Formate dehydrogenase mutant and application thereof in catalytic synthesis of D-mannitol

Abstract

The invention discloses a formate dehydrogenase mutant and application thereof in catalytic synthesis of D-mannitol, and belongs to the technical field of bioengineering. According to the formate dehydrogenase mutant provided by the invention, the 18th-site glutamic acid of wild-type formate dehydrogenase with an amino acid sequence shown as SEQ ID NO.2 is mutated into proline, the 57th-site asparagine is mutated into glutamic acid, the 70th-site histidine is mutated into tryptophan, the 235th-site serine is mutated into threonine, and the 316th-site valine is mutated into threonine, so that the formate dehydrogenase mutant is high in enzyme activity and catalytic activity; the coenzyme circulation efficiency during the production of D-mannitol can be obviously improved, so that the yield of D-mannitol can be effectively improved. When the genetically engineered bacterium co-expressed by the formate dehydrogenase mutant and the mannitol dehydrogenase mutant is used for catalyzing 150 g / L fructose to produce D-mannitol, the yield of D-mannitol within 15 h can reach 140 g / L or above.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of bioengineering, and particularly relates to a formic acid dehydrogenase mutant and application thereof in catalyzing synthesis of D-mannitol. BACKGROUND

[0002] The information disclosed in the background of the present application is only intended to increase the understanding of the overall background of the present application and should not necessarily be regarded as acknowledging or implicitly suggesting that this information constitutes prior art known to those of ordinary skill in the art.

[0003] The molecular formula of D-mannitol is C6H 14 O6, the structural general formula is HOCH2(CHOH)4CH2OH, and the chiral carbon configuration is mainly in the D type. As an important functional sugar alcohol, D-mannitol has irreplaceable application value in the fields of medicine, food and the like due to its unique physicochemical properties and physiological activities.

[0004] At present, the synthesis methods of D-mannitol mainly include plant extraction, chemical synthesis and enzyme method. Among them, the plant extraction method and the chemical synthesis method have problems of high energy consumption, complex purification, serious pollution and the like, and the enzyme method has advantages of mild conditions, environmental friendliness, high specificity and the like by using fructose as a raw material and catalyzing and synthesizing by mannitol dehydrogenase in one step, and has become a mainstream synthesis method.

[0005] Mannitol dehydrogenase is an NADH (reduced nicotinamide adenine dinucleotide) dependent oxidoreductase, and its catalytic reaction needs to add exogenous coenzyme, which leads to high production cost, so the construction of an efficient coenzyme circulation system becomes the key to industrial application. In the existing coenzyme circulation system, although the glucose dehydrogenase-glucose system has high circulation efficiency, it generates a by-product gluconic acid to interfere with product purification; the formic acid dehydrogenase-formic acid system has more application potential because no by-product is generated, but its coenzyme circulation efficiency is low, which limits the actual application. However, the reported formic acid dehydrogenases and mutants thereof generally have problems of low enzyme activity and insufficient catalytic efficiency, which are difficult to meet the requirements of industrial production on efficiency and cost, and become a key bottleneck restricting the large-scale application of the enzyme method for synthesizing D-mannitol. SUMMARY

[0006] Therefore, the present application provides a formic acid dehydrogenase mutant and application thereof in synthesizing D-mannitol. Based on the theory of directed evolution, the formic acid dehydrogenase mutant with high enzyme activity and high catalytic efficiency is obtained by simultaneous mutation of multiple sites, which can be used as a coenzyme to catalyze the synthesis of D-mannitol together with mannitol dehydrogenase.

[0007] In a first aspect, the present application provides a mutant of formate dehydrogenase, which is obtained by mutating glutamic acid at position 18, aspartic acid at position 57, histidine at position 70, serine at position 235, and valine at position 316 of wild-type formate dehydrogenase with proline, glutamic acid, tryptophan, threonine and threonine, respectively, as shown in SEQ ID NO. 2.

[0008] In a second aspect, the present application provides a nucleic acid molecule encoding the mutant of formate dehydrogenase according to the first aspect.

[0009] In the present application, the nucleic acid molecule can be DNA, such as cDNA, genomic DNA or recombinant DNA; or RNA, such as mRNA or hnRNA.

