A mannitol dehydrogenase mutant and its application in synthesis of d-mannitol

Abstract

The present application relates to the technical field of bioengineering, and particularly relates to a mannitol dehydrogenase mutant and application thereof in synthesis of D-mannitol. Weissella paramesenteroides Weissella paramesenteroides Weiss The present application simultaneously mutates amino acid multiple sites of 2nd, 176th, 195th and 325th amino acids of mannitol dehydrogenase WpMDH derived from Weissella cibaria to obtain a mannitol dehydrogenase mutant, and then significantly improves the efficiency of producing D-mannitol from D-fructose, couples a coenzyme cycle system, and catalyzes 150 g / L of D-fructose to produce only 12 h; the yield of D-mannitol reaches 135.6 g / L. Therefore, the present application has great application value in the field of D-mannitol production.

Description

TECHNICAL FIELD

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

[0002] The information disclosed in this Background section is only for the purpose of increasing the understanding of the general background of the application and does not necessarily constitute an admission that the information forms part of the prior art already known to a person of ordinary skill in the art.

[0003] D-mannitol is a natural six-carbon sugar alcohol, which is an isomer of sorbitol, and has been proven to be an important precursor of antitumor drugs and immune stimulants, and is widely used in food, medical and chemical industries. Mannitol dehydrogenase (MDH) as a key enzyme can catalyze the substrate D-fructose to generate D-mannitol under the participation of coenzyme NADH or NADPH.

[0004] However, the reported mannitol dehydrogenase and its mutants have the problems of low enzyme activity and low catalytic efficiency, and therefore cannot meet the requirements of industrial production. SUMMARY

[0005] In order to overcome the problems of low enzyme activity and low catalytic efficiency in the production of D-mannitol in the prior art, the present application provides a mannitol dehydrogenase mutant and application thereof in synthesis of D-mannitol.

[0006] In a first aspect, the present application provides a mannitol dehydrogenase mutant, which is obtained by simultaneously mutating the glutamic acid at position 2, the glutamic acid at position 176, the isoleucine at position 195 and the valine at position 325 of the wild-type mannitol dehydrogenase with the amino acid sequence shown in SEQ ID NO. 2.

[0007] In one or more embodiments, the mannitol dehydrogenase mutant is obtained by mutating the glutamic acid at position 2 to lysine, the glutamic acid at position 176 to glutamine, the isoleucine at position 195 to arginine, and the valine at position 325 to lysine in the wild-type mannitol dehydrogenase with the amino acid sequence shown in SEQ ID NO. 2.

[0008] In a second aspect, the present application provides a gene encoding the mannitol dehydrogenase mutant of the first aspect or simultaneously encoding the mannitol dehydrogenase mutant of the first aspect and glucose dehydrogenase.

[0009] In a third aspect, the present application provides an expression cassette comprising the gene of the second aspect.

[0010] In a fourth aspect, the present application provides a recombinant expression vector comprising the gene of the second aspect.

[0011] In order to improve the catalytic efficiency and economic benefits, the recombinant expression vector provided by the present application uses pET-duet plasmid as an expression vector, clones the mannitol dehydrogenase with the amino acid sequence shown in SEQ ID NO. 2 into the first polyclone site of pET-duet, and constructs the glucose dehydrogenase PlGDH in the second polyclone site of the expression plasmid vector pET-duet after site-directed mutagenesis, wherein the amino acid sequence of the glucose dehydrogenase PlGDH is shown in SEQ ID NO. 8.

[0012] In a fifth aspect, the present application provides a recombinant bacterium comprising the gene of the second aspect or the recombinant expression vector of the fourth aspect.

[0013] The present application also provides a transgenic cell line comprising the gene of the second aspect.

[0014] In a sixth aspect, the present application provides the use of the mannitol dehydrogenase mutant of the first aspect, the gene of the second aspect or the recombinant bacterium of the fifth aspect in catalyzing the synthesis of D-mannitol.

[0015] In a seventh aspect, the present application provides a method for synthesizing D-mannitol, comprising:

[0016] The wet bacterium obtained by inducing the culture of the recombinant bacterium of the fifth aspect or the crude enzyme solution extracted by ultrasonic disruption of the wet bacterium or the immobilized enzyme is used as a catalyst, D-fructose is used as a substrate, and pure water is used as a reaction medium to form a reaction system, and D-mannitol is obtained by reaction.

