and stored at -80°C for subsequent enzyme activity assays. Protein concentration was determined using the Bradford method, with bovine serum albumin (BSA) as a standard.

[0061] 2. Determination of kinetic parameters The reaction conditions were the same as those in the in vitro enzyme catalysis conditions in Example 1. VA was used as the substrate, with a concentration ranging from 0.25 to 12.5 mM. The reaction mixture was incubated at 30°C with shaking at 800 rpm for 30 min. Using the above-mentioned A... 340 The change in absorbance of the supernatant was measured and analyzed using a detection method. GraphPad Prism software was used to fit the data and calculate... K M and k cat Values. All described experimental measurements were performed in triplicate.

[0062] 3. Comparison of dynamic parameters As shown in Table 14, the results indicate that the mutant Mm (A)_M4- Mav (PR)_WT and Mm (A)_M5-Mav (PR)_M2 compared to Mm CAR_WT and Mm (A)_WT- Mav (PR)_WT both showed higher performance k cat and k cat / K M The value indicates that it has a higher catalytic rate and catalytic efficiency in the process of converting VA to VAN.

[0063] Table 14 Kinetic parameters of carboxylic acid reductase Example 7: Application of chimeric carboxylic acid reductase in the preparation of 3-methoxy-4-hydroxybenzaldehyde 1. Whole-cell catalytic reaction This embodiment selects wild-type carboxylic acid reductase. Mm CAR_WT, wild-type chimeric carboxyl reductase Mm (A)_WT- Mav (PR)_WT, chimeric carboxylate reductase mutant Mm (A)_M4- Mav (PR)_WT and chimeric carboxylate reductase mutation Mm (A)_M5- Mav (PR)_M2 whole-cell catalysis prepares 3-methoxy-4-hydroxybenzaldehyde from 3-methoxy-4-hydroxybenzoic acid. Induced expression and whole-cell catalysis were performed as in Example 1, with the substrate concentration of 3-methoxy-4-hydroxybenzoic acid at 15 g / L and the catalytic reaction time at 12 h. Samples were taken periodically during the reaction.

[0064] 2. Establish a standard curve 3-Methoxy-4-hydroxybenzaldehyde standards at concentrations of 0.75 g / L, 0.5 g / L, 0.4 g / L, 0.3 g / L, 0.2 g / L, and 0.1 g / L were prepared, and their absorption peaks were measured at 270 nm. A standard curve was established for the standards: y = 14442x, R 2 =0.9999 (y is the peak area of ​​3-methoxy-4-hydroxybenzaldehyde obtained directly by HPLC, and x is the concentration of 3-methoxy-4-hydroxybenzaldehyde obtained directly by HPLC).

[0065] 3. Create a product generation quantity-time curve. Samples were taken during and after the catalytic reaction, and the same pretreatment method was used. The peak area of ​​the product 3-methoxy-4-hydroxybenzaldehyde was detected by high-performance liquid chromatography (HPLC). The sample concentration at each sampling time point was calculated based on the dilution factor and the established product concentration-peak area calculation formula (y=14442x). The following product formation-time curve was plotted.

[0066] like Figure 9 As shown, when 3-methoxy-4-hydroxybenzoic acid is used as the substrate, the optimal mutant is... Mm (A)_M5- Mav (PR)_M2 reached 12 g / L in 6 hours, while the wild type Mm CAR_WT was only 2.4 g / L after 12 h. This indicates that the optimal mutant not only has high total activity but also a much faster catalytic rate. This demonstrates that the mutant of this invention not only significantly improves catalytic activity but also significantly accelerates the reaction rate, showing excellent potential for industrial application.