[0010] In a third aspect, the present application provides a biological material containing the nucleic acid molecule according to the second aspect, which includes recombinant DNA, expression cassette, transposon, plasmid vector, viral vector or engineered bacteria.

[0011] In one or more embodiments of the present application, the plasmid vector is pET-duet plasmid.

[0012] In a fourth aspect, the present application provides a recombinant microorganism, which is constructed by introducing the nucleic acid molecule according to the second aspect into Escherichia coli via plasmid or integrating into the chromosome of Escherichia coli by genetic engineering means.

[0013] In one or more embodiments of the present application, the Escherichia coli is Escherichia coli BL21 (DE3). E. coli BL21 (DE3).

[0014] In a fifth aspect, the present application provides use of the mutant of formate dehydrogenase according to the first aspect, the nucleic acid molecule according to the second aspect, the biological material according to the third aspect or the recombinant microorganism according to the fourth aspect in catalyzing synthesis of D-mannitol.

[0015] In a sixth aspect, the present application provides a method for catalyzing synthesis of D-mannitol, comprising: introducing the mutant of formate dehydrogenase according to the first aspect and mannitol dehydrogenase or mutant of mannitol dehydrogenase into a host bacterium to construct an engineered bacterium, inducing culture of the engineered bacterium to obtain wet bacterium, and using the wet bacterium or crude enzyme solution extracted from the wet bacterium by ultrasonic disruption as a catalyst, and using fructose as a substrate and sodium formate as a co-substrate to react in a liquid phase environment to obtain D-mannitol.

[0016] Preferably, the nucleotide sequence of the mutant of mannitol dehydrogenase is shown in SEQ ID NO. 37.

[0017] Preferably, the solvent of the liquid phase environment is phosphate buffer, and the pH is 6.8-7.2.

[0018] Preferably, the temperature of the reaction is 32-38℃, and the reaction time is 12-20h.

[0019] Preferably, in the catalyst, the amount of wet bacteria is 15-30g / L, more preferably 20-30g / L; the final concentration of fructose is 100-180g / L, more preferably 130-170g / L; the final concentration of sodium formate is 50-80g / L, more preferably 60-75g / L; the liquid phase environment further contains zinc sulfate, and the final concentration of zinc sulfate is 0.05-3mM.

[0020] Compared with the prior art, the present application has the following beneficial effects: The present application obtains formate dehydrogenase mutant by simultaneously mutating the 18th, 57th, 70th, 235th and 316th amino acids of formate dehydrogenase OpFDH derived from Hansenula polymorpha, Ogataea polymorpha The present application significantly improves the coenzyme cycle efficiency, and further improves the efficiency of producing D-mannitol from fructose by using mannitol dehydrogenase; when the genetically engineered bacteria co-expressing the formate dehydrogenase mutant and the mannitol dehydrogenase mutant are used to catalyze 150g / L of fructose to produce D-mannitol, the yield of D-mannitol can reach more than 140g / L within 15h. BRIEF DESCRIPTION OF DRAWINGS

[0021] The drawings accompanying the specification of the present application serve to provide further understanding of the present application, and the schematic embodiments of the present application and the description thereof serve to explain the present application, and do not constitute improper limitations on the present application. Obviously, other drawings can be obtained by those skilled in the art without creative labor based on these drawings.

[0022] Figure 1 is the D-mannitol yield graph of different reaction times in Example 6 of the present application. DETAILED DESCRIPTION

[0023] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art to which the present application belongs.

[0024] The technical solutions of the present application will be further described below in combination with specific embodiments. The reagents used in the following embodiments of the present application are not specially limited, and commercially available goods known to those skilled in the art can be used.

[0025] The culture medium formula used in the following examples is as follows: LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, solvent: water, pH 7.4.

[0026] LB plate: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 18 g / L agar, solvent is water, pH 7.4.

[0027] The concentration of the product D-mannitol was detected by high performance liquid chromatography (HPLC). The chromatographic conditions were as follows: the chromatographic column model was a calcium-type cation chromatographic column; the mobile phase was water, the injection volume was 10 μL, the detector was a differential detector, the detection time was 25 min, the flow rate was 0.6 mL / min; and the column temperature was 80°C.

