A 2,3-butanediol dehydrogenase mutant and its application

Abstract

The invention discloses a 2,3-butanediol dehydrogenase mutant and its application, and relates to the field of genetic engineering. The present invention carries out site-directed mutagenesis to SmBDH sequence, and improves its oxidoreductase activity. Compared with wild type, the mutant to which the present invention relates, with D245C mutant activity improvement is the most significant, its oxidase activity reaches 128.77U / mg, which is 7.64 times of wild type enzyme activity (16.86U / mg); reductase activity can reach up to 200.28U / mg, which is 4.42 times of wild type enzyme activity (45.33U / mg). The inventive method effectively improves the redox ability of 2,3-butanediol dehydrogenase, is conducive to solving the problem of low efficiency of enzymatic synthesis of 2,3-butanediol / acetoin, alleviates the problems such as high cost, serious pollution, and cumbersome steps brought by chemical synthesis, and is conducive to the industrial application of biological synthesis of 2,3-butanediol / acetoin.

Description

Technical Field

[0001] The invention relates to the field of genetic engineering, and in particular to a 2,3-butanediol dehydrogenase mutant and application thereof. Background Art

[0002] 2,3-Butanediol is a valuable platform compound. Acetoin and diacetyl synthesized by its decarboxylation reaction can be used as food flavor additives and are widely used in food, medicine, energy and other fields. The structure of acetoin is 3-hydroxy-2-butanone (acetoin), which is the smallest natural chiral α-hydroxyketone with one chiral center and exists in two stereoisomers, (R)-acetoin and (S)-acetoin. At present, acetoin has been used as an additive, preservative, and plant growth promoter. At the same time, acetoin has been listed as one of the 30 potential compounds for priority development by the US Department of Energy and is widely used in food, cosmetics, especially in the asymmetric synthesis of optically active drugs and liquid crystals. Acetoin has a wide range of application value in food flavoring, biochemistry and pharmacology. At present, there are relatively in-depth studies on its synthesis and production at home and abroad. Some bacteria in nature have the ability to produce acetoin, including Klebisella, Enterobacter, Bacillus, Serratia, and Lactococcus. However, in the metabolic process of most strains, acetoin exists as a byproduct of the metabolism of 2,3-butanediol and diacetyl, and the accumulation concentration is low, which directly leads to the difficulty of using these microbial strains to produce acetoin by industrial fermentation.

[0003] With the scarcity of petroleum fuels, bio-based chemicals and bioproducts have attracted more and more attention. 2,3-Butanediol / acetoin and its derivatives are a very important class of chiral compounds, which have extremely important application value in the synthesis of fine chemicals and chiral drugs.

[0004] At present, the production methods of acetoin in industry are chemical synthesis and biosynthesis. Among them, the chemical synthesis method has problems such as high cost, serious environmental pollution, and long preparation cycle. In comparison, the biosynthesis method for preparing 2,3-butanediol / acetoin is more in line with the principle of sustainable green development, and has attracted widespread attention from researchers in recent years. The method of producing 2,3-butanediol / acetoin by microbial fermentation greatly reduces the production cost, but the fermentation method has problems such as by-product accumulation and low yield. Therefore, the enzymatic method of cloning and expressing 2,3-butanediol dehydrogenase or acetoin reductase from natural strains has a broader development space, and 2,3-butanediol / acetoin produced by biological enzyme catalysis has a very large application prospect.

[0005] The existing research on the enzymatic production of 2,3-butanediol / acetoin is still in the development stage. The existing 2,3-butanediol dehydrogenase has problems such as low enzyme activity and is still difficult to meet the requirements of industrial preparation of 2,3-butanediol / acetoin. Therefore, it is necessary to develop bio-enzyme catalysts with significantly improved industrial applicability to further increase the yield of the target product and promote the industrial application process of enzymatic synthesis of 2,3-butanediol / acetoin. Summary of the invention

[0006] The present invention starts from 2,3-butanediol dehydrogenase (BDH for short, ec1.1.1.4) from Serratia marcescens, and aims to modify the gene through site-directed mutagenesis to improve the enzyme activity of SmBDH, so as to further increase the output of 2,3-butanediol / acetoin and lay a foundation for industrial production.