[0017] In one or more embodiments, the amount of the catalyst is 5-30 g / L based on the total weight of the wet bacterium, the final concentration of the substrate D-fructose is 100 g / L-180 g / L, and the final concentration of the auxiliary substrate glucose is 110 g / L-200 g / L.

[0018] Compared with the prior art, the present application has the following beneficial effects:

[0019] The present application provides a mannitol dehydrogenase mutant and its application. The present application is derived from Weissella cibaria (WIBR1) and has the amino acid sequence shown in SEQ ID NO. 2. Weissella paramesenteroidesThe simultaneous mutation of the amino acid multiple sites of the 2nd, 176th, 195th and 325th amino acids of the mannitol dehydrogenase WpMDH obtains a mannitol dehydrogenase mutant, and further significantly improves the efficiency of producing D-mannitol from D-fructose, couples a coenzyme cycle system, and catalyzes 150 g / L of D-fructose to produce only 12 h; the yield of D-mannitol reaches 135.6 g / L, which is much higher than the prior art. It has great application value in the field of D-mannitol production. BRIEF DESCRIPTION OF DRAWINGS

[0020] The drawings accompanying the specification of the present application serve to provide further understanding of the present application, and the illustrative embodiments of the present application and the description thereof serve to explain the present application, and do not constitute an improper limitation on the present application.

[0021] Figure 1 For E. coli Reaction progress chart of BL21(DE3) / pETduet-WpMDH-E2K-E176Q-I195R-V325K-OpFDH catalyzed synthesis of D-mannitol. DETAILED DESCRIPTION

[0022] 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 in the present application have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0023] It should be noted that the terms used herein are only for the purpose of describing specific embodiments, and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form, and in addition, it should be understood that when the terms "comprise" and / or "include" are used in the specification, they indicate the presence of a feature, step, operation, device, component and / or combination thereof.

[0024] In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below with specific examples.

[0025] The medium formula used in the following examples is as follows:

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

[0027] LB plate: 10 g / L of tryptone, 5 g / L of yeast extract, 10 g / L of sodium chloride, 18 g / L of agar, solvent is water, pH is 7.4.

[0028] The product D-mannitol concentration was detected by high performance liquid chromatography (HPLC), and the analysis method was as follows:

[0029] Column type: calcium cation exchange column; column temperature: 80 ℃; mobile phase: water; mobile phase flow rate: 0.6 mL / min; sample injection amount: 10 μL; differential detector; detection time: 20 min.

[0030] Sample treatment: 100 μL of the reaction ended sample was diluted 20 times with water solution, filtered through a 0.22 μm filter membrane, and subjected to HPLC detection.

[0031] Example 1, construction of expression vector and engineering bacteria:

[0032] Through gene library mining, a mannitol dehydrogenase WpMDH derived from Weissella cibaria (WP_131474179.1) was screened. Weissella paramesenteroides The full gene synthesis was entrusted to Nanjing Kingsriver Biotechnology Co., Ltd., the nucleotide sequence is shown in SEQ ID NO. 1, and the amino acid sequence is shown in SEQ ID NO. 2.

[0033] According to the nucleotide sequence shown in SEQ ID NO. 1 and the pET-duet vector sequence, primers F1, R1, F2 and R2 (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) were designed, and WpMDH was cloned into the first multiple cloning site of pET-duet.

[0034] F1: 5'-ctttaagaaggagatataccATGGAAGCTCTAGTATTAACAGGAATAAA-3';

[0035] R1: 5'-cttaagcattatgcggccgcTTACGCCTCTTCACCACCCA-3';

[0036] F2: 5'-GCGGCCGCATAATGCTTA-3';

[0037] R2: 5'-GGTATATCTCCTTCTTAAAGTTAAACAAAAT-3';

[0038] Using pET-duet plasmid as an expression vector, Escherichia coli E. coliBL21(DE3) / pETduet-WpMDH.

[0039] Construction of expression plasmid: Under the initiation of primers F1 / R1 and F2 / R2, the target gene was amplified using high-fidelity Pfu DNA polymerase to obtain the mannitol dehydrogenase gene sequence with homologous arms. Using pET-duet plasmid as template, the linearized vector sequence was amplified using high-fidelity Pfu DNA polymerase to obtain the linearized vector sequence. Homologous recombination was then performed between the target gene and the linearized vector using homologous recombination enzyme to construct the plasmid pETduet-WpMDH.