[0067] The nucleotide / amino acid sequence involved in this invention is: 1. Mm CAR wild-type full-length amino acid sequence (SEQ ID NO:1)

[0068] 2. Mm CAR wild-type full-length DNA sequence (SEQ ID NO:2) 3. Mm Amino acid sequence of CAR A domain (1-650AA, SEQ ID NO:3) MSPITREERLERRIQDLYANDPQFAAAKPATAITAAIERPGLPLPQIIETVMTGYADRPALAQRSVEFVTDAGTGHTTLRLLPHFETISYGELWDRISALADVLSTEQTVKPGDRVCLLGFNSVDYATIDMTLARLGAVAVPLQTSAAITQLQPIVAETQPTMIAASVDALADATELALSGQTATRVLVFDHHRQVDAHRAAVESARERLAGSAVVETLAEAIARGDVPRGASAGSAPGTDVSDDSLALLIYTSGSTGAPKGAMYPRRNVATFWRKRTWFEGGYEPSITLNFMPMSHVMGRQILYGTLCNGGTAYFVAKSDLSTLFEDLALVRPTELTFVPRVWDMVFDEFQSEVDRRLVDGADRVALEAQVKAEIRNDVLGGRYTSALTGSAPISDEMKAWVEELLDMHLVEGYGSTEAGMILIDGAIRRPAVLDYKLVDVPDLGYFLTDRPHPRGELLVKTDSLFPGYYQRAEVTADVFDADGFYRTGDIMAEVGPEQFVYLDRRNNVLKLSQGEFVTVSKLEAVFGDSPLVRQIYIYGNSARAYLLAVIVPTQEALDAVPVEELKARLGDSLQEVAKAAGLQSYEIPRDFIIETTPWTLENGLLTGIRKLARPQLKKHYGELLEQIYTDLAHGQADELRSLRQSGAD。

[0069] 4. Mm DNA sequence of CAR A domain (SEQ ID NO:4)

[0070] 5 , Mm CAR A domain mutant Mm CAR_M1 amino acid sequence (1-650AA, SEQ ID NO:5) MSPITREERLERRIQDLYANDPQFAAAKPATAITAAIERPGLPLPQIIETVMTGYADRPALAQRSVEFVTDAGTGHTTLRLLPHFETISYGELWDRISALADVLSTEQTVKPGDRVCLLGFNSVDYATIDMTLARLGAVAVPLQTSAAITQLQPIVAETQPTMIAASVDALADATELALSGQTATRVLVFDHHRQVDAHRAAVESARERLAGSAVVETLAEAIARGDVPRGASAGSAPGTDVSDDSLALLIYTSGSTGAPKGAMYPRRNVATFWRKRTWFEGGYEPSITLNFMPMSHVMGRQILYGTLCNGGTAYFVAKSDLSTLFEDLALVRPTELTFVPRVWDMVFDEFQSEVDRRLVDGADRVALEAQVKAEIRNDVLGGRYTSALAGSAPISDEMKAWVEELLDMHLVEGYGSTEAGMILIDGAIRRPAVLDYKLVDVPDLGYFLTDRPHPRGELLVKTDSLFPGYYQRAEVTADVFDADGFYRTGDIMAEVGPEQFVYLDRRNNVLKLSQGEFVTVSKLEAVFGDSPLVRQIYIYGNSARAYLLAVIVPTQEALDAVPVEELKARLGDSLQEVAKAAGLQSYEIPRDFIIETTPWTLENGLLTGIRKLARPQLKKHYGELLEQIYTDLAHGQADELRSLRQSGAD。

[0071] 6、 Mm CAR A domain mutant Mm CAR_M1 DNA sequence (SEQ ID NO:6)

