Synthesis method of 3-methoxy-4-hydroxybenzaldehyde

Abstract

The invention provides a carboxylic acid reductase mutant and an application of the carboxylic acid reductase mutant in catalytic synthesis of 3-methoxy-4-hydroxybenzaldehyde. According to the present invention, the carboxylic acid reductase derived from Segniliparus rugosus is modified so as to obtain the mutant with characteristics of high 3-methoxy-4-hydroxybenzaldehyde yield, the 3-methoxy-4-hydroxybenzaldehyde is produced from a recombinant vector and a recombinant genetically engineered bacterium constructed by the mutant in a resting cell catalysis manner, and the yield of the 3-methoxy-4-hydroxybenzaldehyde is remarkably improved and is 1.9-4.7 times that of a wild type. The method for producing the 3-methoxy-4-hydroxybenzaldehyde, provided by the invention, has the advantages of mild reaction conditions, simplicity in operation, short production period, environmental friendliness, reduction of production cost and the like.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly relates to a carboxylic acid reductase mutant and its application in the catalytic synthesis of 3-methoxy-4-hydroxybenzaldehyde. Background Art

[0002] 3-Methoxy-4-hydroxybenzaldehyde mainly exists in the plant vanilla, has the aroma of vanilla and a strong milk fragrance, and is one of the most important spices in the world. Most of the 3-methoxy-4-hydroxybenzaldehyde is applied to the food industry and is an indispensable flavoring raw material for high-grade foods. The global annual demand exceeds 18,000 tons, and most of it is obtained by chemical synthesis. The price of 3-methoxy-4-hydroxybenzaldehyde synthesized by chemical methods is less than $15 per kilogram, while the price of naturally extracted 3-methoxy-4-hydroxybenzaldehyde is expensive, with a selling price of over $1,200 per kilogram.

[0003] There are mainly three methods for the chemical synthesis of 3-methoxy-4-hydroxybenzaldehyde, namely the lignin route, the guaiacol-formaldehyde route, and the guaiacol-glyoxylic acid route. In the process, conditions such as high temperature and high pressure under alkaline conditions are required, and raw materials such as guaiacol, formaldehyde, and p-nitrosodiphenylamine are needed, which have disadvantages such as strong toxicity and environmental pollution. The natural product extraction method is restricted by plant resources, and there are problems such as low content of plant-derived aromatic compounds and high costs in the distillation / extraction process. In this context, developing white biotechnology to synthesize natural flavors and fragrances using microorganisms is an extremely economical production method that can replace traditional plant-derived and petroleum-based aromatic spice compounds.In 1998, Li et al. first achieved the synthesis of 3-methoxy-4-hydroxybenzaldehyde from glucose (Synthesis of vanillin from glucose. Li K, et al. 1998); in 2009, Hansen et al. obtained 0.065 g / L of 3-methoxy-4-hydroxybenzaldehyde in yeast (De novo biosynthesis of vanillin in fission yeast (Schizosaccharomyces pombe) and baker’s yeast (Saccharomyces cerevisiae). Hansen EH, et al. 2009); in 2010, Hansen et al. introduced AtUGT into yeast to convert 3-methoxy-4-hydroxybenzaldehyde into less toxic vanillin glucoside, with a 5-fold increase in production (Improved vanillin production in baker’s yeast through in silico design. Brochado AR, et al. 2010); in 2012, Ana Rita Brochado et al. overexpressed HsOMT in yeast, doubling the production of vanillin glucoside to 400 mg / L (Overexpression of O-methyltransferase leads to improved vanillin production in baker’s yeast only when complemented with model-guided network engineering. Brochado, et al. 2012); in 2021, Joanna C Sadler et al. directly converted plastic derivative monomers into 3-methoxy-4-hydroxybenzaldehyde in Escherichia coli, with a maximum synthesis titer of 119 mg / L (Microbial synthesis of vanillin from waste poly(ethylene terephthalate). Sadler, et al. 2021). Modern research uses biotechnological methods to produce high-quality vanillin derivatives, which include a series of metabolites produced by vanillin plants. Using plant or microbial cells as biocatalysts, the biotransformation of inexpensive substrates is carried out to produce high-value products.

[0004] In summary, there are currently various ways to synthesize 3-methoxy-4-hydroxybenzaldehyde. However, when using 3-methoxy-4-hydroxybenzoic acid as a substrate and catalyzed by carboxylic acid reductase, the enzyme activity is low, and high yields of 3-methoxy-4-hydroxybenzaldehyde cannot be produced, so it cannot be applied to industrial production. Summary of the Invention

[0005] Currently, there are problems in industrial production of 3-methoxy-4-hydroxybenzoic acid, such as low yield, harsh conditions, and easy environmental pollution. In view of the deficiencies in the existing technology, the present invention uses genetic engineering technology and computer-aided design to construct a carboxylic acid reductase mutant with higher activity, thereby greatly increasing the yield of 3-methoxy-4-hydroxybenzaldehyde.

[0006] The present invention provides a method for synthesizing 3-methoxy-4-hydroxybenzaldehyde using a carboxylic acid reductase or its mutant.

[0007] Therefore, the first object of the present invention is to provide a mutant obtained by mutating the carboxylic acid reductase SrCAR (NCBI accession number WP_007468889.1) derived from Segniliparus rugosus. The mutation is based on the amino acid sequence of the carboxylic acid reductase SrCAR and has the following mutations: selected from one or several of the following amino acid residue sites: 130, 181, 291, 292, 317, 356, 430, 440, 505, 519, 524, 533, 627, 873, 936, 1121, and / or 1133.

[0008] All of the above amino acid mutants have the function of catalyzing 3-methoxy-4-hydroxybenzoic acid of the wild-type carboxylic acid reductase as shown by SrCAR.

