A vanillin dehydrogenase mutant and a method for synthesizing furan carboxylic acid by biotransformation of high-concentration 5-hydroxymethylfurfural

Abstract

The present application relates to a kind of vanilla aldehyde dehydrogenase mutant and its method for the biological conversion of high-concentration 5-methylol furfural (HMF) synthesis furan carboxylic acid, vanilla aldehyde dehydrogenase mutant is to the mutation of any one site of 118,245,257,305,275,96 of vanilla aldehyde dehydrogenase Deh, obtain vanilla aldehyde dehydrogenase mutant H118A, L245A, A157T, E305D, Y275W, S96A.Compared with prior art, the vanilla aldehyde dehydrogenase mutant obtained by the present application can catalyze 100mM HMF to be converted into furan carboxylic acid, greatly improve the biosynthesis efficiency of furan carboxylic acid, provide a feasible technical scheme for large-scale biological preparation of furan carboxylic acid, and have important industrial application value.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology and relates to a vanillin dehydrogenase mutant and a method for the biotransformation of high concentrations of 5-hydroxymethylfurfural (HMF) to synthesize furan carboxylic acid. Background Technology

[0002] Furan carboxylic acids (mainly referring to 2-furan carboxylic acid, commonly known as furoic acid) are a class of heterocyclic carboxylic acids produced from furfural, a biomass platform chemical, through air oxidation, electrocatalysis, or enzyme catalytic oxidation. They are widely used in food flavorings, pharmaceutical and pesticide intermediates, and functional materials, and are currently a hot topic in the research of biomass high-value utilization and heterocyclic drugs.

[0003] Currently, the preparation methods of furan carboxylic acid include: (1) Traditional heavy metal oxidation method: using furfural as raw material, using oxidants such as K2Cr2O7 and KMnO4 to react, but a large amount of sludge containing metal ions is generated, which puts great pressure on environmental protection; (2) Alkali-air catalytic oxidation: in an alkaline solution under certain conditions, a catalyst is selected, and air or oxygen is introduced to react, but the amount of salt wastewater is large; (3) Converting the biomass platform molecule 5-hydroxymethylfurfural into furan carboxylic acid through oxidation reaction is the most important research and production route at present, and the technology is becoming increasingly mature.

[0004] Whole-cell catalysis is a method that utilizes dormant or dead cells as catalysts to achieve biocatalytic reactions. The catalytic efficiency of whole-cell catalysis primarily depends on various intracellular enzymes. Unlike traditional chemical catalysis or pure enzyme catalysis, whole-cell catalysis does not require cumbersome enzyme purification or the addition of extra cofactors. It relies on the integrity of the cell and the enzymes and related coenzymes present within the cell to complete the catalytic reaction. Therefore, through cell culture, large quantities of whole-cell catalysts can be obtained in a short time, significantly reducing costs and improving efficiency. Whole-cell catalysis features mild reaction conditions and environmental friendliness. Furthermore, engineered bacteria can be constructed according to different catalytic requirements, exhibiting good specificity and selectivity. Most reaction conditions are relatively mild and can be carried out in water, providing a novel approach to biocatalysis.

[0005] Currently, the biocatalytic production of 2,5-furandicarboxylic acid (FDCA) from 5-hydroxymethylfurfural (HMF) mainly involves two methods: whole-cell catalysis and multi-enzyme combined systems. The core of both intracellular and in vitro enzyme catalysis lies in the catalytic efficiency and stability of the enzyme protein. However, existing HMF oxidases have certain shortcomings in both aspects, leading to problems such as low reaction efficiency, difficulty in control, low yield of the target product, and high pressure for subsequent separation and purification in current biocatalytic FDCA production methods. Summary of the Invention

[0006] The purpose of this invention is to overcome the problems of low reaction efficiency, difficulty in control, and low yield of target products in the existing technology, and to provide a method for synthesizing furan carboxylic acid from a vanillin dehydrogenase mutant and its biotransformation of high concentration of 5-hydroxymethylfurfural (HMF).

[0007] The objective of this invention can be achieved through the following technical solutions: One of the technical solutions of the present invention is to provide a vanillin dehydrogenase mutant, wherein the vanillin dehydrogenase Deh is mutated, and the mutant is selected from any one of the following 1) to 6). 1) Vanillin dehydrogenase mutant H118A: Histidine (H) at position 118 is mutated to alanine (A). 2) Vanillin dehydrogenase mutant L245A: Leucine (L) at position 245 is mutated to alanine (A). 3) Vanillin dehydrogenase mutant A157T: The alanine (A) at position 157 is mutated to threonine (T). 4) Vanillin dehydrogenase mutant E305D: Glutamic acid (E) at position 305 is mutated to aspartic acid (D). 5) Vanillin dehydrogenase mutant Y275W: Tyrosine (Y) at position 275 is mutated to tryptophan (W); 6) Vanillin dehydrogenase mutant S96A: The serine (S) at position 96 is mutated to alanine (A). The amino acid sequences of vanillin dehydrogenase Deh, vanillin dehydrogenase mutant H118A, vanillin dehydrogenase mutant L245A, vanillin dehydrogenase mutant A157T, vanillin dehydrogenase mutant E305D, vanillin dehydrogenase mutant Y275W, and vanillin dehydrogenase mutant S96A are shown in SEQ ID NO. 1~7, respectively.

[0008] The second technical solution of the present invention is to provide a biomaterial related to the vanillin dehydrogenase mutant as described in one of the above technical solutions, characterized in that the biomaterial is any one of the following: a) Nucleotides encoding vanillin dehydrogenase mutants as described in one of the above technical solutions; b) A recombinant expression vector containing the nucleotides described in a); c) Bioengineered bacteria containing the nucleotides described in a), or bioengineered bacteria containing the recombinant expression vector described in b).