[0072] 7, Mm CAR A domain mutant Mm CAR_M2 amino acid sequence (1-650AA, SEQ ID NO:7) MSPITREERLERRIQDLYANDPQFAAAKPATAITAAIERPGLPLPQIIETVMTGYADRPALAQRSVEFVTDAGTGHTTLRLLPHFETISYGELWDRISALADVLSTEQTVKPGDRVCLLGFNSVDYATIDMTLARLGAVAVPLQTSAAITQLQPIVAETQPTMIAASVDALADATELALSGQTATRVLVFDHHRQVDAHRAAVESARERLAGSAVVETLAEAIARGDVPRGASAGSAPGTDVSDDSLALLIYTSGSTGAPKGAMYPRRNVATFWRKRTWFEGGYEPSITLNFMPMSHVMGRCILYGTLCNGGTAYFVAKSDLSTLFEDLALVRPTELTFVPRVWDMVFDEFQSEVDRRLVDGADRVALEAQVKAEIRNDVLGGRYTSALAGSAPISDEMKAWVEELLDMHLVEGYGSTEAGMILIDGAIRRPAVLDYKLVDVPDLGYFLTDRPHPRGELLVKTDSLFPGYYQRAEVTADVFDADGFYRTGDIMAEVGPEQFVYLDRRNNVLKLSQGEFVTVSKLEAVFGDSPLVRQIYIYGNSARAYLLAVIVPTQEALDAVPVEELKARLGDSLQEVAKAAGLQSYEIPRDFIIETTPWTLENGLLTGIRKLARPQLKKHYGELLEQIYTDLAHGQADELRSLRQSGAD.

[0073] 8, Mm CAR A domain mutant Mm CAR_M2 DNA sequence (SEQ ID NO:8)

[0074] 9、 Mm CAR A domain mutant Mm CAR_M3 amino acid sequence (1-650AA, SEQ ID NO:9) MSPITREERLERRIQDLYANDPQFAAAKPATAITAAIERPGLPLPQIIETVMTGYADRPALAQRSVEFVTDAGTGHTTLRLLPHFETISYGELWDRISALADVLSTEQTVKPGDRVCLLGFNSVDYATIDMTLARLGAVAVPLQTSAAITQLQPIVAETQPTMIAASVDALADATELALSGQTATRVLVFDHHRQVDAHRAAVESARERLAGSAVVETLAEAIARGDVPRGASAGSAPGTDVSDDSLALLIYTSGSTGAPKGAMYPRRNVATFWRKLTWFEGGYEPSITLNFMPMSHVMGRCILYGTLCNGGTAYFVAKSDLSTLFEDLALVRPTELTFVPRVWDMVFDEFQSEVDRRLVDGADRVALEAQVKAEIRNDVLGGRYTSALAGSAPISDEMKAWVEELLDMHLVEGYGSTEAGMILIDGAIRRPAVLDYKLVDVPDLGYFLTDRPHPRGELLVKTDSLFPGYYQRAEVTADVFDADGFYRTGDIMAEVGPEQFVYLDRRNNVLKLSQGEFVTVSKLEAVFGDSPLVRQIYIYGNSARAYLLAVIVPTQEALDAVPVEELKARLGDSLQEVAKAAGLQSYEIPRDFIIETTPWTLENGLLTGIRKLARPQLKKHYGELLEQIYTDLAHGQADELRSLRQSGAD。

[0075] 10、 Mm CAR A domain mutant Mm CAR_M3 DNA sequence (SEQ ID NO:10)

[0076] 11、 Mm CAR A domain mutant Mm CAR_M4 amino acid sequence (1-650AA, SEQ ID NO:11) MSPITREERLERRIQDLYANDPQFAAAKPATAITAAIERPGLPLPQIIETVMTGYADRPALAQRSVEFVTDAGTGHTTLRLLPHFETISYGELWDRISALADVLSTEQTVKPGDRVCLLGFNSVDYATIDMTLARLGAVAVPLQTSAAITQLQPIVAETQPTMIAASVDALADATELALSGQTATRVLVFDHHRQVDAHRAAVESARERLAGSAVVETLAEAIARGDVPRGASAGSAPGTDVSDDSLALLIYTSGSTGAPKGAMYPRRNVATFWRKLTWFEGGYEPSITLNFMPMSHVMGRCILYGTLCNGGTAYFVAKSDLSTLFEDLALVRPTELNFVPRVWDMVFDEFQSEVDRRLVDGADRVALEAQVKAEIRNDVLGGRYTSALAGSAPISDEMKAWVEELLDMHLVEGYGSTEAGMILIDGAIRRPAVLDYKLVDVPDLGYFLTDRPHPRGELLVKTDSLFPGYYQRAEVTADVFDADGFYRTGDIMAEVGPEQFVYLDRRNNVLKLSQGEFVTVSKLEAVFGDSPLVRQIYIYGNSARAYLLAVIVPTQEALDAVPVEELKARLGDSLQEVAKAAGLQSYEIPRDFIIETTPWTLENGLLTGIRKLARPQLKKHYGELLEQIYTDLAHGQADELRSLRQSGAD。