[0028] Example 1 This example provides the construction of expression vectors and engineered bacteria.

[0029] Through the mining of gene library, a gene from Hansenula polymorpha ( Ogataea polymorpha The NCBI accession number for formate dehydrogenase OpFDH is XP_018212858.1. Nanjing GenScript Biotechnology Co., Ltd. was commissioned to perform full gene synthesis. The nucleotide sequence is shown in SEQ ID NO. 1, and the amino acid sequence is shown in SEQ ID NO. 2.

[0030] SEQ ID NO. 1 is as follows:

[0031] SEQ ID NO. 2 is as follows: MKVVLVLYDAGKHAQDEERLYGCTENALGIRDWLEKQGHELVVTSDKEGQNSVLEKNISDADVIISTPFHPAYITKERIDKAKKLKLLVVAGVGSDHIDLDYINQSGRDISVLEVTGSNVVSVAEHVVMTMLVLVRNFVPAHEQIISGGWNVAEIAKDSFDIEGKVIATIGAGRIGYRVLE RLVAFNPKELLYYDYQSLSKEAEEKVGARRVHDIKELVAQADIVTINCPLHAGSKGLVNAELLKHFKKGAWLVNTARGAICVAEDVAAAVKSGQLRGYGGDVWFPQPAPKDHPWRSMANKYGAGNAMTPHYSGSVIDAQVRYAQGTKNILESFFTQKFDYRPQDIILLNGKYKTKSYGADK.

[0032] Primers F1, R1, F2, and R2 were designed based on the nucleotide sequence shown in SEQ ID NO. 1 and the pET-duet vector sequence. The nucleotide sequence of F1 is shown in SEQ ID NO. 3, the nucleotide sequence of R1 is shown in SEQ ID NO. 4, the nucleotide sequence of F2 is shown in SEQ ID NO. 5, and the nucleotide sequence of R2 is shown in SEQ ID NO. 6. OpFDH was cloned into the first multiple cloning site of pET-duet.

[0033] F1: 5'-ctttaagaaggagatataccATGAAAGTAGTTCTAGTCTTATATGATGCTG-3'; R1: 5'-cttaagcattatgcggccgcTTACTTGTCCGCACCATAGCTCT-3'; F2: 5'-GCGGCCGCATAATGCTTA-3'; R2: 5'- GGTATATCTCCTTCTTAAAGTTAAACAAAAT-3'.

[0034] Using pET-duet plasmid as expression vector, Escherichia coli was constructed E. coli BL21(DE3) / pETduet-OpFDH.

[0035] Construction of expression vector: the sequence of formate dehydrogenase gene with homologous arms was obtained by amplification with high-fidelity Pfu DNA polymerase using the primer F1 / R1 and F2 / R2 to initiate and the target gene as template, the linearized vector sequence was obtained by amplification with high-fidelity Pfu DNA polymerase using the pET-duet plasmid as template, and the target gene was homologously recombined with the linearized vector by using a homologous recombination enzyme to construct the plasmid pETduet-OpFDH.

[0036] Construction of recombinant E. coli: the recombinant product stored at -80°C was first thawed, and then 5 μL of the recombinant product was added to the competent cells of BL21 (DE3) in an ice bath at 0°C for 10 min, followed by 30 min of ice bath at 0°C, 90 s of heat shock in a water bath at 42°C, 2 min of ice bath at 0°C, and addition of 600 μL of LB medium for culture at 37°C and 200 rpm for 1 h; the culture was spread on an LB plate containing 50 μg / mL of kanamycin resistance and cultured at 37°C for 10 h, and plasmids were extracted from randomly selected clones for sequencing identification, and a recombinant E. coli containing the expression recombinant plasmid was screened and obtained E. coli BL21 (DE3) / pETduet-OpFDH. E. coli BL21 (DE3) / pETduet-OpFDH.

[0037] Example 2 This example provides induced expression of formate dehydrogenase.