[0009] In some specific embodiments, the nucleotide sequences of the encoding genes of the vanillin dehydrogenase mutants H118A, L245A, A157T, E305D, Y275W, and S96A are shown in SEQ ID NO. 8-13, respectively.

[0010] The third technical solution of the present invention is to provide a vanillin dehydrogenase mutant as described in the first technical solution above, or a biomaterial as described in the second technical solution above, for the biotransformation of 5-hydroxymethylfurfural into furan carboxylic acid.

[0011] The fourth technical solution of the present invention provides a method for the biotransformation of high-concentration 5-hydroxymethylfurfural to synthesize furan carboxylic acid, comprising the following steps: S1. Culture bioengineered bacteria containing nucleotides encoding vanillin dehydrogenase mutants, add IPTG as an inducer, collect the bacterial cells, and centrifuge to collect the bacterial cells; S2. The bacterial cells obtained in step S1 are resuspended in buffer solution to prepare a cell suspension, 5-hydroxymethylfurfural is added, and a transformation reaction is carried out to obtain furan carboxylic acid.

[0012] In some specific embodiments, in step S1, the bioengineered bacteria containing the nucleotide encoding the vanillin dehydrogenase mutant is Escherichia coli; When the bacterial solution OD 600 When the value reaches 0.6-0.8, add the inducing agent IPTG to 0.4 mM.

[0013] In some specific implementations, in step S1, the centrifugation conditions are 4°C and 8000 rpm.

[0014] In some specific embodiments, in step S2, when converting 5-hydroxymethylfurfural to a final concentration of 100 mM, the concentration range of the cell suspension is 10~140 g / L.

[0015] In some specific implementations, the conversion temperature in step S2 is 20~45℃.

[0016] In some specific implementations, the pH during the conversion in step S2 is 5.5 to 9.0.

[0017] Compared with the prior art, the present invention has the following advantages: This invention modifies the vanillin dehydrogenase gene Deh from Klebsiella pneumoniae, constructing vanillin dehydrogenase mutants H118A, L245A, A157T, E305D, Y275W, and S96A, successfully creating engineered bacteria that produce high levels of adenine. The whole-cell catalytic conditions of this engineered bacteria were optimized to achieve HMF tolerance of 100 mM. Existing wild-type dehydrogenases, oxidases, and engineered bacterial systems generally exhibit HMF tolerance of 10–30 mM; above this concentration, enzyme activity rapidly declines, bacterial cell inhibition is severe, and byproducts increase. The tolerance concentration of this invention is 3–10 times higher than conventional levels, significantly reducing substrate costs and increasing unit yield. This provides a feasible technical solution for the large-scale bioproduction of furanyl carboxylic acids and has significant industrial application value. Attached Figure Description

[0018] Figure 1 To illustrate the product accumulation of the vanillin dehydrogenase mutant S96A at different cell concentrations in this invention; Figure 2 The product accumulation of the vanillin dehydrogenase mutant S96A at different temperatures in this invention; Figure 3 This shows the product accumulation of the vanillin dehydrogenase mutant S96A at different pH values ​​according to the present invention. Figure 4 This shows the product accumulation of different vanillin dehydrogenase mutants of the present invention. Detailed Implementation

[0019] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0021] Unless otherwise specified, the materials and processes described in the following embodiments or examples are conventional materials and processes used in the art to achieve the corresponding functions.

[0022] Example 1: This embodiment provides vanillin dehydrogenase mutants H118A, L245A, A157T, E305D, Y275W, and S96A. Taking vanillin dehydrogenase mutant H118A as an example, the preparation method includes the following: (1) The 118th histidine (H) of the vanillin dehydrogenase Deh with the amino acid sequence as described in SEQ ID NO.1 was used as the target mutation site and mutated to alanine (A). Therefore, a pair of complementary primers H118A-F and H118A-R with the desired codon change were designed.

[0023] (2) Using the gene encoding vanillin dehydrogenase Deh as a template, site-directed mutagenesis PCR was performed, and the methylated template was removed. The digestion product was then directly converted to... E. coli BL21 (DE3) was selected as a single clone and sent for sequencing to confirm that the target site had been correctly mutated, thus obtaining the vanillin dehydrogenase mutant H118A.

[0024] The methods for preparing vanillin dehydrogenase mutants L245A, A157T, E305D, Y75W, and S96A are the same as those for vanillin dehydrogenase mutant H118A, with the only difference being the mutation sites and complementary primers.

[0025] Vanillin dehydrogenase mutant L245A: The 245th leucine (L) of the vanillin dehydrogenase with the amino acid sequence as described in SEQ ID NO.1 was used as the target mutation site and mutated to alanine (A). Complementary primers L245A-F and L245A-R were designed.

[0026] Vanillin dehydrogenase mutant A157T: The alanine (A) at position 157 of the vanillin dehydrogenase as described in SEQ ID NO.1 was used as the target mutation site and mutated to threonine (T). Complementary primers A157T-F and A157T-R were designed.

[0027] Vanillin dehydrogenase mutant E305D: The glutamic acid (E) at position 305 of the vanillin dehydrogenase as described in SEQ ID NO.1 was used as the target mutation site and mutated to aspartic acid (D). Complementary primers E305D-F and E305D-R were designed.