[0009] More specifically, the SrCAR carboxylic acid reductase mutant is a protein obtained by any one, any two, any three, any four, any five, any six, any seven, any eight, any nine, any ten, any eleven, any twelve, any thirteen, any fourteen, any fifteen, any sixteen, or all of the following seventeen modifications of SrCAR:

[0010] X1. Mutating the isoleucine at position 130 of the carboxylic acid reductase SrCAR to aspartic acid, cysteine, or lysine;

[0011] X2. Mutating the histidine at position 181 of the carboxylic acid reductase SrCAR to cysteine, methionine, or serine;

[0012] X3. Mutating the histidine at position 291 of the carboxylic acid reductase SrCAR to threonine, isoleucine, or lysine;

[0013] X4. Mutate the asparagine at position 292 of the carboxylic acid reductase SrCAR to histidine or lysine;

[0014] X5. Mutate the alanine at position 317 of the carboxylic acid reductase SrCAR to valine, leucine or asparagine;

[0015] X6. Mutate the phenylalanine at position 356 of the carboxylic acid reductase SrCAR to proline, glutamic acid or aspartic acid;

[0016] X7. Mutate the glycine at position 430 of the carboxylic acid reductase SrCAR to glutamine, proline or lysine;

[0017] X8. Mutate the tryptophan at position 440 of the carboxylic acid reductase SrCAR to glutamic acid, histidine or alanine;

[0018] X9. Mutate the threonine at position 505 of the carboxylic acid reductase SrCAR to alanine, methionine or valine;

[0019] X10. Mutate the tyrosine at position 519 of the carboxylic acid reductase SrCAR to valine, threonine or histidine;

[0020] X11. Mutate the lysine at position 524 of the carboxylic acid reductase SrCAR to proline, methionine or serine;

[0021] X12. Mutate the aspartic acid at position 533 of the carboxylic acid reductase SrCAR to histidine, arginine or tryptophan;

[0022] X13. Mutate the alanine at position 627 of the carboxylic acid reductase SrCAR to threonine, valine or leucine;

[0023] X14. Mutate the aspartic acid at position 873 of the carboxylic acid reductase SrCAR to asparagine or proline;

[0024] X15. Mutate the valine at position 936 of the carboxylic acid reductase SrCAR to leucine, serine or lysine;

[0025] X16. Mutate the glutamic acid at position 1121 of the carboxylic acid reductase SrCAR to glutamine or tryptophan;

[0026] X17. Mutate the histidine at position 1133 of the carboxylic acid reductase SrCAR to glutamine or leucine.

[0027] In one embodiment of the present invention, the SrCAR carboxylic acid reductase mutant is:

[0028] A protein obtained by mutating the 130th site of the carboxylic acid reductase SrCAR to cysteine;

[0029] A protein obtained by mutating the 181st site of carboxylic acid reductase SrCAR to cysteine;

[0030] A protein obtained by mutating the 291st site of carboxylic acid reductase SrCAR to lysine;

[0031] A protein obtained by mutating the 292nd site of carboxylic acid reductase SrCAR to histidine;

[0032] A protein obtained by mutating the 317th site of carboxylic acid reductase SrCAR to valine;

[0033] A protein obtained by mutating the 356th site of carboxylic acid reductase SrCAR to proline;

[0034] A protein obtained by mutating the 430th site of carboxylic acid reductase SrCAR to asparagine;

[0035] A protein obtained by mutating the 440th site of carboxylic acid reductase SrCAR to glycine;

[0036] A protein obtained by mutating the 505th site of carboxylic acid reductase SrCAR to alanine;

[0037] A protein obtained by mutating the 519th site of carboxylic acid reductase SrCAR to valine;

[0038] A protein obtained by mutating the 524th site of carboxylic acid reductase SrCAR to proline;

[0039] A protein obtained by mutating the 524th and 627th sites of carboxylic acid reductase SrCAR to serine and leucine respectively;

[0040] A protein obtained by mutating the 524th, 627th and 800th sites of carboxylic acid reductase SrCAR to serine, leucine and alanine respectively;

[0041] A protein obtained by mutating the 524th, 627th and 1133rd sites of carboxylic acid reductase SrCAR to serine, leucine and glutamine respectively;

[0042] A protein obtained by mutating the 524th, 627th and 1133rd sites of carboxylic acid reductase SrCAR to serine, leucine and leucine respectively;

[0043] A protein obtained by mutating the 524th, 627th and 1121st sites of carboxylic acid reductase SrCAR to serine, leucine and tryptophan respectively;

[0044] A protein obtained by mutating the 291st, 524th, and 627th sites of carboxylic acid reductase SrCAR into lysine, serine, and leucine respectively;

[0045] A protein obtained by mutating the 292nd, 524th, and 627th sites of carboxylic acid reductase SrCAR into histidine, serine, and leucine respectively;

[0046] A protein obtained by mutating the 524th, 627th, and 873rd sites of carboxylic acid reductase SrCAR into serine, leucine, and asparagine respectively;

[0047] A protein obtained by mutating the 130th, 181st, 524th, and 627th sites of carboxylic acid reductase SrCAR into cysteine, cysteine, serine, and leucine respectively;

[0048] A protein obtained by mutating the 292nd, 524th, 627th, and 1121st sites of carboxylic acid reductase SrCAR into histidine, serine, leucine, and tryptophan respectively.

[0049] The present invention discloses the following technical achievements:

[0050] The present invention discloses a newly modified carboxylic acid reductase mutant, which obtains a plurality of mutants by performing site-specific mutations on the amino acid sequence of carboxylic acid reductase such as SrCAR; and the obtained mutants all have the function of catalyzing 3-methoxy-4-hydroxybenzoic acid to generate 3-methoxy-4-hydroxybenzaldehyde with the wild type of SrCAR.