[0077] 12、 Mm CAR A domain mutant Mm CAR_M4 DNA sequence (SEQ ID NO:12)

[0078] 13. Mm CAR A domain mutant Mm CAR_M5 amino acid sequence (1-650AA, SEQ ID NO:13) MSPITREERLERRIQDLYANDPQFAAAKPATAITAAIERPGLPLPQIIETVMTGYADRPALAQRSVEFVTDAGTGHTTLRLLPHFETISYGELWDRISALADVLSTEQTVKPGDRVCLLGFNSVDYATIDMTLARLGAVAVPLQTSAAITQLQPIVAETQPTMIAASVDALADATELALSGQTATRVLVFDHHRQVDAHRAAVESARERLAGSAVVETLAEAIARGDVPRGASAGSAPGTDVSDDSLALLIYTSGSTGAPKGAMYPRRNVATFWRKLTWFSGGYEPSITLNFMPMSHVMGRCILYGTLCNGGTAYFVAKSDLSTLFEDLALVRPTELNFVPRVWDMVFDEFQSEVDRRLVDGADRVALEAQVKAEIRNDVLGGRYTSALAGSAPISDEMKAWVEELLDMHLVEGYGSTEAGMILIDGAIRRPAVLDYKLVDVPDLGYFLTDRPHPRGELLVKTDSLFPGYYQRAEVTADVFDADGFYRTGDIMAEVGPEQFVYLDRRNNVLKLSQGEFVTVSKLEAVFGDSPLVRQIYIYGNSARAYLLAVIVPTQEALDAVPVEELKARLGDSLQEVAKAAGLQSYEIPRDFIIETTPWTLENGLLTGIRKLARPQLKKHYGELLEQIYTDLAHGQADELRSLRQSGAD.

[0079] 14. Mm CAR A domain mutant Mm CAR_M5 DNA sequence (SEQ ID NO:14)

[0080] 15. Mav Full-length amino acid sequence of wild-type CAR (SEQ ID NO:15)

[0081] 16. Mav CAR wild-type full-length DNA sequence (SEQ ID NO:16)

[0082] 17、 Mav Amino acid sequence of CAR PR domain (651-1173AA, SEQ ID NO:17) APVLQTVSRAAGAMLGSAASDLSPDAHFTDLGGDSLSALTFGNLLREIFDVDVPVGVIVSPANDLAAIASYIEAERQGSKRPTFASVHGRDATVVRAADLTLDKFLDADTLASAPNLPKPATEVRTVLLTGATGFLGRYLALEWLERMDMVDGKVIALVRARSDEEARARLDKTFDSGDPKLLAHYQQLAADHLEVIAGDKGEANLGLRQDVWQRLADTVDVIVDPAALVNHVLPYSELFGPNALGTAELIRLALTSKQKPYTYVSTIGVGDQIEPGKFVENADIRQMSATRAINDSYANGYGNSKWAGEVLLREAHDLCGLPVAVFRCDMILADTTYAGQLNLPDMFTRLMLSLVATGIAPGSFYELDADGNRQRAHYDGLPVEFIAAAISTLGSQITDSDTGFQTYHVMNPYDDGIGLDEYVDWLVDAGYSIERIADYSEWLRRFETSLRALPDRQRQYSLLPLLHNYRTPEKPINGSIAPTDVFRAAVQEAKIGPDKDIPHVSPPVIVKYITDLQLLGLL。