[0028] Vanillin dehydrogenase mutant Y275W: The tyrosine (Y) at position 275 of the vanillin dehydrogenase as described in SEQ ID NO.1 was used as the target mutation site and mutated to tryptophan (W). Complementary primers Y275W-F and Y275W-R were designed. Vanillin dehydrogenase mutant S96A: The 96th serine (S) of the vanillin dehydrogenase with the amino acid sequence as described in SEQ ID NO.1 was used as the target mutation site and mutated to alanine (A). Complementary primers S96A-F and S96A-R were designed.

[0029] The nucleotide sequence of the gene encoding vanillin dehydrogenase Deh is shown in SEQ ID NO.14. The nucleotide sequences of H118A-F and H118A-R are shown in SEQ ID NO.15 and 16, respectively; The nucleotide sequences of L245A-F and L245A-R are shown in SEQ ID NO.17 and 18, respectively; The nucleotide sequences of A157T-F and A157T-R are shown in SEQ ID NO.19 and 20, respectively; The nucleotide sequences of E305D-F and E305D-R are shown in SEQ ID NO.21 and 22, respectively; The nucleotide sequences of Y275W-F and Y275W-R are shown in SEQ ID NO.23 and 24, respectively; The nucleotide sequences of S96A-F and S96A-R are shown in SEQ ID NO.25 and 26, respectively.

[0030] Example 2: In this embodiment, the vanillin dehydrogenase mutants H118A, L245A, A157T, E305D, Y275W, and S96A obtained in Example 1 were used to construct bioengineered bacteria. E. coli BL21 (DE3), with a wild-type (WT) control, was used for whole-cell catalysis. The method was as follows: (1) 10 μL of the bacterial culture containing nucleotides encoding the vanillin dehydrogenase mutant was inoculated into liquid LB medium (4.7 mL LB, 5 μL kanamycin) and cultured with shaking at 30 °C and 220 rpm for 16 h. Then, 1% (1.66 mL) of the seed culture was transferred to a 250 mL baffle flask containing 150 mL TB medium (prepared with 16.5 mL phosphate buffer and 167 μL kanamycin before inoculation). The culture was incubated at 30 °C and 220 rpm until the bacterial culture reached OD... 600 When the concentration reaches approximately 0.7, add IPTG to a final concentration of 0.4 mM and continue culturing at 160 rpm for about 3 minutes. Collect the cells by centrifugation at 8000 rpm at 4°C, and wash twice with 0.85% (w / v) sterile physiological saline. Then, collect samples by centrifugation (12000 rpm, 4°C, 10 minutes). Resuspend the cells in buffer at a specific OD value. 600 Dispense the solution into 25mL flat-bottomed conical flasks, with each flask containing 10mL of liquid.

[0031] (2) After centrifugation, the cells were resuspended in 50 mM sodium phosphate buffer (pH 7.0) to a dry weight of 100 g / L. 5-hydroxymethylfurfural (HMF) was added to a final concentration of 100 mM. The cells were transformed at 30 °C for 24 h. The yields of the products 2,5-furandicarboxylic acid (FDCA), 5-hydroxymethyl-2-furancarboxylic acid (HMFCA), and 5-formyl-2-furancarboxylic acid (FFCA) were determined.

[0032] like Figure 4 As shown, the vanillin dehydrogenase mutant S96A catalyzed the highest yield of HMF to FDCA synthesis, at 42.57 mmol / L, while the intermediate products HMFCA and FFCA were also produced in lower amounts, at 39.32 mmol / L and 3.25 mmol / L, respectively. This indicates that the vanillin dehydrogenase mutant S96A is the most suitable mutant.

[0033] Example 3: This embodiment uses the vanillin dehydrogenase mutant S96A as an example to optimize the whole-cell catalytic conditions, including optimization of cell concentration, temperature, and pH.

[0034] (1) Optimization of cell concentration: After centrifugation, the cells were resuspended in 50 mM sodium phosphate buffer (pH 7.0) to concentrations of 10, 20, 40, 60, 80, 100, 120 and 140 g / L (corresponding to OD values ​​respectively). 600 =30, 40, 50, 60, 70, 80, 90, 100), add HMF to a final concentration of 100 mM, convert at 30℃ for 24 h, and determine the yield of products FDCA, HMFCA, and FFCA.

[0035] like Figure 1 As shown, under conditions of approximately 100 g / L cell dry weight (OD) 600 At a concentration of 80%, the highest yield of FDCA was observed (56.56 mmol / L), while the lowest yields of intermediates HMFCA and FFCA were observed (36.40 mmol / L and 3.38 mmol / L, respectively). These results indicate that at this cell concentration, the whole-cell catalytic system exhibited the highest oxidation efficiency for HMF and the least accumulation of byproducts.

[0036] (2) Temperature optimization: Under the above optimal cell concentration conditions, the cells collected by centrifugation were resuspended in 50 mM sodium phosphate buffer (pH 7.0), and HMF was added to a final concentration of 100 mM. Then, the cells were transformed at 20, 25, 30, 35, 40 and 45 °C for 24 h, respectively, and the yields of FDCA, HMFCA and FFCA were measured.

[0037] like Figure 2As shown, at 30℃, the yield of product FDCA was the highest, at 65.25 mmol / L, while the yields of intermediate products HMFCA and FFCA were the lowest, at 30.55 mmol / L and 2.57 mmol / L, respectively.

[0038] (3) pH optimization: Under the optimal cell concentration and temperature conditions described above, the centrifuged cells were resuspended in buffers with different pH values ​​(5.0~9.0). Specifically, acetate-sodium acetate buffer was used for pH 5.0, NaH2PO4-Na2HPO4 buffer for pH 7.0, Tris-HCl buffer for pH 8.0, and glycine-sodium hydroxide buffer for pH 9.0. HMF was added to a final concentration of 100 mM, and the cells were transformed at 30°C for 24 h. The yields of FDCA, HMFCA, and FFCA were then measured.