[0051] The present invention also discloses a recombinant vector and a recombinant strain containing the carboxylic acid reductase gene and its mutant gene. The gene encoding the carboxylic acid reductase in the recombinant vector is expressed by means of shake flask fermentation culture to obtain the corresponding enzyme. By mutating specific sites of the wild-type enzyme, the yield of the obtained mutant is significantly increased, which is 1.2 - 4.7 times that of the starting strain, and can significantly increase the yield of 3-methoxy-4-hydroxybenzaldehyde.

[0052] The application of the carboxylic acid reductase or its carboxylic acid reductase mutant in the synthesis of 3-methoxy-4-hydroxybenzaldehyde belongs to the protection scope of the present invention. Description of the Drawings

[0053] Figure 1 It is a plasmid map for the construction of the carboxylic acid reductase recombinant vector.

[0054] Figure 2 It is a standard curve graph for the HPLC liquid chromatography detection of the 3-methoxy-4-hydroxybenzaldehyde standard product.

[0055] Figure 3For the yield ratio of the recombinant engineering strain with advantageous mutant to 3 - methoxy - 4 - hydroxybenzaldehyde. Detailed implementation manners

[0056] The present invention will be further described in detail below in conjunction with the specific implementation manners. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0057] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. The materials, reagents, instruments, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0058] Example 1. Obtaining of carboxylic acid reductase or its mutant

[0059] The present invention modifies the carboxylic acid reductase (NCBI accession number: WP_007468889.1, hereinafter simply referred to as SrCAR) derived from Segniliparus rugosus through techniques such as molecular docking, three - codon saturation mutagenesis, and HotSpot prediction, to obtain mutants with higher yields of 3 - methoxy - 4 - hydroxybenzaldehyde, so as to more efficiently produce 3 - methoxy - 4 - hydroxybenzaldehyde using industrial microorganisms.

[0060] 1.1 Construction of the SrCAR single - point saturation mutant library

[0061] Based on gene mining, molecular docking, rational analysis, etc. of the wild - type carboxylic acid reductase, and according to the substrate - binding pocket and the interaction between the substrate molecules, 10 sites of the amino acid sequence (SEQ ID No: 1) of the wild - type carboxylic acid reductase derived from Segniliparus rugosus (NCBI accession number: WP_007468889.1) are rationally designed. The 10 sites are 317th, 356th, 430th, 440th, 505th, 519th, 524th, 533rd, 627th, 936th, 998th, 999th, and 1015th positions. Based on the above 10 sites, a single - point saturation mutant library is constructed, as shown in Table 1.

[0062] Table 1. Single - point saturation mutant library constructed based on carboxylic acid reductase SrCAR

[0063]

[0064]

[0065] Note: In Table 1, there are primers of multiple single-stranded DNAs, namely primer A317-F, F356-F, G430-F, W440-F, T505-F, Y519-F, K524-F, E533-F, A627-F, V936-F, D998-F, M999-F, Q1015-F. Each single-stranded DNA is mixed in a molar ratio of 1:1:6:12 in the order of TGG:ATG:VMA:NDT in the table; V represents G or A or C, M represents A or C, N represents A or T or G or C, and D represents G or A or T.

[0066] To obtain the carboxylic acid reductase SrCAR mutant, the following experiments were carried out:

[0067] PCR amplification reaction system (50 μL): PrimeSTAR (2×) 25 μL, template SrCAR 1 μL, forward primer (10 μM) 2 μL, reverse primer (10 μM) 2 μL, ddH2O 20 μL. The specific sequences of the forward and reverse primers are shown in Table 1.

[0068] PCR amplification reaction conditions: 98 °C: 2 min, (98 °C: 10 s, 65 °C: 15 s, 72 °C: 4 min 30 s) 30 cycles, 72 °C: 4 min.

[0069] The obtained PCR product was subjected to the following operations: 1 μL of Dpn I enzyme was added to 20 μL of the PCR product for digesting the plasmid template, and the treatment was carried out at a constant temperature of 37 °C for 2 h. 5 μL of the digested PCR product was electrotransformed into 100 μL of E.coli BAP1 competent cells. The electrotransformed E.coli BAP1 bacterial solution was evenly spread on an LB plate with kanamycin resistance (concentration: 50 μg / mL). After culturing at a constant temperature of 37 °C for 14 h, single colonies grew out, which were the engineering strains of the carboxylic acid reductase SrCAR gene mutant. The recombinant plasmid containing the target nucleotide with random or directed mutation in this strain was the expression vector for expressing the carboxylic acid reductase SrCAR mutant.

[0070] 1.2 Screening of the SrCAR single-site saturation mutant library

[0071] A single copy of the sfp gene under the control of the T7 RNA polymerase promoter was integrated into the prp operon of BL21(DE3) to obtain Escherichia coli BAP1. After successful sequencing of the single-site saturation mutant library, 5 μL of the digested PCR product was electrotransformed into competent E. coli BAP1 and spread onto a Kan-resistant plate. Using the SrCAR wild type as a control, sterile toothpicks were used to transfer the positive and negative control monoclonal colonies into a 96-well deep-well culture plate containing 400 μL of LB medium. The 400 μL of LB contained 50 μg / mL kanamycin (8 mL / L glycerol). After sealing the membrane, the cultures were shaken at 37 °C and 800 rpm for 12 h.

[0072] 700 μL of TB medium was added to the 96-well deep-well culture plate, and IPTG and kanamycin were added to final concentrations of 0.1 mM and 50 μg / mL, respectively. The cultures were shaken at 30 °C and 800 rpm for 14 h for protein expression and catalyzing the conversion of 3-methoxy-4-hydroxybenzoic acid to 3-methoxy-4-hydroxybenzaldehyde.