[0007] In order to solve the problems of low efficiency in the enzymatic conversion and synthesis of 2,3-butanediol / acetoin and low activity of 2,3-butanediol dehydrogenase, the present invention provides a method for improving the activity of 2,3-butanediol dehydrogenase by site-directed mutagenesis.

[0008] In order to solve the problem of low enzyme activity of 2,3-butanediol dehydrogenase, the present invention obtains multiple single-point mutant 2,3-butanediol dehydrogenases through site-directed mutagenesis, and measures their activity through NADH generation / consumption, and screens out mutants with significantly improved catalytic activity: V90I, V92I, A93Q, R106K, K185R, G191D, E195D, I196L, R197L, A209P, A241L, D245N, D245S, D245C and D245T. Among them, the mutants are represented by the following abbreviations: V90I represents: the 90th amino acid is mutated from valine to isoleucine; the same applies to the others.

[0009] The present invention provides a 2,3-butanediol dehydrogenase mutant, which is obtained by mutating a wild-type 2,3-butanediol dehydrogenase from Serratia marcescens, wherein the amino acid sequence of the wild-type 2,3-butanediol dehydrogenase is shown in SEQ ID NO.2, and the specific mutation is any one of the following:

[0010] (1) The amino acid at position 90 mutated from valine to isoleucine;

[0011] (2) amino acid 92 was mutated from valine to isoleucine;

[0012] (3) amino acid position 93 was mutated from alanine to glutamine;

[0013] (4) amino acid 106 was mutated from arginine to lysine;

[0014] (5) The amino acid at position 185 was mutated from lysine to arginine;

[0015] (6) The amino acid at position 191 was mutated from glycine to aspartic acid;

[0016] (7) The amino acid at position 195 was mutated from glutamic acid to aspartic acid;

[0017] (8) The amino acid at position 196 mutated from isoleucine to leucine;

[0018] (9) The amino acid at position 197 mutated from arginine to leucine;

[0019] (10) The amino acid at position 209 was mutated from alanine to proline;

[0020] (11) The amino acid at position 241 was mutated from alanine to leucine;

[0021] (12) The amino acid at position 245 was mutated from aspartic acid to asparagine;

[0022] (13) The amino acid at position 245 was mutated from lysine to serine;

[0023] (14) The amino acid at position 245 was mutated from lysine to cysteine;

[0024] (15) The amino acid at position 245 was mutated from lysine to threonine.

[0025] The present invention also provides application of the 2,3-butanediol dehydrogenase mutant in producing 2,3-butanediol / acetoin.

[0026] The present invention also provides a gene encoding the 2,3-butanediol dehydrogenase mutant.

[0027] The invention also provides application of the gene in producing 2,3-butanediol / acetoin.

[0028] The present invention also provides an expression vector comprising the encoding gene.

[0029] The invention also provides application of the expression vector in producing 2,3-butanediol / acetoin.

[0030] The present invention also provides a genetically engineered bacterium for expressing the 2,3-butanediol dehydrogenase.

[0031] The invention also provides application of the genetically engineered bacteria in producing 2,3-butanediol / acetoin.

[0032] Beneficial effects of the present invention:

[0033] The present invention performs site-directed mutagenesis on the SmBDH sequence to improve its oxidoreductase activity. Compared with the wild type, the mutants of the present invention, D245C and D245S mutants, have the most significant improvement in activity, with oxidase activity reaching 128.77U / mg and 56.44U / mg, which are 7.64 times and 3.34 times that of the wild type enzyme activity (16.86U / mg); the reductase activity can reach up to 200.28U / mg and 198.03U / mg, which are 4.42 times and 4.37 times that of the wild type enzyme activity (45.33U / mg).