[0039] like Figure 3 As shown, under pH 7.0 conditions, the yield of product FDCA was the highest at 72.08 mmol / L, while the yields of intermediate products HMFCA and FFCA were the lowest at 16.68 mmol / L and 3.71 mmol / L, respectively.

[0040] The sequence information involved in this invention is as follows: SEQ ID NO.1 (Amino acid sequence of vanillin dehydrogenase Deh): MATLRAGLLGVGMMGRHHARVLGEVEGVELVAVADPGGDRFGVAGDLPILPDVDALIDAGIDIAVVAVPTGFHEEAALRLAAAGVHTLIEKPIAHSVEAGRRIVDAFADANLVGAVGHIERFNPALQELRRRLEAGELGAVYQIQTRRQGPFPARIADVGVAKD LASHDVDLTAWVAQSEYSSVFAQTAFKSGREYEDLIAITGQLESGIIVNHLVNWLNPLKERVTIVTGERGTFVADTGSGDLTFYANGTVATNEWDQVAAFRGVSEGDVTRYAFVKREPLRVEHEAFRDAVLGKSSDVVTLEQGLRTLEVVEAALGSAHKGEVARL SEQ ID NO.2 (Amino acid sequence of vanillin dehydrogenase mutant H118A): MATLRAGLLGVGMMGRHHARVLGEVEGVELVAVADPGGDRFGVAGDLPILPDVDALIDAGIDIAVVAVPTGFHEEAALRLAAAGVHTLIEKPIAHSVEAGRRIVDAFADANLVGAVG A IERFNPALQELRRRLEAGELGAVYQIQTRRQGPFPARIADVGVAKDLASHDVDLTAWVAQSEYSSVFAQTAFKSGREYEDLIAITGQLESGIIVNHLVNWLNPLKERVTIVTGERGTFVADTGSGDLTFYANGTVATNEWDQVAAFRGVSEGDVTRYAFVKREPLRVEHEAFRDAVLGKSSDVVTLEQGLRTLEVVEAALGSAHKGEVARL SEQ ID NO.3 (Amino acid sequence of vanillin dehydrogenase mutant L245A): MATLRAGLLGVGMMGRHHARVLGEVEGVELVAVADPGGDRFGVAGDLPILPDVDALIDAGIDIAVVAVPTGFHEEAALRLAAAGVHTLIEKPIAHSVEAGRRIVDAFADANLVGAVGHIERFNPALQELRRRLEAGELGAVYQIQTRRQGPFPARIADVGVAKDLASHDVDLTAWVAQSEYSSVFAQTAFKSGREYEDLIAITGQLESGIIVNHLVNWLNPLKERVTIVTGERGTFVADTGSGD A TFYANGTVATNEWDQVAAFRGVSEGDVTRYAFVKREPLRVEHEAFRDAVLGKSSDVVTLEQGLRTLEVVEAALGSAHKGEVARL SEQ ID NO.4 (Amino acid sequence of vanillin dehydrogenase mutant A157T): MATLRAGLLGVGMMGRHHARVLGEVEGVELVAVADPGGDRFGVAGDLPILPDVDALIDAGIDIAVVAVPTGFHEEAALRLAAAGVHTLIEKPIAHSVEAGRRIVDAFADANLVGAVGHIERFNPALQELRRRLEAGELGAVYQIQTRRQGPFPARI TDVGVAKDLASHDVDLTAWVAQSEYSSVFAQTAFKSGREYEDLIAITGQLESGIIVNHLVNWLNPLKERVTIVTGERGTFVADTGSGDLTFYANGTVATNEWDQVAAFRGVSEGDVTRYAFVKREPLRVEHEAFRDAVLGKSSDVVTLEQGLRTLEVVEAALGSAHKGEVARL SEQ ID NO.5 (Amino acid sequence of vanillin dehydrogenase mutant E305D): MATLRAGLLGVGMMGRHHARVLGEVEGVELVAVADPGGDRFGVAGDLPILPDVDALIDAGIDIAVVAVPTGFHEEAALRLAAAGVHTLIEKPIAHSVEAGRRIVDAFADANLVGAVGHIERFNPALQELRRRLEAGELGAVYQIQTRRQGPFPARIADVGVAKDLASHDVDLTAWVAQSEYSSVFAQTAFKSGREYEDLIAITGQLESGIIVNHLVNWLNPLKERVTIVTGERGTFVADTGSGDLTFYANGTVATNEWDQVAAFRGVSEGDVTRYAFVKREPLRVEHEAFRDAVLGKSSDVVTL D QGLRTLEVVEAALGSAHKGEVARL SEQ ID NO.6 (Amino acid sequence of vanillin dehydrogenase mutant Y275W): MATLRAGLLGVGMMGRHHARVLGEVEGVELVAVADPGGDRFGVAGDLPILPDVDALIDAGIDIAVVAVPTGFHEEAALRLAAAGVHTLIEKPIAHSVEAGRRIVDAFADANLVGAVGHIERFNPALQELRRRLEAGELGAVYQIQTRRQGPFPARIADVGVAKDLASHDVDLTAWVAQSEYSSVFAQTAFKSGREYEDLIAITGQLESGIIVNHLVNWLNPLKERVTIVTGERGTFVADTGSGDLTFYANGTVATNEWDQVAAFRGVSEGDVTR W AFVKREPLRVEHEAFRDAVLGKSSDVVTLEQGLRTLEVVEAALGSAHKGEVARL SEQ ID NO.7 (Amino acid sequence of vanillin dehydrogenase mutant S96A): MATLRAGLLGVGMMGRHHARVLGEVEGVELVAVAVDPGGDRFGVAGDLPILPDVDALIDAGIDIAVVAVPTGFHEEAALRLAAAGVHTLIEKPIAH A VEAGRRIVDAFADANLVGAVGHIERFNPALQELRRRLEAGELGAVYQIQTRRQGPFPARIADVGVAKDLASHDVDLTAWVAQSEYSSVFAQTAFKSGREYEDLIAITGQLESGIIV NHLVNWLNPLKERVTIVTGERGTFVADTGSGDLTFYANGTVATNEWDQVAAFRGVSEGDVTRYAFVKREPLRVEHEAFRDAVLGKSSDVVTLEQGLRTLEVVEAALGSAHKGEVARL