[0040] Construction of recombinant Escherichia coli: First, bacteria stored at -80 ℃... E. coli BL21(DE3) competent cells were incubated at 0°C on ice for 10 min, then 5 µL of the recombinant product was added in a clean bench, followed by an incubation at 0°C on ice for 30 min, a heat shock at 42°C for 90 s, an incubation at 0°C on ice for 2 min, and then 600 µL of LB medium was added. The cells were then cultured at 37°C and 200 rpm for 1 h. The culture was then spread on LB agar plates containing 50 μg / mL ampicillin resistance and cultured at 37°C for 8–12 h. Clones were randomly selected, plasmids were extracted, and sequenced for identification. Recombinant *E. coli* containing the recombinant plasmid expression were screened for these results. E. coli BL21(DE3) / pETduet-WpMDH.

[0041] Example 2, Induced expression of mannitol dehydrogenase:

[0042] Wet bacterial cells containing the mannitol dehydrogenase gene: The recombinant Escherichia coli obtained in Example 1 were used respectively. E. coli BL21(DE3) / pETduet-WpMDH was inoculated into LB liquid medium containing 50 μg / mL ampicillin resistance and cultured at 37 ℃ and 200 rpm for 12 h. Then, it was inoculated at a 1% (v / v) inoculation rate into fresh LB liquid medium containing 50 μg / mL ampicillin resistance and cultured at 37 ℃ and 200 rpm until the bacterial OD reached the target cell count. 600 When the concentration reaches 0.6-0.8, isopropyl-β-D-thiogalactopyranoside (IPTG) is added to a final concentration of 0.1 mM. After induction culture at 25℃ for 16 h, the mixture is centrifuged at 4℃ and 8000 rpm for 20 min. The supernatant is discarded, and the precipitate is collected to obtain the recombinant strain containing mannitol dehydrogenase. E. coli Wet cells of BL21(DE3) / pETduet-WpMDH.

[0043] Example 3, Establishment of a mannitol dehydrogenase gene mutant library:

[0044] Constructed using Example 2E. coli BL21(DE3) / pETduet-WpMDH was the starting strain.

[0045] According to the software calculation, the mutation sites E2K / H30K / H41K / F123V / Q132K / E176Q / Q183K / I195R / V196S / M235T / M266V / V325K were selected for site-directed mutagenesis by the theory of directed evolution method.

[0046] 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 products were subjected to DpnI enzyme digestion of the template at 37 ℃ for 1 h, 200 rpm, inactivated at 65 ℃ for 1 min, and the PCR products were transformed by heat shock, and the E. coli E. coli BL21(DE3) was activated and cultured at 37 ℃, 200 rpm for 1 h, and then plated on LB plates containing 50 μg / mL kanamycin resistance and incubated at 37 ℃ overnight.

[0047] Table 1 Design of site-directed mutagenesis primers for mannitol dehydrogenase

[0048]

[0049] Example 4, screening of the mannitol dehydrogenase gene mutation library:

[0050] 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 preserved strain was sequenced and verified by a sequencing company. 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%, and cultured at 37 ℃, 200 rpm for 12 h. Then, the culture was inoculated into fresh 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. The culture was induced by adding 1 mM IPTG, and the culture was continued at 37 ℃, 200 rpm for 16 h. 600The wet bacteria containing the mutant library of mannitol dehydrogenase gene are obtained by adding isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM, inducing the culture at 25°C for 16 h, centrifuging at 4°C and 8000 rpm for 20 min, and discarding the supernatant.

[0051] 1. Primary screening:

[0052] The reaction solution (200 μL) is prepared by adding D-fructose as a substrate to a final concentration of 100 mM, coenzyme NADH to a final concentration of 120 mM, zinc sulfate to a final concentration of 1 mM, and the catalyst to a concentration of 5 g / L based on the total weight of the wet bacteria, and using phosphate buffer at pH 7 as the reaction medium. The reaction is carried out at 35°C and 500 rpm for 1 h, and then 20 μL of the reaction sample is taken, diluted 20 times, filtered through a 0.22 μm filter, and subjected to HPLC detection. The detection results are shown in Table 2.