[0083] 18、 Mav DNA sequence of CAR PR domain (SEQ ID NO:18)

[0084] 19、 Mav CAR PR domain mutant Mav CAR_M2 amino acid sequence (651-1173AA, SEQ ID NO:19) APVLQTVSRAAGAMLGSAASDLSPDAHFTDLGGDSLSALTFGNLLREIFDVDVPVGVIVSPANDLAAIASYIEAERQGSKRPTFASVHGRDATVVRAADLTLDKFLDADTLASAPNLPKPATEVRTVLLTGATGFLGRYLALEWLERMDMVDGKVIALVRARSDEEARARLDKTFDSGDPKLLAHYQQLAADHLEVIAGDKGEANLGLRQDVWQRLADTVDVIVDPAALVNHVLPYSELFGPNALGTAELIRLALTSKQKPYTYVSTIGVGDQIEPGKFVENADIRQMSATRAINDSYANGYGNSKWAGEVLLREAHDLCGLPVAVFRCDMILADTTYAGQLNLEQMFTRLMLSLVATGIAPGSFYELDADGNRQRAHYDGLPVEFIAAAISTLGSQITDSDTGFQTYHVMNPYDDGIGLDEYVDWLVDAGYSIERIADYSEWLRRFETSLRALPDRQRQYSLLPLLHNYRTPEKPINGSIAPTDVFRAAVQEAKIGPDKDIPHVSPPVIVKYITDLQLLGLL。

[0085] 20、 Mav CAR PR domain mutant Mav CAR_M2 DNA sequence (SEQ ID NO:20)

[0086] twenty one, Mm (A)_WT-Mav (PR)_WT chimeric carboxyl reductase mutant amino acid sequence (SEQ ID NO: 21)

[0087] twenty two, Mm (A)_WT- Mav (PR)_WT chimeric carboxylate reductase mutant DNA sequence (SEQ ID NO:22)

[0088] twenty three, Mm (A)_M4- Mav (PR)_WT chimeric carboxylate reductase mutant amino acid sequence (SEQ ID NO:23)

[0089] twenty four, Mm (A)_M4- Mav (PR)_WT chimeric carboxylate reductase mutant DNA sequence (SEQ ID NO:24)

[0090] 25. Mm (A)_M5- Mav (PR)_WT chimeric carboxylate reductase mutant amino acid sequence (SEQ ID NO:25)

[0091] 26. Mm (A)_M5- Mav (PR)_WT chimeric carboxylate reductase mutant DNA sequence (SEQ ID NO:26)

[0092] 27. Mm (A)_M5- Mav (PR)_M2 chimeric carboxylate reductase mutant amino acid sequence (SEQ ID NO:27)

[0093] 28. Mm (A)_M5- Mav (PR)_M2 chimeric carboxylate reductase mutant DNA sequence (SEQ ID NO:28)