SEQ ID NO.14 (nucleotide sequence of the gene encoding vanillin dehydrogenase Deh): ATGGCAACTCTGAGAGCAGGATTGCTTGGCGTCGGCATGATGGGCAGGCACCATGCACGTGTGTTAGGTGAGGTAGAGGGGGTTGAACTCGTAGCTGTTGCTGATCCAGGAGGAGATAGATTCGGGGTTGCGGGAGACCTTCCTATCCTGCCAGATGTTGACGCATTGATCGATGCTGGCATCGATATTGCGGTTGTGGCTGTGCCTACGGGATTTCACGAGGAGGCTGCACTTCGGTTGGCCGCTGCAGGCGTGCATACGTTAATTGAGAAGCCGATTGCACACTCGGTAGAAGCTGGACGGCGTATCGTCGATGCGTTCGCAGACGCTAATCTTGTTGGGGCGGTTGGTCATATTGAGCGCTTTAACCCTGCACTGCAGGAACTGCGTCGTCGGTTGGAAGCAGGAGAATTGGGTGCTGTCTATCAGATTCAGACCCGTAGGCAAGGACCGTTCCCTGCACGCATTGCGGATGTTGGAGTGGCCAAAGATCTGGCTTCACATGATGTGGATTTGACTGCTTGGGTGGCCCAGTCAGAATATTCTTCCGTTTTCGCGCAAACCGCCTTCAAATCAGGCCGGGAGTACGAGGACCTGATTGCTATTACTGGCCAGCTCGAATCAGGAATTATTGTCAATCATCTCGTTAACTGGCTCAACCCTCTTAAAGAACGGGTAACGATTGTTACGGGTGAACGGGGAACCTTTGTAGCTGACACTGGATCGGGTGACCTCACCTTCTACGCCAATGGAACTGTTGCCACCAATGAGTGGGATCAAGTCGCCGCGTTTCGTGGTGTCAGCGAGGGCGATGTGACACGGTATGCGTTTGTCAAGCGAGAGCCACTACGGGTCGAACATGAGGCATTCCGCGATGCCGTGCTTGGCAAGTCATCGGATGTGGTGACGCTGGAGCAGGGGTTGCGAACACTTGAAGTAGTTGAGGCGGCACTGGGATCAGCGCATAAAGGCGAAGTAGCAAGGTTATAG SEQ ID NO.8 (nucleotide sequence of the gene encoding vanillin dehydrogenase mutant H118A): ATGGCAACTCTGAGAGCAGGATTGCTTGGCGTCGGCATGATGGGCAGGCACCATGCACGTGTGTTAGGTGAGGTAGAGGGGGTTGAACTCGTAGCTGTTGCTGATCCAGGAGGAGATAGATTCGGGGTTGCGGGAGACCTTCCTATCCTGCCAGATGTTGACGCATTGATCGATG CTGGCATCGATATTGCGGTTGTGGCTGTGCCTACGGGATTTCACGAGGAGGCTGCACTTCGGTTGGCCGCTGCAGGCGTGCATACGTTAATTGAGAAGCCGATTGCACACTCGGTAGAAGCTGGACGGCGTATCGTCGATGCGTTCGCAGACGCTAATCTTGTTGGGGCGGTTGGT GCGATTGAGCGCTTTAACCCTGCACTGCAGGAACTGCGTCGTCGGTTGGAAGCAGGAGAATTGGGTGCTGTCTATCAGATTCAGACCCGTAGGCAAGGACCGTTCCCTGCACGCATTGCGGATGTTGGAGTGGCCAAAGATCTGGCTTCACATGATGTGGATTTGACTGCTTGGGTGGCCCAGTCAGAATATTCTTCCGTTTTCGCGCAAACCGCCTTCAAATCAGGCCGGGAGTACGAGGACCTGATTGCTATTACTGGCCAGCTCGAATCAGGAATTATTGTCAATCATCTCGTTAACTGGCTCAACCCTCTTAAAGAACGGGTAACGATTGTTACGGGTGAACGGGGAACCTTTGTAGCTGACACTGGATCGGGTGACCTCACCTTCTACGCCAATGGAACTGTTGCCACCAATGAGTGGGATCAAGTCGCCGCGTTTCGTGGTGTCAGCGAGGGCGATGTGACACGGTATGCGTTTGTCAAGCGAGAGCCACTACGGGTCGAACATGAGGCATTCCGCGATGCCGTGCTTGGCAAGTCATCGGATGTGGTGACGCTGGAGCAGGGGTTGCGAACACTTGAAGTAGTTGAGGCGGCACTGGGATCAGCGCATAAAGGCGAAGTAGCAAGGTTATAG SEQ ID NO.9 (Nucleotide sequence of the coding gene of vanillin dehydrogenase mutant L245A): ATGGCAACTCTGAGAGCAGGATTGCTTGGCGTCGGCATGATGGGCAGGCACCATGCACGTGTGTTAGGTGAGGTAGAGGGGGTTGAACTCGTAGCTGTTGCTGATCCAGGAGGAGATAGATTCGGGGTTGCGGGAGACCTTCCTATCCTGCCAGATGTTGACGCATTGATCGATGCTGGCATCGATATTGCGGTTGTGGCTGTGCCTACGGGATTTCACGAGGAGGCTGCACTTCGGTTGGCCGCTGCAGGCGTGCATACGTTAATTGAGAAGCCGATTGCACACTCGGTAGAAGCTGGACGGCGTATCGTCGATGCGTTCGCAGACGCTAATCTTGTTGGGGCGGTTGGTCATATTGAGCGCTTTAACCCTGCACTGCAGGAACTGCGTCGTCGGTTGGAAGCAGGAGAATTGGGTGCTGTCTATCAGATTCAGACCCGTAGGCAAGGACCGTTCCCTGCACGCATTGCGGATGTTGGAGTGGCCAAAGATCTGGCTTCACATGATGTGGATTTGACTGCTTGGGTGGCCCAGTCAGAATATTCTTCCGTTTTCGCGCAAACCGCCTTCAAATCAGGCCGGGAGTACGAGGACCTGATTGCTATTACTGGCCAGCTCGAATCAGGAATTATTGTCAATCATCTCGTTAACTGGCTCAACCCTCTTAAAGAACGGGTAACGATTGTTACGGGTGAACGGGGAACCTTTGTAGCTGACACTGGATCGGGTGAC GCG