[0073] Through preliminary screening, 13 mutants with relatively high activities, namely A317L, F356P, G430Q, W440G, T505A, Y519V, K524P, E533H, A627T, V936L, D998A, M999T, and Q1015Y, were obtained. Their conversion rates of the 3-methoxy-4-hydroxybenzoic acid substrate were all higher than that of the wild type. The results are shown in Table 2.

[0074] Table 2. Yield ratios of 3-methoxy-4-hydroxybenzaldehyde of mutants

[0075] Strain Conversion rate (%) Relative yield (fold) WT 16.05 1.00 A317L 37.37 2.3 F356P 44.93 2.8 G430Q 25.93 1.6 W440G 40.96 2.6 T505A 24.63 1.5 Y519V 34.88 2.2 K524P 38.40 2.4 E533H 22.59 1.4 A627T 22.36 1.4 V936L 27.02 1.7 D998A 18.98 1.2 M999T 16.33 1.0 Q1015Y 20.64 1.3

[0076] In summary, based on the mutation of specific sites of the wild-type carboxylic acid reductase SrCAR, the yield of the obtained mutants was significantly increased, which was 1.0 - 2.8 times that of the starting strain, and the yield of 3-methoxy-4-hydroxybenzaldehyde could be significantly increased.

[0077] Example 2. Construction of a combinatorial saturation mutant library of carboxylic acid reductase SrCAR or obtaining its mutants

[0078] 2.1 Construction of the SrCAR combinatorial mutant library

[0079] Catalytic detection was carried out on single-site saturation mutants at 13 sites. The mutant sites included A317, F356, G430, W440, T505, Y519, K524, E533, A627, V936, D998, M999, and Q1015, which catalyzed the reduction of 3-methoxy-4-hydroxybenzoic acid to 3-methoxy-4-hydroxybenzaldehyde. There were five dominant sites, namely F356, W440, K524, A317, and Y519, with a significant increase in the conversion rate. However, the mutant sites D998, M999, and Q1015 showed poor performance, and the number of mutant residues with a conversion rate higher than that of WT was less than three. According to the spatial structure of the SrCAR protein and the distribution of mutant sites, the 10 previously selected mutant sites were grouped. Near the substrate, Y519, K524, E533, and A627 were divided into Library A; near AMP, G430, W440, and F356 were divided into Library B; in the overall layout, the relatively distant A317, T505, and V936 were divided into Library C. The primer design is shown in Table 3 below.

[0080] Table 3. Combinatorial saturation mutant library constructed based on carboxylic acid reductase SrCAR

[0081]

[0082]

[0083]

[0084] Note: In Table 3, for primers with multiple single-stranded DNAs, M represents A or C, Y represents C or T, S represents C or G, H represents A or C or T, V represents G or A or C, R represents A or G, K represents G or T, W represents A or T, D represents G or A or T, and B represents C or G or T.

[0085] To obtain the carboxylic acid reductase SrCAR mutant Library A, the following experiment was carried out:

[0086] PCR amplification reaction system (50 μL): PrimeSTAR (2×) 25 μL, template SrCAR 1 μL, forward primer (10 μM) 2 μL, reverse primer (10 μM) 2 μL, ddH2O 20 μL. The specific sequences of the forward and reverse primers are shown in Table 1.

[0087] PCR amplification reaction conditions: 98°C: 2 min, (98°C: 10 s, 55°C: 15 s, 72°C: 4 min 30 s) 30 cycles, 72°C: 4 min.

[0088] Overlap extension PCR procedure:

[0089] Round 1: 519-524-533-1, 519-524-533-2, 519-524-533-3, 519-524-533-4, 519-524-533-5, 519-524-533-6, 519-524-533-7, 519-524-533-8, 519-524-533-9, 519-524-533-10, 519-524-533-11, 519-524-533-12, 519-524-533-13, 519-524-533-14, 519-524-533-15, 519-524-533-16, 19-524-533-17, 519-524-533-18 were mixed into the upstream primer F1 in a ratio of 4:2:2:4:2:2:2:1:1:2:1:1:2:1:1:2:1:1. 627-1 and 627-2 were mixed into the downstream primer R1 in a ratio of 1:1. Fragment 1 (about 361 bp) was amplified by F1 and R1.

[0090] Round 2: The gel-extracted fragment 1 was used as a primer, and the whole plasmid was amplified using the plasmid as a template.

[0091] The obtained PCR product was subjected to the following operations: 1 μL of Dpn I enzyme was added to 20 μL of the PCR product to digest the plasmid template, and the treatment was carried out at a constant temperature of 37 °C for 2 h. 5 μL of the digested PCR product was electrotransformed into 100 μL of E. coli BAP1 competent cells. The electrotransformed E. coli BAP1 bacterial solution was evenly spread on an LB plate with kanamycin resistance (concentration 50 μg / mL). After culturing at a constant temperature of 37 °C for 14 h, single colonies grew, which were the engineered strains of the carboxylic acid reductase SrCAR gene mutant. The recombinant plasmid containing the target nucleotide with random or directed mutations in this strain was the expression vector for expressing the carboxylic acid reductase SrCAR gene mutant.

[0092] To obtain the carboxylic acid reductase SrCAR mutant Library B, the following experiment was carried out:

[0093] PCR amplification reaction system (50 μL): PrimeSTAR (2×) 25 μL, template SrCAR 1 μL, forward primer (10 μM) 2 μL, reverse primer (10 μM) 2 μL, ddH2O 20 μL. The specific sequences of the forward and reverse primers are shown in Table 1.

[0094] PCR amplification reaction conditions: 98 °C: 2 min, (98 °C: 10 s, 55 °C: 15 s, 72 °C: 4 min 30 s) 30 cycles, 72 °C: 4 min.