[0038] Wet bacteria containing formate dehydrogenase gene: the recombinant E. coli obtained in Example 1 E. coli BL21 (DE3) / pETduet-OpFDH was inoculated into LB liquid medium containing 50 μg / mL of ampicillin resistance and cultured at 37°C and 200 rpm for 12 h, and then inoculated into fresh LB liquid medium containing 50 μg / mL of ampicillin resistance at a 1% (v / v) inoculation amount, and cultured at 37°C and 200 rpm until the OD 600 of the bacteria reached 0.6-0.8, and then isopropyl-β-D-thiogalactopyranoside (IPTG) was added at a final concentration of 0.1 mM, and the culture was induced at 25°C for 16 h, followed by centrifugation at 4°C and 8000 rpm for 20 min, removal of the supernatant, and collection of the precipitate, thereby obtaining the recombinant strain containing formate dehydrogenase E. coli BL21 (DE3) / pETduet-OpFDH wet bacteria.

[0039] Example 3 This example provides establishment of a mutation library of formate dehydrogenase gene.

[0040] In this example, the recombinant E. coli BL21 (DE3) / pETduet-OpFDH constructed in Example 2 was used as the host strain. E. coliBL21(DE3) / pETduet-OpFDH as the starting strain, and the strain was modified by the theory of directed evolution.

[0041] According to the software calculation, the sites 8, 10, 11, 18, 50, 57, 70, 133, 198, 213, 235, 271, 304, 316, 337 were selected for site-directed mutation.

[0042] The primer design is shown in Table 1.

[0043] Table 1 Site-directed mutation primer design of formate dehydrogenase

[0044] The mutation PCR system (100 μL) was as follows: 2×Phanta Max buffer 25 μL, dNTPs 1 μL, mutation upper and lower primers 1 μL each, template (starting strain) 1 μL, Pfu DNA polymerase 0.5 μL, and ddH2O was added to 50 μL. The PCR conditions were as follows: 95℃ pre-denaturation for 3 min, 30 cycles of 95℃ for 15 s, 60℃ for 15 s, 72℃ for 4 min, and finally 72℃ for 5 min. The PCR results were verified by DNA agarose gel electrophoresis, and the PCR product was subjected to DpnI enzyme digestion of the template, which was digested at 37℃, 200 rpm for 1 hour, inactivated at 65℃ for 1 minute, and then transformed by heat shock. The E. coli E. E. coli The BL21(DE3) was activated and cultured at 37℃, 200 rpm for 1 hour, and then plated on LB plates containing 50 μg / mL ampicillin resistance and cultured at 37℃ overnight.

[0045] Example 4 This example provides screening of the gene mutation library of formate dehydrogenase.

[0046] The single colonies on the plate obtained in Example 3 were picked and inoculated into LB liquid medium containing 50 μg / mL ampicillin resistance, and cultured at 37℃, 200 rpm for 12 h. The strain was preserved and sent to a sequencing company for sequencing verification. After the sequencing verification was correct, the preserved strain was inoculated into LB liquid medium containing 50 μg / mL ampicillin resistance at an inoculation amount of 0.2% (v / v), and cultured at 37℃, 200 rpm for 12 h. Then, the culture was inoculated into LB liquid medium containing 50 μg / mL ampicillin resistance at an inoculation amount of 1% (v / v), and cultured at 37℃, 200 rpm until the OD600 of the bacterial cells reached 0.6-0.8. 600The wet bacteria containing the mutation library of formate dehydrogenase gene were obtained by adding isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM, culturing at 25°C for 16 h, centrifuging at 4°C and 8000 rpm for 20 min, and discarding the supernatant.

[0047] 1. Primary screening The reaction solution (200 μL) was prepared as follows: 50 mM substrate NAD (nicotinamide adenine dinucleotide) at a final concentration, 60 mM sodium formate at a final concentration, 5 g / L of catalyst (based on the total weight of the wet bacteria), and a phosphate buffer at pH 6.5 as the reaction medium. The reaction was carried out at 35°C and 500 rpm for 1 h. After the reaction, 20 μL of the reaction sample was taken, diluted 20 times, and the absorbance was detected at 340 nm (NADH has an absorbance peak at 340 nm) using an enzyme marker. The detection results are shown in Table 2. +

[0048] Table 2. Primary screening reaction results

[0049] As can be seen from Table 2, the absorbance of the mutants E18P, N57E, H70W, S235T, and V316T is higher than that of the parent strain. Therefore, these mutant strains were selected for subsequent rescreening.