ACCTTCTACGCCAATGGAACTGTTGCCACCAATGAGTGGGATCAAGTCGCCGCGTTTCGTGGTGTCAGCGAGGGCGATGTGACACGGTATGCGTTTGTCAAGCGAGAGCCACTACGGGTCGAACATGAGGCATTCCGCGATGCCGTGCTTGGCAAGTCATCGGATGTGGTGACGCTGGAGCAGGGGTTGCGAACACTTGAAGTAGTTGAGGCGGCACTGGGATCAGCGCATAAAGGCGAAGTAGCAAGGTTATAG SEQ ID NO.10 (Nucleotide sequence of the coding gene of vanillin dehydrogenase mutant A157T): ATGGCAACTCTGAGAGCAGGATTGCTTGGCGTCGGCATGATGGGCAGGCACCATGCACGTGTGTTAGGTGAGGTAGAGGGGGTTGAACTCGTAGCTGTTGCTGATCCAGGAGGAGATAGATTCGGGGTTGCGGGAGACCTTCCTATCCTGCCAGATGTTGACGCATTGATCGATGCTGGCATCGATATTGCGGTTGTGGCTGTGCCTACGGGATTTCACGAGGAGGCTGCACTTCGGTTGGCCGCTGCAGGCGTGCATACGTTAATTGAGAAGCCGATTGCACACTCGGTAGAAGCTGGACGGCGTATCGTCGATGCGTTCGCAGACGCTAATCTTGTTGGGGCGGTTGGTCATATTGAGCGCTTTAACCCTGCACTGCAGGAACTGCGTCGTCGGTTGGAAGCAGGAGAATTGGGTGCTGTCTATCAGATTCAGACCCGTAGGCAAGGACCGTTCCCTGCACGCATT ACCGATGTTGGAGTGGCCAAAGATCTGGCTTCACATGATGTGGATTTGACTGCTTGGGTGGCCCAGTCAGAATATTCTTCCGTTTTCGCGCAAACCGCCTTCAAATCAGGCCGGGAGTACGAGGACCTGATTGCTATTACTGGCCAGCTCGAATCAGGAATTATTGTCAATCATCTCGTTAACTGGCTCAACCCTCTTAAAGAACGGGTAACGATTGTTACGGGTGAACGGGGAACCTTTGTAGCTGACACTGGATCGGGTGACCTCACCTTCTACGCCAATGGAACTGTTGCCACCAATGAGTGGGATCAAGTCGCCGCGTTTCGTGGTGTCAGCGAGGGCGATGTGACACGGTATGCGTTTGTCAAGCGAGAGCCACTACGGGTCGAACATGAGGCATTCCGCGATGCCGTGCTTGGCAAGTCATCGGATGTGGTGACGCTGGAGCAGGGGTTGCGAACACTTGAAGTAGTTGAGGCGGCACTGGGATCAGCGCATAAAGGCGAAGTAGCAAGGTTATAG SEQ ID NO.11 (Nucleotide sequence of the encoding gene of vanillin dehydrogenase mutant E305D): ATGGCAACTCTGAGAGCAGGATTGCTTGGCGTCGGCATGATGGGCAGGCACCATGCACGTGTGTTAGGTGAGGTAGAGGGGGTTGAACTCGTAGCTGTTGCTGATCCAGGAGGAGATAGATTCGGGGTTGCGGGAGACCTTCCTATCCTGCCAGATGTTGACGCATTGATCGATGCTGGCATCGATATTGCGGTTGTGGCTGTGCCTACGGGATTTCACGAGGAGGCTGCACTTCGGTTGGCCGCTGCAGGCGTGCATACGTTAATTGAGAAGCCGATTGCACACTCGGTAGAAGCTGGACGGCGTATCGTCGATGCGTTCGCAGACGCTAATCTTGTTGGGGCGGTTGGTCATATTGAGCGCTTTAACCCTGCACTGCAGGAACTGCGTCGTCGGTTGGAAGCAGGAGAATTGGGTGCTGTCTATCAGATTCAGACCCGTAGGCAAGGACCGTTCCCTGCACGCATTGCGGATGTTGGAGTGGCCAAAGATCTGGCTTCACATGATGTGGATTTGACTGCTTGGGTGGCCCAGTCAGAATATTCTTCCGTTTTCGCGCAAACCGCCTTCAAATCAGGCCGGGAGTACGAGGACCTGATTGCTATTACTGGCCAGCTCGAATCAGGAATTATTGTCAATCATCTCGTTAACTGGCTCAACCCTCTTAAAGAACGGGTAACGATTGTTACGGGTGAACGGGGAACCTTTGTAGCTGACACTGGATCGGGTGACCTCACCTTCTACGCCAATGGAACTGTTGCCACCAATGAGTGGGATCAAGTCGCCGCGTTTCGTGGTGTCAGCGAGGGCGATGTGACACGGTATGCGTTTGTCAAGCGAGAGCCACTACGGGTCGAACATGAGGCATTCCGCGATGCCGTGCTTGGCAAGTCATCGGATGTGGTGACGCTG GAC CAGGGGTTGCGAACACTTGAAGTAGTTGAGGCGGCACTGGGATCAGCGCATAAAGGCGAAGTAGCAAGGTTATAG SEQ ID NO.12 (Nucleotide sequence of the coding gene of vanillin dehydrogenase mutant Y275W): ATGGCAACTCTGAGAGCAGGATTGCTTGGCGTCGGCATGATGGGCAGGCACCATGCACGTGTGTTAGGTGAGGTAGAGGGGGTTGAACTCGTAGCTGTTGCTGATCCAGGAGGAGATAGATTCGGGGTTGCGGGAGACCTTCCTATCCTGCCAGATGTTGACGCATTGATCGATGCTGGCATCGATATTGCGGTTGTGGCTGTGCCTACGGGATTTCACGAGGAGGCTGCACTTCGGTTGGCCGCTGCAGGCGTGCATACGTTAATTGAGAAGCCGATTGCACACTCGGTAGAAGCTGGACGGCGTATCGTCGATGCGTTCGCAGACGCTAATCTTGTTGGGGCGGTTGGTCATATTGAGCGCTTTAACCCTGCACTGCAGGAACTGCGTCGTCGGTTGGAAGCAGGAGAATTGGGTGCTGTCTATCAGATTCAGACCCGTAGGCAAGGACCGTTCCCTGCACGCATTGCGGATGTTGGAGTGGCCAAAGATCTGGCTTCACATGATGTGGATTTGACTGCTTGGGTGGCCCAGTCAGAATATTCTTCCGTTTTCGCGCAAACCGCCTTCAAATCAGGCCGGGAGTACGAGGACCTGATTGCTATTACTGGCCAGCTCGAATCAGGAATTATTGTCAATCATCTCGTTAACTGGCTCAACCCTCTTAAAGAACGGGTAACGATTGTTACGGGTGAACGGGGAACCTTTGTAGCTGACACTGGATCGGGTGACCTCACCTTCTACGCCAATGGAACTGTTGCCACCAATGAGTGGGATCAAGTCGCCGCGTTTCGTGGTGTCAGCGAGGGCGATGTGACACGG