[0095] Overlap extension PCR procedure:

[0096] First round: Mix 356-1, 356-2, and 356-3 in a ratio of 2:1:1 to form the upstream primer F1, and mix 430-440-1, 430-440-2, 430-440-3, 430-440-4, 430-440-5, 430-440-6, 430-440-7, 430-440-8, 430-440-9 in a ratio of 4:2:2:2:1:1:2:1:1 to form the downstream primer R1. Amplify fragment 1 (about 295 bp) with F1 and R1.

[0097] Second round: Gel-purify fragment 1 as the large primer and amplify the entire plasmid using the plasmid as the template.

[0098] Perform the following operations on the obtained PCR product: Add 1 μL of Dpn I enzyme to 20 μL of the PCR product to digest the plasmid template, and incubate at 37 °C for 2 h. Take 5 μL of the digested PCR product and electrotransform it into 100 μL of E. coli BAP1 competent cells. Spread the electrotransformed E. coli BAP1 bacterial solution evenly on an LB plate with kanamycin resistance (concentration 50 μg / mL) and culture at 37 °C for 14 h to grow single colonies, which are the engineering strains of the carboxylic acid reductase SrCAR gene mutant. The recombinant plasmid containing the target nucleotide with random or directed mutations in this strain is the expression vector for expressing the carboxylic acid reductase SrCAR gene mutant.

[0099] To obtain the carboxylic acid reductase SrCAR mutant Library C, perform the following experiment:

[0100] PCR amplification reaction system (50 μL): PrimeSTAR (2×) 25 μL, template SrCAR 1 μL, forward primer (10 μM) 2 μL, reverse primer (10 μM) 2 μL, ddH2O 20 μL. The specific sequences of the forward and reverse primers are shown in Table 1.

[0101] PCR amplification reaction conditions: 98 °C: 2 min, (98 °C: 10 s, 55 °C: 15 s, 72 °C: 4 min 30 s) 30 cycles, 72 °C: 4 min.

[0102] Overlap extension PCR procedure:

[0103] First round: 317-1 and 317-2 are mixed at a ratio of 1:1 to form the upstream primer F1, and 505-1 and 505-2 are mixed at a ratio of 1:2 to form the downstream primer R1. F1 and R1 amplify fragment 1 (about 606 bp); 936-1, 936-2, and 936-3 are mixed at a ratio of 2:1:1 to form the downstream primer R2, and 505-F and R2 amplify fragment 2 (about 1313 bp).

[0104] Second round: Fragment 1 and fragment 2 are recovered by gel cutting as templates, and the upstream primer F1 and the mixed downstream primer R2 are used as the upstream and downstream primers respectively, and fragment 3 is amplified by overlap extension PCR.

[0105] Third round: Fragment 3 is recovered by gel cutting as a large primer, and the whole plasmid is amplified using the plasmid as a template.

[0106] The obtained PCR product is subjected to the following operations: 1 μL of Dpn I enzyme is added to 20 μL of the PCR product to digest the plasmid template, and the treatment is carried out at a constant temperature of 37°C for 2 h. 5 μL of the digested PCR product is electrotransformed into 100 μL of E.coli BAP1 competent cells, and the electrotransformed E.coli BAP1 bacterial solution is evenly spread on an LB plate with kanamycin resistance (concentration 50 μg / mL). After culturing at a constant temperature of 37°C for 14 h, single colonies grow out, which are the engineering strains of the carboxylic acid reductase SrCAR gene mutant. The recombinant plasmid in which the target nucleotide contained in this strain undergoes random mutation or directed mutation is the expression vector for expressing the carboxylic acid reductase SrCAR gene mutant.

[0107] 2.2 Screening of the SrCAR combinatorial mutant library

[0108] After successful sequencing of the combinatorial mutant library, 5 μL of the digested PCR product is electrotransformed into E.coli BAP1 competent cells and spread on a Kan-resistant plate. Using the SrCAR wild type as a control, sterile toothpicks are used to transfer the positive and negative control monoclonal colonies to a 96-well deep-well culture plate containing 400 μL of LB medium. The 400 μL of LB contains 50 μg / mL of kanamycin. After sealing the film, the culture is shaken at 800 rpm at 37°C for 12 h.

[0109] 700 μL of TB medium (the TB medium contains tryptophan with a final concentration of 5 g / L) is added to the 96-well deep-well culture plate, and IPTG and kanamycin are added to make the final concentrations 0.1 mM and 50 μg / mL respectively. The culture is shaken at 800 rpm at 30°C for 14 h for protein expression and catalyzing the conversion of 3-methoxy-4-hydroxybenzoic acid to 3-methoxy-4-hydroxybenzaldehyde.

[0110] After 14 hours of culture, the 96-well deep-well culture plate was centrifuged at 4000 rpm for 30 minutes to carry out high-throughput screening and detection. The reaction solution was added for reaction. After the reaction was completed, the plate was centrifuged and the supernatant was taken for screening of dominant mutants using the glucose oxidase method (△ = OD505 空白 -OD505 Mutant ). Glucose oxidase will oxidize the remaining glucose in the reaction system to generate hydrogen peroxide. The generated hydrogen peroxide will catalyze phenol to generate quinone imine under the catalytic action of phenol oxidase. The maximum absorption peak of quinone imine is at 505nm, and the change in absorbance is proportional to the glucose concentration in the sample.