[0094] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A carboxylic acid reductase mutant, characterized in that, It includes any of the following mutants (1)-(3): (1) Carboxyl reductases derived from marine mycobacteria Mm The CAR is obtained by substituting one or more of the following substitution mutations into the amino acid sites I303, T390, Y415, I425, R277, E281, M299, Q302, E336, and T338 in the A domain: R277A, R277C, R277S, R277L, R277G, R277V, E281F, E281T, E281L, E281D, E281G, E281V, E2 81S, M299A, M299G, M299C, M299L, M299D, M299I, M299V, M299N, M299T, Q302A, Q302G, Q302S , Q302C, Q302L, Q302T, Q302P, I303A, I303D, I303E, I303I, I303K, I303L, I303M, I303N, I3 03P, I303R, I303Y, I303G, I303H, E336A, E336P, E336G, E336I, E336V, E336C, T338S, T338N , T338L, T338V, T390A, T390D, T390H, T390C, T390I, T390H, T390L, T390M, T390N, T390P, T 390E, T390Q, T390R, T390W, Y415C, Y415H, Y415P, Y415W, Y415E, Y415F, Y415G, Y415I, Y415 M, Y415N, Y415H, Y415P, Y415Q, Y415S, Y415T, Y415V, I425A, I425C, I425F, I425N, I425Q, I425E, I425F, I425G, I425K, I425N, I425K, I425N, I425R, I425S, I425W, and I425Y; wherein, the carboxyl reductase Mm The amino acid sequence of CAR is shown in SEQ ID NO:1; (2) Carboxyl reductase from Mycobacterium avium Mav The CAR is obtained by substituting the amino acid sites P995 and / or D996 in the PR domain with the following substitution mutations: P995L, P995E, P995G, P995H, P995M, P995Q, P995R, D996E, D996G, D996L, D996M, D996P, D996Q, D996S, and D996T; wherein, the carboxylic acid reductase... Mav The amino acid sequence of CAR is shown in SEQ ID NO:15; (3) Carboxyl reductase derived from marine mycobacteria Mm CAR A domain mutants and Mycobacterium avium carboxylate reductase Mav The CAR PR domain mutant was fused to obtain the carboxylic acid reductase. Mm The A domain mutant of CAR is at least one of the mutants shown in item (1), the carboxylic acid reductase Mav The PR domain mutant of CAR is at least one of the mutants shown in item (2).

2. The carboxylic acid reductase mutant as described in claim 1, characterized in that, In the mutant of item (1), the carboxylic acid reductase Mm The A domain of CAR contains the following combinations of mutations: T390A / Q302C, T390A / Q302C / R277L, T390A / T338S / R277G, T390A / T338I / R277A, T390A / T338N / R277L, T390A / T338S / R277C, T390A / T338V / R277C, T390A / T338G / R277V, and T390A / T338N / R277L. / Q302C, T390A / T338S / R277G / E336P, T390A / T338N / R277L / E336P, T390A / T338N / R277 A / E336G / M299C, T390A / T338S / R277G / Q302A / E336P, T390A / T338N / R277L / M299T, T39 0A / T338N / R277L / M299A, T390A / T338N / R277L / M299G, T390A / T338N / R277L / M299C, T3 90A / T338N / R277L / M299L, T390A / T338N / R277L / M299D, T390A / T338N / R277L / M299N, T3 90A / T338N / R277L / M299I, T390A / T338N / R277L / M299V, T390A / T338N / R277L / Q302C / R 301L, T390A / T338N / R277L / Q302C / E281G, T390A / T338N / R277L / Q302C / E281L, T390A / T 338N / R277L / Q302C / E281D, T390A / T338N / R277L / Q302C / E281C, T390A / T338N / R277L / Q302C / E281V, T390A / T338N / R277L / Q302C / E281T, T390A / T338N / R277L / Q302C / E281S.

3. The carboxylic acid reductase mutant as described in claim 1, characterized in that, In the mutant of item (2), the carboxylic acid reductase Mav The PR domain of CAR includes the following combination of mutations: P995E / D996Q, P995H / D996E, P995G / D996Q, P995R / D996Q, P995H / D996Q, P995Q / D996E, P995G / D996E, P995M / D996E, P995G / D996L, P995H / D996L.