TGGGCGTTTGTCAAGCGAGAGCCACTACGGGTCGAACATGAGGCATTCCGCGATGCCGTGCTTGGCAAGTCATCGGATGTGGTGACGCTGGAGCAGGGGTTGCGAACACTTGAAGTAGTTGAGGCGGCACTGGGATCAGCGCATAAAGGCGAAGTAGCAAGGTTATAG SEQ ID NO.13 (Nucleotide sequence of the coding gene of vanillin dehydrogenase mutant S96A): ATGGCAACTCTGAGAGCAGGATTGCTTGGCGTCGGCATGATGGGCAGGCACCATGCACGTGTGTTAGGTGAGGTAGAGGGGGTTGAACTCGTAGCTGTTGCTGATCCAGGAGGAGATAGATTCGGGGTTGCGGGAGACCTTCCTATCCTGCCAGATGTTGACGCATTGATCGATGCTGGCATCGATATTGCGGTTGTGGCTGTGCCTACGGGATTTCACGAGGAGGCTGCACTTCGGTTGGCCGCTGCAGGCGTGCATACGTTAATTGAGAAGCCGATTGCACAC GCGGTAGAAGCTGGACGGCGTATCGTCGATGCGTTCGCAGACGCTAATCTTGTTGGGCGGTTGGTCATATTGAGCGCTTTAACCCTGCACTGCAGGAACTGCGTCGGTTGGAAGCAGGAATTGGGTGCTGTCTATCAGATTCAGACCCGTAGGCAAGGACCGTTCCCTGCCAGCATTGCGGATGTTGGAGTGGGCCAAAGATCTGGGCTTCACATGATGTGATTTGACTGCTTGGTGGCCAGTCAGAATATTTCTCCGTTTTCGCGCAAACCGCCTTCAAATCAGGCCGGGAGTCAGAGACCTGATTGCTATTACTGGGCCAGCTCGAATCAGGAATTATTGTCAAT CATCTCGTTAACTGGCTCAACCCTCTTAAAGAACGGGTAACGATTGTTACGGGTGAACGGGGAACCTTTGTAGCTGACACTGGATCGGGTGACCTCACCTTCTACGCCAATGGAACTGTTGCCACCAATGAGTGGGATCAAGTCGCCGCGTTCGTGGTGTCAGCGAGGGCGATG TGACACGGTATGCGTTTGTCAAGCGAGAGCCACTACGGGTCGAACATGAGGCATTCCGCGATGCCGTGCTTGGCAAGTCATCGGATGGTGACGCTGGAGCAGGGGTTGCGAACACTTGAAGTAGTTGAGGCGGCACTGGGATCAGCGCATAAAGGCGAAGTAGCAAGGTTATAG SEQ ID NO.15 (H118A-F): 5′GCTGTTGGGCGGTTGGTGCGATTGAGCGCTTT3′ SEQ ID NO.16 (H118A-R): 5′AAAGCGCTCAATCGCACCAACCGCCCCAACAGC3′ SEQ ID NO.17 (L245A-F): 5′GACACTGGATCGGGTGACGCGACCTTCTACGCC3′ SEQ ID NO.18 (L245A-R): 5′GGCGTAGAAGGTCGCGTCACCCGATCCAGTGTC3′ SEQ ID NO.19 (A157T-F): 5′CCCTGCAGCATTACCGATGTTGGAGTGGCC3′ SEQ ID NO.20 (A157T-R): 5′GGCCACTCCAACATCGGTAATGCGCAGGG3′ SEQ ID NO.21 (E305D-F): 5′ACGCTGGACCAGGGGGTTGCGAACACTTGAAG3′ SEQ ID NO.22 (E305D-R): 5′CTTCAAGTGTTCGCAACCCCTGGTCCAGCGT3′ SEQ ID NO.23 (Y275W-F): 5′ACACGGTGGGCGTTTGTCAAGCGAGAG3′ SEQ ID NO.24 (Y275W-R): 5′CTCTCGCTTGACAAACGCCCACCGTGTC3′ SEQ ID NO.25 (S96A-F): 5′ATTGCACACGCGGTAGAAGCTGGACGGCGT3′ SEQ ID NO.26 (S96A-R): 5′ACGCCGTCCAGCTTCTACCGCGTGTC3′ The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A vanillin dehydrogenase mutant, characterized in that, Mutate vanillin dehydrogenase Deh using any one of the mutants selected from 1) to 6) below. 1) Vanillin dehydrogenase mutant H118A: Histidine at position 118 is mutated to alanine; 2) Vanillin dehydrogenase mutant L245A: Leucine at position 245 is mutated to alanine; 3) Vanillin dehydrogenase mutant A157T: The alanine at position 157 is mutated to threonine; 4) Vanillin dehydrogenase mutant E305D: Glutamic acid at position 305 is mutated to aspartic acid; 5) Vanillin dehydrogenase mutant Y275W: Tyrosine at position 275 is mutated to tryptophan; 6) Vanillin dehydrogenase mutant S96A: The serine (S) at position 96 is mutated to alanine (A). The amino acid sequences of vanillin dehydrogenase Deh, vanillin dehydrogenase mutant H118A, vanillin dehydrogenase mutant L245A, vanillin dehydrogenase mutant A157T, vanillin dehydrogenase mutant E305D, vanillin dehydrogenase mutant Y275W, and vanillin dehydrogenase mutant S96A are shown in SEQ ID NO. 1~7, respectively.