[0111] The strains that were higher than the wild type and sequenced were activated by streaking on a plate, and a single colony was picked and inoculated into 5 mL of LB test tube medium (containing a final concentration of 50 μg / mL kanamycin), cultured at 37°C 220rpm overnight, and inoculated into 50 mL of TB medium (12 g / L peptone, 24 g / L yeast powder, 8 mL / L glycerol, 2.31 g KH2PO4, 16.43 g K2HPO4) at a 5% inoculation amount, and kanamycin was added at a final concentration of 50 μg / mL. The OD600 was cultured at 37°C 220rpm until it reached about 0.8, and IPTG was added at a final concentration of 0.1 mM. The cells were cultured at 20°C for 14 hours, and the bacterial cells were collected by centrifugation for whole-cell resting catalysis. A reaction solution containing 3-methoxy-4-hydroxybenzoic acid was added to carry out the reaction, and an equal volume of acetonitrile was added after the reaction was completed to terminate the reaction.

[0112] Liquid chromatography (HPLC) detection: 100 μL of fermentation broth was added to 900 μL of ddH2O, centrifuged at 12000 rpm for 5 min, and the supernatant was filtered through a 0.22 μm filter membrane. The content of 3-methoxy-4-hydroxybenzaldehyde was detected using Shimadzu LC-2030.

[0113] Liquid phase detection conditions: chromatographic column Zorbax SB-C18, StableBond Analytical 4.6×150mm, detection wavelength 220nm, column temperature 30°C, flow rate 1mL / min, sample volume 10μL, detection time 14min, mobile phase AddH2O (0.1% TFA), mobile phase B acetonitrile (0.1% TFA), A:B=87:13.

[0114] 3-Methoxy-4-hydroxybenzaldehyde standard to establish a standard curve: prepare 5mmol / L, 2.5mmol / L, 1.25mmol / L, 0.625mmol / L, 0.3125mmol / L, 0.156mmol / L of 3-methoxy-4-hydroxybenzaldehyde standard, measure its absorption peak at 220nm, and establish a standard curve ( Figure 2 ).

[0115] By high performance liquid chromatography (HPLC) detection, the peak emergence times of 3-methoxy-4-hydroxybenzaldehyde were all between 9 min and 9.5 min. The results showed that the ability of the reconstructed recombinant engineering strains to ferment and produce 3-methoxy-4-hydroxybenzaldehyde was improved compared with that of the wild type. Calculate the yield of 3-methoxy-4-hydroxybenzaldehyde according to the standard curve of the standard product. Compared with the wild type, the yield of the mutant was higher than that of the wild type strain. Single colonies were picked and sequenced for identification.

[0116] Through preliminary screening, nine mutants with relatively high activities, namely K524S / A627L, Y519T-K524S-E533W-A627T, Y519V-K524M-E533W, Y519T-K524M-E533H-A627T, Y519T-A627T, Y519T-K524M-E533H-A627T, A317L-V936S, A317Q-V936S, and A317L-V936S, were obtained. Their substrate conversion rates for 3-methoxy-4-hydroxybenzoic acid were higher than that of SrCAR. The results are shown in Table 4.

[0117] Table 4. Yield ratio of 3-methoxy-4-hydroxybenzaldehyde of recombinant engineering strains

[0118] Strain Conversion rate (%) Relative yield (fold) WT 16.10 1.0 K524S / A627L 74.86 4.6 Y519T / K524S / E533W / A627T 27.10 1.7 Y519V / K524M / E533W 25.69 1.6 Y519T / K524M / E533H / A627T 44.25 2.7 Y519T / A627T 39.21 2.4 A317Q / V936S 37.01 2.3 A317L / V936S 67.22 4.2

[0119] In summary, based on the combinatorial saturation mutagenesis of the wild-type carboxylic acid reductase SrCAR, nine multi-site combinatorial mutants were obtained respectively in the present invention. The yields of the obtained mutants were significantly improved, which were 1.6 - 4.6 times that of the starting strain, and the yield of 3-methoxy-4-hydroxybenzaldehyde could be significantly increased.

[0120] Example 3. Obtaining of dominant mutants of carboxylic acid reductase SrCAR by software prediction

[0121] 3.1 Use prediction methods such as Hotspot and Fireprot to predict the dominant mutants of carboxylic acid reductase. Specifically, the HotSpot and Fireprot websites give comprehensive prediction results by integrating several protein crystal data of the same protein structure and the key catalytic residues reported in relevant articles. Select mutants with higher scores according to the prediction scoring results for construction and detection. After resting cell catalysis, it is then analyzed and identified by HPLC, and it is judged whether it is a dominant mutant according to the activity level.

[0122] Select mutants with better performance from the prediction results for construction. The constructed mutants are as follows: K524S / A627L / S800A, K524S / A627L / D873N, H291K / K524S / A627L, N292H / K524S / A627L, K524S / A627L / E1121W, K524S / A627L / H1133Q, K524S / A627L / H1133L, I130C / H181C / K524S / A627L, N292H / K524S / A627L / E1121W. The primer design is shown in Table 5 below.

[0123] Table 5. Combinatorial saturated mutant library constructed based on carboxylic acid reductase SrCAR

[0124]

[0125]

[0126] To obtain the mutant of carboxylic acid reductase SrCAR, the following experiments were carried out:

[0127] PCR amplification reaction system (50 μL): PrimeSTAR (2×) 25 μL, template SrCAR 1 μL, forward primer (10 μM) 2 μL, reverse primer (10 μM) 2 μL, ddH2O 20 μL. The specific sequences of the forward and reverse primers are shown in Table 1.

[0128] PCR amplification reaction conditions: 98°C: 2 min, (98°C: 10 s, 65°C: 15 s, 72°C: 4 min 30 s) 30 cycles, 72°C: 4 min.