[0034] The method of the invention effectively improves the redox ability of 2,3-butanediol dehydrogenase, is conducive to solving the problem of low efficiency of enzymatic synthesis of 2,3-butanediol / acetoin, alleviates the problems of high cost, serious pollution, complicated steps and the like brought by chemical synthesis, and is conducive to the industrial application of biological synthesis of 2,3-butanediol / acetoin. DETAILED DESCRIPTION

[0035] The present invention will be further described in detail below in conjunction with the embodiments of the present invention, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. The experimental methods in the present invention are all conventional methods unless otherwise specified.

[0036] Reagents used in upstream genetic engineering: the genome extraction kit and DpnI used in the examples of the present invention were purchased from TaKaRa, Takara Biotechnology (Dalian) Co., Ltd.; ExnaseII seamless cloning kit was purchased from Nanjing Novagen Biotechnology Co., Ltd.; plasmid extraction kit and DNA recovery and purification kit were purchased from Axygen Hangzhou Co., Ltd.; E.coliBL21 (DE3) was purchased from Novagen; DNA marker, low molecular weight standard protein, and agarose gel electrophoresis reagent were purchased from Beijing Quanshijin Biotechnology Co., Ltd.; primer synthesis, sequence sequencing and full plasmid pET28a-SmBDH were completed by Qingke Biotechnology Co., Ltd.

[0037] The present invention is explained in detail below with reference to the embodiments.

[0038] Example 1

[0039] Construction of E. coli expression vector for BDH mutants.

[0040] (1) BDH whole plasmid PCR: Using the nucleotide sequence shown in SEQ ID NO.1 as a template and the mutant primer sequences in Table 1, PCR was performed to obtain the recombinant genes shown in SEQ ID NO.17 to SEQ ID NO.31.

[0041] Table 1 Mutant primer sequences

[0042]

[0043]

[0044] The PCR amplification system is shown in Table 2:

[0045] Table 2 PCR amplification system

[0046] Element Volume / μL PrimeSTAR Max DNA Polymerase 25 Forward Primer 0.5 Reverse primer 0.5 pET28a-SmBDH 1 <![CDATA[ddH2O]]> Top up to 50

[0047] PCR amplification conditions are as shown in Table 3: 30 cycles in total:

[0048] Table 3 PCR amplification program

[0049]

[0050] (2) Template digestion:

[0051] The PCR product was subjected to agarose gel electrophoresis to detect the band size. After the size was correct, it was recovered and purified using a PCR product purification kit. After purification, the remaining plasmid template was digested with DpnI enzyme. The digestion system is as shown in Table 4:

[0052] Table 4 DpnI digestion system

[0053]

[0054]

[0055] Digestion conditions: 37°C for 1 h, 70°C for 15 min.

[0056] (3) Competent cell transformation and screening:

[0057] The digested product was verified by agarose gel electrophoresis and then transformed into E.coli BL21 (DE3) competent cells using the heat shock method. The specific method is as follows:

[0058] 1) Take out the competent cells from -80℃ and thaw them on ice for 2 minutes;

[0059] 2) Take 10 μL of the ligation product and mix it with the competent E. coli BL21 (DE3) and incubate it on ice for 30 minutes;

[0060] 3) After incubation for 30 minutes, heat shock the centrifuge tube in a 42°C water bath for 1 minute and immediately take it out and incubate it on ice for 3 minutes.

[0061] 4) In a sterile operating table, add 600 μL of LB medium preheated at 37°C to the competent cells, mix well, and place in a 37°C shaker at 200 rpm for 60 min to revive the cells;

[0062] 5) After the cells are revived, centrifuge at 6000 rpm at room temperature for 30 seconds, pour off part of the supernatant in the clean bench, mix the remaining liquid (about 100 μL) with the cells, and evenly spread it on the LB solid culture medium containing Kan antibiotics with a disposable coating rod. After the liquid is completely absorbed, place the plate upside down in a 37°C constant temperature incubator and culture for about 16 hours.