[0050] 2. Rescreening The strains obtained by primary screening were rescreened. The rescreening combined mutants were sent to a sequencing company for sequencing verification. After the sequencing verification was correct, the activity verification was performed. The reaction solution (10 mL) for rescreening was prepared as follows: 50 mM substrate NAD at a final concentration, 60 mM sodium formate at a final concentration, 5 g / L of catalyst (based on the total weight of the wet bacteria), and a phosphate buffer at pH 6.5 as the reaction medium. The reaction was carried out at 35°C and 500 rpm for 1 h. After the reaction, 20 μL of the reaction sample was taken, diluted 20 times, and the absorbance was detected at 340 nm using an enzyme marker. The detection results are shown in Table 3. +

[0051] Table 3. Rescreening reaction results

[0052] As can be seen from the results in Table 3, the mutant with the mutation sites of E18P+N57E+H70W+S235T+V316T has the highest activity.

[0053] Example 5 This example provides the construction of a mannitol dehydrogenase mutant and a formate dehydrogenase mutant co-expressed strain. This example provides the construction of a mannitol dehydrogenase mutant and a formate dehydrogenase mutant co-expressed strain.​

[0054] Mannitol dehydrogenase is an NADH coenzyme-dependent enzyme. In order to improve the catalytic efficiency and economic benefit, a formate dehydrogenase coenzyme circulation system is constructed in the embodiment. The mannitol dehydrogenase used in the embodiment is derived from the mannitol dehydrogenase WpMDH of Weissella cibaria (NCBI accession number WP_131474179.1), and a site-directed mutation is performed on the mannitol dehydrogenase to obtain a mannitol dehydrogenase mutant. The nucleotide sequence of the mannitol dehydrogenase mutant is shown in SEQ ID NO. 37. Weissella paramesenteroides

[0055] SEQ ID NO. 37: ​

[0056] Primers F3, R3, F4 and R4 were designed according to the nucleotide sequence shown in SEQ ID NO. 37 and the pETduet-OpFDH-E18P-N57E-H70W-S235T-V316T vector sequence, wherein the nucleotide sequence of F3 is shown in SEQ ID NO. 38, the nucleotide sequence of R3 is shown in SEQ ID NO. 39, the nucleotide sequence of F4 is shown in SEQ ID NO. 40, and the nucleotide sequence of R4 is shown in SEQ ID NO. 41. WpMDH-E2K-E176Q-I195R-V325K was cloned into the second multiple cloning site of pETduet-OpFDH-E18P-N57E-H70W-S235T-V316T.

[0057] F3: 5'-tataagaaggagatatacatATGAAAGCTCTAGTATTAACAGGAATAAAA-3'; R3: 5'-gtttctttaccagactcgagTTACGCCTCTTCACCACCCA-3'; F4: 5'-CTCGAGTCTGGTAAAGAAACCGC-3'; R4: 5'-ATGTATATCTCCTTCTTATACTTAACTAATATACTAAGA-3'.

[0058] The co-expression plasmid pETduet-OpFDH-E18P-N57E-H70W-S235T-V316T-WpMDH-E2K-E176Q-I195R-V325K was constructed using pET-duet plasmid as an expression vector.

[0059] The strain BL21(DE3) / pETduet-OpFDH-E18P-N57E-H70W-S235T-V316T-WpMDH-E2K-E176Q-I195R-V325K was obtained by transformation according to Example 1, and verified by sequencing by a sequencing company. E. coli BL21(DE3) / pETduet-OpFDH-E18P-N57E-H70W-S235T-V316T-WpMDH-E2K-E176Q-I195R-V325K.