[0129] Perform the following operations on the obtained PCR product: Add 1 μL of Dpn I enzyme to 20 μL of the PCR product for digesting the plasmid template, and incubate at 37°C for 2 h. Take 5 μL of the digested PCR product and electrotransform it into 100 μL of E. coli BAP1 competent cells. Spread the electrotransformed E. coli BAP1 bacterial solution evenly on an LB plate with kanamycin resistance (concentration 50 μg / mL), and culture at 37°C for 14 h to grow single colonies, which are the engineering strains of the carboxylic acid reductase SrCAR gene mutant. The recombinant plasmid containing the target nucleotide with random or directed mutation in this strain is the expression vector for expressing the carboxylic acid reductase SrCAR gene mutant.

[0130] The sequenced strains with a titer higher than the wild type were activated by streak plating on a plate. Single colonies were picked and inoculated into a 5 mL LB test tube medium (containing kanamycin at a final concentration of 50 μg / mL), and cultured overnight at 37°C with shaking at 220 rpm. Then, they were inoculated into 50 mL of TB medium (12 g / L peptone, 24 g / L yeast extract, 8 mL / L glycerol, 2.31 g KH2PO4, 16.43 g K2HPO4) at an inoculation amount of 5%, and kanamycin was added at a final concentration of 50 μg / mL. The culture was continued at 37°C with shaking at 220 rpm until the OD600 reached about 0.8. Then, IPTG was added at a final concentration of 0.1 mM, and the culture was continued at 20°C for 14 hours. The cells were collected by centrifugation for whole-cell resting catalysis. A reaction solution containing 3-methoxy-4-hydroxybenzoic acid was added to carry out the reaction. After the reaction was completed, an equal volume of acetonitrile was added to terminate the reaction.

[0131] Detected by high-performance liquid chromatography (HPLC), the peak elution time of 3-methoxy-4-hydroxybenzaldehyde was between 9 min and 9.5 min. The results showed that the ability of the recombinant engineered strains to ferment and produce 3-methoxy-4-hydroxybenzaldehyde was improved compared with the wild type. The yield of 3-methoxy-4-hydroxybenzaldehyde was calculated according to the standard curve of the standard product. Compared with the wild type, the yield of the mutants was higher than that of the wild type strains. Monoclonal colonies with higher yields were picked and sequenced for identification.

[0132] Nine mutants with relatively high activities were obtained by screening. Their substrate conversion rates for 3-methoxy-4-hydroxybenzoic acid were higher than that of SrCAR. The results are shown in Table 6 and the attached figures. Figure 3 It is the yield ratio of the recombinant engineered strain with dominant mutations for 3-methoxy-4-hydroxybenzaldehyde.

[0133] Table 6. Yield ratio of recombinant engineered strains for 3-methoxy-4-hydroxybenzaldehyde

[0134] Strain Conversion rate (%) Relative yield (fold) WT 12.16 1.0 K524S / A627L 34.64 2.8 K524S / A627L / S800A 37.00 3.0 K524S / A627L / D873N 29.89 2.5 H291K / K524S / A627L 23.54 1.9 N292H / K524S / A627L 48.00 3.9 K524S / A627L / E1121W 43.92 3.6 K524S / A627L / H1133Q 33.38 2.7 K524S / A627L / H1133L 28.74 2.4 I130C / H181C / K524S / A627L 27.6 2.3 N292H / K524S / A627L / E1121W 56.94 4.7

[0135] In summary, based on the combinatorial saturation mutagenesis of the wild-type carboxylic acid reductase SrCAR, nine multi-site combinatorial mutants were obtained in this invention. The yields of the obtained mutants were significantly improved, which were 1.9 - 4.7 times that of the starting strain, and could significantly increase the yield of 3-methoxy-4-hydroxybenzaldehyde.

[0136] The above has described the present invention in detail. For those skilled in the art, without departing from the gist and scope of the present invention and without the need for unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modifications, uses, or improvements to the present invention, including those that depart from the scope disclosed in this application but are made by using conventional techniques known in the art. The application of some basic features can be made according to the scope of the appended claims below.

Claims

1. A carboxylic acid reductase mutant, characterized in that, It is a protein obtained by mutating a protein derived from Segniliparus rugosus ), and the mutation is based on the amino acid sequence of the carboxylic acid reductase Sr CAR and has the following mutations: selected from one or several of the following amino acid residue sites: position 130, position 181, position 291, position 292, position 317, position 356, position 430, position 440, position 505, position 519, position 524, position 533, position 627, position 873, position 936, position 1121 and / or position 1133; The carboxylic acid reductase Sr The NCBI accession number of CAR is WP_007468889.

1.

2. The carboxylic acid reductase mutant according to claim 1, wherein There are proteins obtained by any one, any two, any three, any four, any five, any six, any seven, any eight, any nine, any ten, any eleven, any twelve, any thirteen, any fourteen, any fifteen, any sixteen or all of the following seventeen types of transformation mutations: X1. Mutate the isoleucine at position 130 of the carboxylic acid reductase Sr to aspartic acid, cysteine, or lysine; X2. Reduce the carboxylic acid reductase Sr Mutate the histidine at position 181 of CAR to cysteine, methionine, or serine; X3. Reduce carboxylic acid reductase Sr Mutate the histidine at position 291 of CAR to threonine, isoleucine, or lysine; X4. Reduce carboxylic acid reductase Sr Mutate the asparagine at position 292 of CAR to histidine or lysine; X5. Reduce carboxylic acid reductase Sr Mutate the alanine at position 317 of CAR to valine, leucine, or asparagine; X6. Mutate the phenylalanine at position 356 of the carboxylic acid reductase Sr to proline, glutamic acid, or aspartic acid; X7. The carboxylic acid reductase mutates the glycine at position 430 of Sr CAR into glutamine, proline, or lysine; X8, Carboxylic acid reductase mutates the tryptophan at position 440 of Sr CAR to glutamic acid, histidine, or alanine; X9, the carboxylic acid reductase converts Sr the threonine at position 505 of CAR into alanine, methionine, or valine; X10. Mutate the tyrosine at position 519 of the carboxylic acid reductase Sr to valine, threonine, or histidine; X11. Mutate the lysine at position 524 of the carboxylic acid reductase Sr to proline, methionine, or serine; X12. Reduce carboxylic acid reductase Sr Mutate the aspartic acid at position 533 of CAR to histidine, arginine, or tryptophan; X13. Reduce carboxylic acid reductase Sr Mutate the alanine at position 627 of CAR to threonine, valine, or leucine; X14, the aspartic acid at position 873 of the carboxylic acid reductase SrCAR is mutated to asparagine or proline; X15, the valine at position 936 of the carboxylic acid reductase SrCAR is mutated to leucine, serine or lysine; X16, the glutamic acid at position 1121 of the carboxylic acid reductase SrCAR is mutated to glutamine or tryptophan; X17, the histidine at position 1133 of the carboxylic acid reductase SrCAR is mutated to glutamine or leucine.