[0063] 3-4 single colonies were picked from each plate for sequencing verification, and mutants V90I, V92I, A93Q, R106K, K185R, G191D, E195D, I196L, R197L, A209P, A241L, D245N, D245S, D245C and D245T were obtained.

[0064] Example 2

[0065] Expression and purification of BDH mutant proteins.

[0066] After the mutant strain sequenced successfully in Example 1 was activated by streaking on a plate, a single colony was picked and placed in 5 mL of LB liquid culture medium containing 100 μg / mL kanamycin, and cultured at 37°C and 180 rpm for 16 h to prepare a recombinant engineering bacterial solution;

[0067] Transfer 2 mL of bacterial seed solution to 200 mL of LB liquid medium containing 100 μg / mL kanamycin (1% inoculum), and continue to culture in a shaker at 37°C and 220 rpm until the OD reaches 600 =0.6-0.8 (about 3-4h);

[0068] Add 200 μL of 1 mol / L IPTG (working concentration is 1 mmol / L) sterilized by filtration through a 0.22 μm pinhole filter, and induce the culture at 18°C ​​and 100 rpm for 16 h;

[0069] Transfer the induced bacterial solution to a 100 mL centrifuge cup, centrifuge at 4°C 6000 rpm for 20 min, discard the supernatant, and add 30 mL of sterilized PBS buffer (pH 7.4) to the centrifuge cup to fully resuspend the bacteria;

[0070] The suspension was centrifuged at 4°C and 6000 rpm for 20 min, the supernatant was discarded, and 60 mL of sterilized PBS buffer was added to the centrifuge cup and transferred to a clean beaker. The beaker was placed on ice to prevent heat generation during ultrasound and inactivation of the protein. The horn was adjusted to the optimal position and ultrasound was used to repeatedly break the protein. The ultrasound conditions were: total ultrasound duration of 20 min, working time of 2 s, interval time of 6 s, and breaking power of 60%;

[0071] The broken bacterial solution was centrifuged at 4°C and 10,000 rpm for 10 min, and the precipitate was discarded and repeated once. The final supernatant was the crude enzyme solution, and the precipitate was the cell fragments. The supernatant was used for subsequent purification immediately or temporarily stored at 4°C;

[0072] Take 30mL of crude enzyme solution, add 1mL of affinity Ni-NTA resin filler, and add imidazole to make the final concentration 3mmol / L, and incubate slowly on ice at 60rpm to allow nickel ions to fully bind to the 6×His tag of the target protein;

[0073] After incubation for 1-2 hours, purify on an empty chromatography column and collect the filtrate (Flow-through, denoted as FT). The flow-through solution can be repeatedly loaded onto the column 2-3 times to increase the yield of the protein.

[0074] Add 10-20 times the volume of the column bed to wash the packing repeatedly and collect it, which is recorded as W, Wash;

[0075] After washing with washing buffer, use 3 column volumes of Elution buffer (about 3 mL) to elute the target protein, collect the eluate, and record it as E, Elution (add Elution buffer along the tube wall to prevent the filler from being washed up);

[0076] After elution, add excess Elution buffer to wash the filler, then add ddH2O to wash away excess Elution buffer, add 20% ethanol to rinse the filler, recover the filler, and store at 4°C;

[0077] The purified sample eluate was desalted in Tris-HCl (pH 8.0) using an ultrafiltration tube, centrifuged at 4°C, 4000 rpm for 30 min, and buffer was added to wash and replace imidazole ions until the final imidazole concentration in the protein was less than 10 mM.

[0078] Washing buffer (20mmol / L imidazole): Take 20mL Tris-HCl (pH8.0) stock solution, 100mL 3mol / L NaCl stock solution, 20mL 1mol / L imidazole, and dilute to 1L with ddH2O.