[0053] Table 2. Primary screening reaction results

[0054]

[0055] 2. Secondary screening:

[0056] The strains obtained by the primary screening are subjected to secondary screening, the combined mutants are subjected to sequencing verification by a sequencing company, and after the sequencing verification is correct, the activity verification is carried out. The reaction solution (10 mL) for secondary screening is prepared by adding D-fructose as a substrate to a final concentration of 100 mM, coenzyme NADH to a final concentration of 120 mM, zinc sulfate to a final concentration of 1 mM, and the catalyst to a concentration of 5 g / L based on the total weight of the wet bacteria, and using phosphate buffer at pH 7 as the reaction medium. The reaction is carried out at 35°C and 500 rpm for 1 h, and then 20 μL of the reaction sample is taken, diluted 20 times, filtered through a 0.22 μm filter, and subjected to HPLC detection. The detection results are shown in Table 3.

[0057] Table 3. Secondary screening reaction results

[0058]

[0059] Example 5. Construction of coenzyme system:

[0060] Mannitol dehydrogenase is an NADH coenzyme-dependent enzyme. In order to improve the catalytic efficiency and economic benefit, a glucose dehydrogenase coenzyme circulation system is constructed. The glucose dehydrogenase is derived from chitinolytic Bacillus (Bacillus chitinosolvens) and the mannitol dehydrogenase is derived from Bacillus sp. (Bacillus sp.). Paenibacillus lemnae), NCBI Accession No. WP_377596702.1, the nucleotide sequence is shown as SEQ ID NO. 7, and the amino acid sequence is shown as SEQ ID NO. 8.

[0061] Primers F3, R3, F4 and R4 (the nucleotide sequence of F3 is shown as SEQ ID NO. 9, the nucleotide sequence of R3 is shown as SEQ ID NO. 10, the nucleotide sequence of F4 is shown as SEQ ID NO. 11, and the nucleotide sequence of R4 is shown as SEQ ID NO. 12) were designed according to the nucleotide sequence shown in SEQ ID NO. 7 and the pET-duet-E2K-E176Q-I195R-V325K vector sequence, and PlGDH was cloned into the second multiple cloning site of pET-duet-E2K-E176Q-I195R-V325K.

[0062] F3: 5'-tataagaaggagatatacatATGTATAAAGATCTAGAAGGAAAGACAGTAGTC-3';

[0063] R3: 5'-gtttctttaccagactcgagTTAACCGCGACCACCCTGG-3';

[0064] F4: 5'-CTCGAGTCTGGTAAAGAAACCGC-3';

[0065] R4: 5'-ATGTATATCTCCTTCTTATACTTAACTAATATACTAAGA-3';

[0066] The co-expression plasmid pETduet-WpMDH-E2K-E176Q-I195R-V325K-PlGDH was constructed using pET-duet plasmid as an expression vector.

[0067] The transformation process was according to Example 1, and the strain E. coli BL21(DE3) / pETduet-WpMDH-E2K-E176Q-I195R-V325K-PlGDH.

[0068] Example 6, Application of mannitol dehydrogenase in catalyzing synthesis of D-mannitol:

[0069] The co-expression strain obtained in Example 4 E. coliBL21(DE3) / pETduet-WpMDH-E2K-E176Q-I195R-V325K-PlGDH was inoculated into LB liquid medium containing 50 μg / mL ampicillin at a final concentration, and cultured at 37 ℃ for 9 h 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 bacteria were cultured at 37 ℃ and 500 rpm for 3-4 h until the optical density (OD) reached 6-8. Then, the temperature of the fermenter was reduced to 25 ℃, and 10 g / L of lactose was added as an inducer. The bacteria were then cultured at 25 ℃ and 500 rpm for 12 h. The fermentation liquid was centrifuged at 8000 rpm for 10 min to obtain a wet bacterial cell containing the co-expressed strain E. coli BL21(DE3) / pETduet-WpMDH-E2K-E176Q-I195R-V325K-PlGDH was inoculated into LB liquid medium containing 50 μg / mL ampicillin at a final concentration, and cultured at 37 ℃ for 9 h 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 bacteria were cultured at 37 ℃ and 500 rpm for 3-4 h until the optical density (OD) reached 6-8. Then, the temperature of the fermenter was reduced to 25 ℃, and 10 g / L of lactose was added as an inducer. The bacteria were then cultured at 25 ℃ and 500 rpm for 12 h. The fermentation liquid was centrifuged at 8000 rpm for 10 min to obtain a wet bacterial cell containing the co-expressed strain

[0070] The fermentation medium was composed of 45 g of tryptone, 36 g of yeast extract, 30 g of sodium chloride, 4.08 g of potassium dihydrogen phosphate, 45 g of glycerol (glycerin), 6.84 g of potassium phosphate dibasic trihydrate, 15 g of ammonium sulfate, 1.125 g of magnesium sulfate, and 3 g of antifoaming agent, and was dissolved in distilled water to a final volume of 3 L.