3. The carboxylic acid reductase mutant according to claim 2, wherein It is one of the following mutants: Reduce carboxylic acid reductase Sr A protein obtained by mutating the 130th site of CAR to cysteine; Reduce carboxylic acid reductase Sr A protein obtained by mutating the 181st site of CAR to cysteine; Reduce carboxylic acid reductase Sr A protein obtained by mutating the 291st site of CAR to lysine; The carboxylic acid reductase Sr A protein obtained by mutating the 292nd site of CAR to histidine; The carboxylic acid reductase Sr A protein obtained by mutating the 317th site of CAR to valine; Carboxylic acid reductase Sr A protein obtained by mutating the 356th site of CAR to proline; A carboxylic acid reductase Sr A protein obtained by mutating the 430th site of CAR to asparagine; Carboxylic acid reductase Sr A protein obtained by mutating the 440th site of CAR to glycine; The carboxylic acid reductase Sr A protein obtained by mutating the 505th site of CAR to alanine; The carboxylic acid reductase Sr A protein obtained by mutating the 519th site of CAR to valine; The carboxylic acid reductase Sr A protein obtained by mutating the 524th site of CAR to proline.

4. The carboxylic acid reductase mutant according to claim 3, wherein It is one of the following mutants: Reduce carboxylic acid reductase Sr A protein obtained by mutating the 519th, 524th, 533rd, and 627th positions of CAR to threonine, serine, tryptophan, and threonine, respectively; Reduce carboxylic acid reductase Sr A protein obtained by mutating the 519th, 524th, and 533rd sites of CAR to valine, methionine, and tryptophan, respectively; Carboxylic acid reductase Sr A protein obtained by mutating the 519th, 524th, 533rd, and 627th sites of CAR to threonine, methionine, histidine, and threonine, respectively; The carboxylic acid reductase Sr A protein obtained by mutating the 519th and 627th positions of CAR to threonine respectively; Carboxylic acid reductase Sr A protein obtained by mutating the 317th and 936th sites of CAR to leucine and serine, respectively; Carboxylic acid reductase Sr A protein obtained by mutating the 317th and 936th sites of CAR to glutamine and serine, respectively; The carboxylic acid reductase Sr A protein obtained by mutating the 524th and 627th sites of CAR to serine and leucine, respectively; Reduce carboxylic acid reductase Sr A protein obtained by mutating the 524th, 627th, and 800th sites of CAR to serine, leucine, and alanine, respectively; Carboxylic acid reductase Sr A protein obtained by mutating the 524th, 627th, and 873rd sites of CAR to serine, leucine, and asparagine, respectively; Carboxylic acid reductase Sr A protein obtained by mutating the 291st, 524th, and 627th sites of CAR to lysine, serine, and leucine, respectively; The carboxylic acid reductase Sr A protein obtained by mutating the 292nd, 524th, and 627th sites of CAR to histidine, serine, and leucine respectively; Reduce carboxylic acid reductase Sr A protein obtained by mutating the 524th, 627th, and 1121st sites of CAR to serine, leucine, and tryptophan, respectively; Carboxylic acid reductase Sr A protein obtained by mutating the 524th, 627th, and 1133rd sites of CAR into serine, leucine, and leucine, respectively; The carboxylic acid reductase Sr A protein obtained by mutating the 524th, 627th, and 1133rd sites of CAR to serine, leucine, and leucine, respectively; Reduce the carboxylic acid reductase Sr A protein obtained by mutating the 130th, 181st, 524th, and 627th sites of CAR to cysteine, cysteine, serine, and leucine, respectively; Reduce carboxylic acid reductase Sr A protein obtained by mutating the 292nd, 524th, 627th, and 1121st sites of CAR to histidine, serine, leucine, and tryptophan, respectively.

5. A coding gene comprising the carboxylic acid reductase mutant according to any one of claims 1 to 4.

6. A recombinant vector containing the coding gene according to claim 4.

7. A recombinant strain containing the recombinant vector according to claim 4.

8. The recombinant strain according to claim 6, characterized in that, The starting bacterium is Escherichia coli, more preferably E.coli BAP1.

9. Use of the carboxylic acid reductase mutant according to any one of claims 1 to 4, the coding gene according to claim 5, the recombinant vector according to claim 6, or the recombinant strain according to claim 7 or 8 in the production of 3-methoxy-4-hydroxybenzaldehyde.

10. A method for producing 3-methoxy-4-hydroxybenzaldehyde, characterized in that, It includes the step of culturing the recombinant strain according to claim 7 or 8 to produce 3-methoxy-4-hydroxybenzaldehyde, and optionally further includes the step of collecting the produced 3-methoxy-4-hydroxybenzaldehyde.

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