[0079] Eluent buffer (containing 250mmol / L imidazole): Take 20mL Tris-HCl (pH8.0) stock solution, 10mL 3mol / L NaCl stock solution, 500mL 1mol / L imidazole, and dilute to 1L with ddH2O.

[0080] Example 3

[0081] Determination of enzyme activity of BDH and its mutants.

[0082] BDH enzyme activity definition: The enzyme activity unit is defined as the reduction of 1 μmol of NAD per minute at room temperature. + The amount of enzyme required is one enzyme activity unit U. The specific activity of an enzyme is defined as the enzyme activity per unit protein U / mg.

[0083] BDH enzyme activity assay method: The enzyme reaction system is 200 μL, containing 12.5 mM substrate, 0.56 mM NAD + The enzymatic reaction starts immediately after a certain amount of enzyme solution is added. The NADH / NAD + concentration and calculated the enzyme activity.

[0084] Table 5 Oxidoreductase activity assay results

[0085]

[0086]

[0087] Among them, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001, and ns indicates p > 0.05.

[0088] The protein obtained above was subjected to oxidoreductase activity determination using an ELISA instrument, and the measured data results are shown in Table 5. It can be seen from Table 5 above that the oxidoreductase activity of BDH was improved after site-directed mutagenesis. Among them, it can be seen from Table 5 that compared with the wild-type SmBDH, the oxidation activity of multiple mutants was improved. Among them, the activity of D245C and D245S mutants was improved most significantly, and their oxidase activities were 128.77U / mg and 56.44U / mg, respectively, which were 7.64 times and 3.34 times the wild-type enzyme activity (16.86U / mg), respectively; from the changes in reductase activity, it can be seen that the reductase activity of multiple mutants was significantly improved, among which the reductase activity of D245C and D245S was improved most significantly, and their reductase activities were 200.28U / mg and 198.03U / mg, respectively, which were 4.42 times and 4.37 times the wild-type enzyme activity (45.33U / mg), respectively.

Claims

1. A 2,3-butanediol dehydrogenase mutant, characterized in that: It is obtained by mutating the wild-type 2,3-butanediol dehydrogenase from Serratia marcescens, the amino acid sequence of the wild-type 2,3-butanediol dehydrogenase is shown in SEQ ID NO.2, and the specific mutation is any one of the following: (1) The amino acid at position 245 was mutated from aspartic acid to asparagine; (2) the amino acid at position 245 was mutated from aspartic acid to serine; (3) the amino acid at position 245 was mutated from aspartic acid to cysteine; (4) The amino acid at position 245 was mutated from aspartic acid to threonine.

2. Use of the 2,3-butanediol dehydrogenase mutant as claimed in claim 1 in catalyzing the oxidation of meso-2,3-butanediol.

3. Use of the 2,3-butanediol dehydrogenase mutant as claimed in claim 1 in catalyzing the reduction of acetoin.

4. A gene encoding the 2,3-butanediol dehydrogenase mutant according to claim 1.

5. Use of the gene as claimed in claim 4 in catalyzing the oxidation of meso-2,3-butanediol.

6. Use of the gene as claimed in claim 4 in catalyzing acetoin reduction.

7. An expression vector comprising the coding gene according to claim 4.

8. Use of the expression vector as claimed in claim 7 in catalyzing the oxidation of meso-2,3-butanediol.

9. Use of the expression vector as claimed in claim 7 in catalyzing the reduction of acetoin.

10. A genetically engineered bacterium expressing the 2,3-butanediol dehydrogenase mutant according to claim 1.

11. Use of the genetically engineered bacteria as claimed in claim 10 in catalyzing the oxidation of meso-2,3-butanediol.

12. Use of the genetically engineered bacteria as claimed in claim 10 in catalyzing acetoin reduction.

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