[0071] The catalyst was used at a dosage of 5 g / L based on the total weight of the wet bacterial cells, and the final concentrations of D-fructose, glucose, and zinc sulfate were 100 g / L, 110 g / L, and 1 mM, respectively. The reaction medium was a phosphate buffer at pH 7, and the total volume of the reaction solution was 1 L. The reaction conditions were 35 ℃ and 300 rpm for 12 h. After the reaction, 20 μL of the reaction sample was diluted 30 times, filtered through a 0.22 μm filter, and subjected to HPLC detection. The concentration of D-mannitol after the reaction was 48.7 g / L.

[0072] The catalyst was used at a dosage of 30 g / L based on the total weight of the wet bacterial cells, and the final concentrations of D-fructose, glucose, and zinc sulfate were 180 g / L, 200 g / L, and 1 mM, respectively. The reaction medium was a phosphate buffer at pH 7, and the total volume of the reaction solution was 1 L. The reaction conditions were 35 ℃ and 300 rpm for 12 h. After the reaction, 20 μL of the reaction sample was diluted 30 times, filtered through a 0.22 μm filter, and subjected to HPLC detection. The concentration of D-mannitol after the reaction was 128.9 g / L.

[0073] The catalyst amount is 25 g / L based on the total weight of the wet bacteria, the final concentration of D-fructose is 150 g / L, the final concentration of glucose is 165 g / L, the final concentration of zinc sulfate is 1 mM, the reaction medium is phosphate buffer with pH 7, and the total volume of the reaction solution is 1 L. The reaction conditions are 35°C, 300 rpm for 12 h. After the reaction is completed, 20 μL of the sample at the end of the reaction is taken, diluted 30 times, filtered through a 0.22 μm filter membrane, and subjected to HPLC detection. The concentration of D-mannitol after the reaction is completed is 135.6 g / L, and the reaction progress is shown in Table 1. Figure 1 .

[0074] The preferred embodiments of the present application have been described above, but the present application is not limited to the above examples, and various modifications and changes can be made by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A mannitol dehydrogenase mutant, characterized in that, The amino acid sequence of wild-type mannitol dehydrogenase, as shown in SEQ ID NO.2, is mutated as follows: glutamic acid at position 2 is replaced by lysine, glutamic acid at position 176 is replaced by glutamine, isoleucine at position 195 is replaced by arginine, and valine at position 325 is replaced by lysine.

2. A gene encoding the mannitol dehydrogenase mutant of claim 1 or simultaneously encoding the mannitol dehydrogenase mutant of claim 1 and glucose dehydrogenase.

3. An expression box, characterized in that, It contains the gene described in claim 2.

4. A recombinant expression vector, characterized in that, It contains the gene described in claim 2.

5. The recombinant expression vector as described in claim 4, characterized in that, Using pET-duet plasmid as an expression vector, mannitol dehydrogenase with the amino acid sequence shown in SEQ ID NO.2 was cloned into the first multiple cloning site of pET-duet. After site-directed mutagenesis, glucose dehydrogenase P1GDH was constructed into the second multiple cloning site of the expression plasmid vector pET-duet. The amino acid sequence of glucose dehydrogenase P1GDH is shown in SEQ ID NO.

8.

6. A recombinant bacterium, characterized in that, It comprises the gene of claim 2 or the recombinant expression vector of any one of claims 4 to 5.

7. The use of the mannitol dehydrogenase mutant of claim 1 or the gene of claim 2 in the catalytic synthesis of D-mannitol.

8. A method for synthesizing D-mannitol, characterized in that, include: Using wet bacterial cells obtained by induction culture of the recombinant bacteria described in claim 6, or crude enzyme solution extracted by ultrasonic disruption of wet bacterial cells, or immobilized enzyme as a catalyst, D-fructose as a substrate, and purified water as a reaction medium, a reaction system is constructed to obtain D-mannitol.

9. The method as described in claim 8, characterized in that, The amount of catalyst used is 5~30 g / L based on the total weight of wet bacterial cells.

10. The method as described in claim 8, characterized in that, The final concentration of the substrate D-fructose is 100 g / L to 180 g / L; the final concentration of the co-substrate glucose is 110 g / L to 200 g / L.

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