2. Biomaterials related to the vanillin dehydrogenase mutant as described in claim 1, characterized in that, The biomaterial is any one of the following: a) Nucleotides encoding the vanillin dehydrogenase mutant as described in claim 1; b) A recombinant expression vector containing the nucleotides described in a); c) Bioengineered bacteria containing the nucleotides described in a), or bioengineered bacteria containing the recombinant expression vector described in b).

3. The biomaterial according to claim 2, characterized in that, The nucleotide sequences encoding the vanillin dehydrogenase mutants H118A, L245A, A157T, E305D, Y275W, and S96A are shown in SEQ ID NO. 8-13, respectively.

4. The application of a vanillin dehydrogenase mutant as described in claim 1 or a biomaterial associated with the vanillin dehydrogenase mutant as described in claim 2 in the biotransformation of 5-hydroxymethylfurfural to furan carboxylic acid.

5. A method for the biotransformation of high-concentration 5-hydroxymethylfurfural to synthesize furan carboxylic acid, characterized in that, Includes the following steps: S1. Culture bioengineered bacteria containing nucleotides encoding vanillin dehydrogenase mutants, add IPTG as an inducer, collect the bacterial cells, and centrifuge to collect the bacterial cells; S2. The bacterial cells obtained in step S1 are resuspended in buffer solution to prepare a cell suspension, 5-hydroxymethylfurfural is added, and a transformation reaction is carried out to obtain furan carboxylic acid.

6. The method for biotransformation of high-concentration 5-hydroxymethylfurfural to synthesize furan carboxylic acid according to claim 5, characterized in that, In step S1, the bioengineered bacterium containing the nucleotide encoding the vanillin dehydrogenase mutant is Escherichia coli; When the bacterial solution OD 600 When the value reaches 0.6-0.8, add the inducing agent IPTG to 0.4 mM.

7. The method for biotransformation of high-concentration 5-hydroxymethylfurfural to synthesize furan carboxylic acid according to claim 5, characterized in that, In step S1, the centrifugation conditions are 4℃ and 8000 rpm.

8. The method for biotransformation of high-concentration 5-hydroxymethylfurfural to synthesize furan carboxylic acid according to claim 5, characterized in that, In step S2, when converting to a final concentration of 100 mM 5-hydroxymethylfurfural, the concentration range of the cell suspension is 10~140 g / L.

9. The method for biotransformation of high-concentration 5-hydroxymethylfurfural to synthesize furan carboxylic acid according to claim 8, characterized in that, In step S2, the conversion temperature is 20~45℃.

10. The method for biotransformation of high-concentration 5-hydroxymethylfurfural to synthesize furan carboxylic acid according to claim 9, characterized in that, In step S2, the pH during conversion is 5.5–9.0.

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