[0060] In order to obtain co-expression strains with higher catalytic efficiency, and at the same time as a control group, WpMDH-E2K-E176Q-I195R-V325K was placed in the first polyclonal site of pETduet, and OpFDH-E18P-N57E-H70W-S235T-V316T was placed in the second polyclonal site of pETduet. For specific construction process, refer to the construction process of pETduet-OpFDH-E18P-N57E-H70W-S235T-V316T-WpMDH-E2K-E176Q-I195R-V325K plasmid. After transformation and sequencing verification by a company, the strain E. coli BL21(DE3) / pETduet-WpMDH-E2K-E176Q-I195R-V325K-OpFDH-E18P-N57E-H70W-S235T-V316T.

[0061] The fermentation process of the strain is referred to Example 4. Two co-expression strains obtained were compared for activity, and the reaction solution was prepared: the final concentration of substrate fructose was 150 g / L, the final concentration of sodium formate was 68 g / L, the final concentration of zinc sulfate was 1 mM, the amount of catalyst (based on the total weight of wet bacterial body) was 20 g / L, and the reaction medium was phosphate buffer with pH = 7. The reaction was carried out at 35°C and 500 rpm in a reactor for 1 hour. After the reaction was completed, 20 μL of the sample at the end of the reaction was taken, diluted 10 times, filtered through a 0.22 μm filter membrane, and subjected to HPLC detection. The detection results are shown in Table 4. The results show that E. coli BL21(DE3) / pETduet-WpMDH-E2K-E176Q-I195R-V325K-OpFDH-E18P-N57E-H70W-S235T-V316T has better activity.

[0062] Table 4 Enzyme activity detection results

[0063] Example 6 This example provides the application of the co-expression strain of Example 5 in the catalytic synthesis of D-mannitol.

[0064] The co-expression strain of Example 5 E. coliBL21(DE3) / pETduet-WpMDH-E2K-E176Q-I195R-V325K-OpFDH-E18P-N57E-H70W-S235T-V316T was inoculated into LB liquid medium containing 50 μg / mL of ampicillin at a final concentration, and cultured at 37°C for 9 hours as a seed liquid. The seed liquid was inoculated into a 5 L fermenter containing 3 L of fermentation medium at a volume concentration of 3.5%. The culture was incubated at 37°C and 500 rpm for about 4 hours until the bacterial density reached OD 6-8. Then, the temperature of the fermenter was reduced to 25°C, and 10 g / L of lactose was added as an inducer. The culture was then incubated at 25°C and 500 rpm for 12 hours to obtain a fermentation broth. The fermentation broth was centrifuged at 8000 rpm for 10 minutes to obtain wet bacterial cells of BL21(DE3) / pETduet-WpMDH-E2K-E176Q-I195R-V325K-OpFDH-E18P-N57E-H70W-S235T-V316T. E. coli BL21(DE3) / pETduet-WpMDH-E2K-E176Q-I195R-V325K-OpFDH-E18P-N57E-H70W-S235T-V316T was inoculated into LB liquid medium containing 50 μg / mL of ampicillin at a final concentration, and cultured at 37°C for 9 hours as a seed liquid. The seed liquid was inoculated into a 5 L fermenter containing 3 L of fermentation medium at a volume concentration of 3.5%. The culture was incubated at 37°C and 500 rpm for about 4 hours until the bacterial density reached OD 6-8. Then, the temperature of the fermenter was reduced to 25°C, and 10 g / L of lactose was added as an inducer. The culture was then incubated at 25°C and 500 rpm for 12 hours to obtain a fermentation broth. The fermentation broth was centrifuged at 8000 rpm for 10 minutes to obtain wet bacterial cells of BL21(DE3) / pETduet-WpMDH-E2K-E176Q-I195R-V325K-OpFDH-E18P-N57E-H70W-S235T-V316T.

[0065] The fermentation medium in the fermenter of this example was composed of the following: tryptone 45 g, yeast extract 36 g, sodium chloride 30 g, potassium dihydrogen phosphate 4.08 g, glycerol (glycerin) 45 g, potassium phosphate dibasic trihydrate 6.84 g, ammonium sulfate 15 g, magnesium sulfate 1.125 g, and antifoaming agent 3 g. Distilled water was added to make up the volume to 3 L for dissolution.