4. The carboxylic acid reductase mutant as described in claim 1, characterized in that, In the mutants of item (3), the fusion mutants include any combination of the following: Mm (A)_WT- Mav (PR)_WT、 Mm (A)_M1- Mav (PR)_WT、 Mm (A)_M2- Mav (PR)_WT、 Mm (A)_M3- Mav (PR)_WT、 Mm (A)_M4- Mav (PR)_WT、 Mm (A)_M5- Mav (PR)_WT、 Mm (A)_M1- Mav (PR)_M2、 Mm (A)_M2- Mav (PR)_M2、 Mm (A)_M3- Mav (PR)_M2、 Mm (A)_M4- Mav (PR)_M2、 Mm (A)_M5- Mav (PR)_M2; where the Mm (A)_WT- Mav (PR)_WT is a carboxylic acid reductase. Mm The A domain of CAR and carboxylic acid reductase Mav Chimeric carboxylate reductase obtained by fusing the PR domain of CAR; Mm (A)_M1- Mav (PR)_WT is a carboxylic acid reductase. Mm T390A mutant of CAR A domain and carboxyl reductase Mav Chimeric carboxylate reductase obtained by fusing the PR domain of CAR; Mm (A)_M2- Mav (PR)_WT is a carboxylic acid reductase. Mm The T390A / Q302C mutant of the A domain of CAR and carboxyl reductase Mav Chimeric carboxylate reductase obtained by fusing the PR domain of CAR; Mm (A)_M3- Mav (PR)_WT is a carboxylic acid reductase. Mm The T390A / Q302C / R277L mutant of the A domain of CAR and carboxyl reductase Mav Chimeric carboxylate reductase obtained by fusing the PR domain of CAR; Mm (A)_M4- Mav (PR)_WT is a carboxylic acid reductase. Mm The T390A / T338N / R277L / Q302C mutant of the A domain of CAR and carboxyl reductase Mav Chimeric carboxylate reductase obtained by fusing the PR domain of CAR; Mm (A)_M5- Mav (PR)_WT is a carboxylic acid reductase. Mm The T390A / T338N / R277L / Q302C / E281S mutant of the A domain of CAR and carboxyl reductase Mav Chimeric carboxylate reductase obtained by fusing the PR domain of CAR; Mm (A)_M1- Mav (PR)_M2、 Mm (A)_M2- Mav (PR)_M2、 Mm (A)_M3- Mav (PR)_M2、 Mm (A)_M4- Mav (PR)_M2、 Mm (A)_M5- Mav (PR)_M2 is a carboxylic acid reductase. Mm T390A mutant of CAR A domain, carboxyl reductase Mm T390A / Q302C mutant of CAR A domain, carboxyl reductase Mm T390A / Q302C / R277L mutant of CAR A domain, carboxyl reductase Mm The T390A / T338N / R277L / Q302C mutant of the A domain of CAR and carboxyl reductase Mm The T390A / T338N / R277L / Q302C / E281S mutants of the A domain of CAR, respectively, are associated with carboxyl reductase. Mav Chimeric carboxylic acid reductase obtained by fusing the P995E / D996Q mutant of the PR domain of CAR.

5. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the carboxylic acid reductase mutant according to any one of claims 1-4.

6. A biomaterial, characterized in that, The biomaterial comprises the nucleic acid molecule of claim 5, and the biomaterial includes a recombinant vector or recombinant engineered bacteria.

7. A whole-cell catalyst, characterized in that, The whole-cell catalyst comprises the recombinant engineered bacteria as described in claim 6.

8. A method for preparing 3-methoxy-4-hydroxybenzaldehyde, characterized in that, Using 3-methoxy-4-hydroxybenzoic acid as a substrate, and catalyzing the reaction with the recombinant engineered bacteria of claim 6 or the whole-cell catalyst of claim 7, 3-methoxy-4-hydroxybenzaldehyde is obtained.

9. The use of the carboxylic acid reductase mutant as described in any one of claims 1-4, the nucleic acid molecule as described in claim 5, the biomaterial as described in claim 6, or the whole-cell catalyst as described in claim 7 in catalyzing the reduction of carboxylic acid substrates to the corresponding aldehydes or alcohols.

10. The application as described in claim 9, characterized in that, The application is in the preparation of 3-methoxy-4-hydroxybenzaldehyde.

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