[0066] Catalyst E.coli BL21(DE3) / pETduet-WpMDH-E2K-E176Q-I195R-V325K-OpFDH-E18P-N57E-H70W-S235T-V316T was inoculated into LB liquid medium containing 50 μg / mL of ampicillin at a final concentration, and cultured at 37°C for 9 hours as a seed liquid. The seed liquid was inoculated into a 5 L fermenter containing 3 L of fermentation medium at a volume concentration of 3.5%. The culture was incubated at 37°C and 500 rpm for about 4 hours until the bacterial density reached OD 6-8. Then, the temperature of the fermenter was reduced to 25°C, and 10 g / L of lactose was added as an inducer. The culture was then incubated at 25°C and 500 rpm for 12 hours to obtain a fermentation broth. The fermentation broth was centrifuged at 8000 rpm for 10 minutes to obtain wet bacterial cells of BL21(DE3) / pETduet-WpMDH-E2K-E176Q-I195R-V325K-OpFDH-E18P-N57E-H70W-S235T-V316T. Figure 1 The results are shown in Table 1. As can be seen, the concentration of D-mannitol was 140.3 g / L at 15 h of reaction time.

[0067] Comparative Example This comparative example refers to the method of Example 5 to construct a co-expression strain of the formate dehydrogenase OpFDH (nucleotide sequence shown as SEQ ID NO. 1) and the mannitol dehydrogenase mutant of H. polymorpha Ogataea polymorpha ​E.coli BL21(DE3) / pETduet-WpMDH-E2K-E176Q-I195R-V325K-OpFDH, and the co-expression strain constructed is referred to Example 6 E.coli BL21(DE3) / pETduet-WpMDH-E2K-E176Q-I195R-V325K-OpFDH is used to catalyze the synthesis of D-mannitol, and the reaction conditions are the same as those in Example 6. When the reaction time is 15 h, the concentration of D-mannitol is 72.8 g / L, which is detected by HPLC.

[0068] The above only describes the preferred embodiments of the present application and is not used to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A formate dehydrogenase mutant, characterized in that In the wild-type formate dehydrogenase shown in the amino acid sequence of SEQ ID NO.2, glutamic acid at position 18 is mutated to proline, asparagine at position 57 is mutated to glutamic acid, histidine at position 70 is mutated to tryptophan, serine at position 235 is mutated to threonine, and valine at position 316 is mutated to threonine.

2. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the formate dehydrogenase mutant according to claim 1.

3. A biomaterial containing the nucleic acid molecule according to claim 2, characterized in that: The biological material includes recombinant DNA, expression cassette, transposon, plasmid vector, virus vector or engineered bacteria.

4. A recombinant microorganism, characterized in that The recombinant microorganism is constructed by introducing the nucleic acid molecule according to claim 2 into Escherichia coli via a plasmid or integrating it into the chromosome of Escherichia coli through genetic engineering means.

5. Use of the formate dehydrogenase mutant according to claim 1, the nucleic acid molecule according to claim 2, the biomaterial according to claim 3 or the recombinant microorganism according to claim 4 in catalytic synthesis of D-mannitol.

6. A method for catalytic synthesis of D-mannitol, characterized in that: include: The formate dehydrogenase mutant according to claim 1 and mannitol dehydrogenase or a mannitol dehydrogenase mutant are co-introduced into a host bacterium to construct a genetically engineered bacterium, the genetically engineered bacterium is induced and cultured to obtain wet cells, the wet cells or a crude enzyme solution extracted from the wet cells by ultrasonic crushing are used as a catalyst, fructose is used as a substrate, and sodium formate is used as a cosubstrate to react in a liquid phase environment to obtain D-mannitol.

7. The method according to claim 6, wherein The nucleotide sequence of the mannitol dehydrogenase mutant is shown in SEQ ID NO.

37.

8. The method according to claim 6, wherein The solvent of the liquid phase environment is phosphate buffer with a pH of 6.8-7.

2.

9. The method according to claim 6, wherein The reaction temperature is 32-38° C., and the reaction time is 12-20 h.

10. The method according to claim 6, wherein In the catalyst, the dosage of wet bacteria is 15-30 g / L, the final concentration of fructose is 100-180 g / L, and the final concentration of sodium formate is 50-80 g / L; the liquid phase environment also contains zinc sulfate, and the final concentration of zinc sulfate is 0.05-3 mM.

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