A recombinant Bacillus subtilis producing olefin reductase and alcohol dehydrogenase and its application

Abstract

The present invention provides a recombinant Bacillus subtilis producing ene reductase and alcohol dehydrogenase and its application, belonging to the field of bioengineering technology. The recombinant Bacillus subtilis producing ene reductase and alcohol dehydrogenase is a fusion protein of ene reductase and alcohol dehydrogenase expressed in Bacillus subtilis; the amino acid sequence of the ene reductase has more than 80% sequence identity with the amino acid sequence shown in SEQ ID No.1; the amino acid sequence of the ene reductase has more than 80% sequence identity with the amino acid sequence shown in SEQ ID No.2; the recombinant Bacillus subtilis uses Bacillus subtilis 168 as the starting strain. The recombinant Bacillus subtilis prepared by the present invention can realize the efficient production of ene reductase and alcohol dehydrogenase, and the fermented recombinant strain or its crude enzyme solution can efficiently catalyze compound II to produce compound I, and has good prospects for industrial application.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to a recombinant Bacillus subtilis producing ene reductase and alcohol dehydrogenase and application thereof. Background Art

[0002] Compound I (chemical formula shown in Formula 1 below) is a natural functional whitening raw material, which is prepared by catalytic hydrogenation of Compound II (chemical formula shown in Formula 2 below). The synthesis method from Compound II to Compound I includes chemical hydrogenation method using transition metal as catalyst and biocatalytic hydrogenation catalyzed by olefin reductase. The chemical method has the problems of high cost, unsatisfactory reaction yield and selectivity, use of highly dangerous hydrogen as reducing agent, harsh reaction conditions and environmental pollution. The biological method uses enzymes or whole cells as catalysts, can react at room temperature and pressure, and has the advantages of fast reaction rate, strong catalytic specificity, and green environmental protection.

[0003] It has been reported that a variety of ene reductases can catalyze compound II to prepare compound I. Such enzymes can use NAD(P)H as a cofactor to provide reducing power, such as the ene reductase VvCurA from Vibrio vulnificus and the ene reductase EcCurA from Escherichia coli. Currently, the expression and application research of ene reductase mainly uses Escherichia coli and Saccharomyces cerevisiae as host cells.

[0004] As an expression host, Escherichia coli has a wide range of applications in the field of biotechnology, but it also has the disadvantage of containing a variety of endotoxins, which may have toxic effects on host cells or organisms. When using the E. coli expression system to produce proteins, appropriate measures need to be taken to remove or reduce the content of these endotoxins to ensure the safety and effectiveness of the product. As a GRAS strain, Saccharomyces cerevisiae plays an important role in food industry and drug production, but it also has the problems of long fermentation cycle and high production cost. Therefore, it is urgent to seek a method for safely and simply producing ene reductase and preparing compound I. Summary of the invention

[0005] In view of the above problems existing in the prior art, the present invention provides a recombinant Bacillus subtilis producing ene reductase and alcohol dehydrogenase and its application. The recombinant Bacillus subtilis can achieve efficient production of ene reductase and alcohol dehydrogenase, and the fermented recombinant strain or its crude enzyme solution can efficiently catalyze compound II to produce compound I, which has good prospects for industrial application.

[0006] The technical solution of the present invention is as follows:

[0007] The first object of the present invention is to provide a recombinant Bacillus subtilis producing alkene reductase and alcohol dehydrogenase, that is, DNA encoding an alkene reductase and an alcohol dehydrogenase is expressed in Bacillus subtilis;

[0008] The amino acid sequence encoded by the olefin reductase DNA has a sequence identity of more than 80% with the amino acid sequence shown in SEQ ID No. 1;

[0009] The amino acid sequence encoded by the alcohol dehydrogenase DNA has a sequence identity of more than 80% with the amino acid sequence shown in SEQ ID No.2.

[0010] Preferably, the recombinant Bacillus subtilis uses Bacillus subtilis 168 as the starting strain.

[0011] Preferably, the alkene reductase and alcohol dehydrogenase are expressed in the form of a fusion protein, with the alkene reductase located at the N-terminus of the fusion protein and the alcohol dehydrogenase located at the C-terminus of the fusion protein, and the two are connected by a flexible connecting peptide.

[0012] The second object of the present invention is to provide a method for improving the total activity of ene reductase and alcohol dehydrogenase in the recombinant Bacillus subtilis, that is, knocking out the genes lytC, pcfA and cwlC in the strain Bacillus subtilis 168. Further, the genes lytC, pcfA and cwlC are related to cell lysis.

[0013] The third object of the present invention is to provide a method for producing alkene reductase and alcohol dehydrogenase, wherein the alkene reductase and alcohol dehydrogenase are obtained by inoculating the recombinant Bacillus subtilis into a fermentation medium for fermentation.

[0014] Preferably, the fermentation temperature is 20-45°C and the fermentation time is 12-60h.

[0015] The fourth object of the present invention is to provide a biocatalytic method for preparing compound I from compound II, wherein the catalytic method uses the olefin reductase and alcohol dehydrogenase to catalyze compound II to obtain compound I;

[0016] The chemical formula of the compound I is shown in Formula 1 below:

[0017]

[0018] The chemical formula of the compound II is shown in Formula 2 below:

[0019]

[0020] Preferably, the catalytic method comprises the following raw materials:

[0021] Broken or unbroken recombinant Bacillus subtilis: dosage is 5-50g / L wet bacteria;

[0022] Compound II: dosage is 1-50g / L;

[0023] Isopropyl alcohol: dosage is 10-70g / L;

[0024] NADP + : The dosage is 0.01-1mM;

[0025] The solution pH of the catalytic system is 5.5-7.5, the temperature of the catalytic reaction is 25-45° C., and the time of the catalytic reaction is 1-72 hours.

[0026] The beneficial technical effects of the present invention are:

[0027] 1. The recombinant Bacillus subtilis producing ene reductase and alcohol dehydrogenase provided by the present invention increases the expression level and total activity of ene reductase and alcohol dehydrogenase. The fermented recombinant strain or its crude enzyme solution can efficiently catalyze compound II to generate compound I, laying a foundation for the industrial production of compound I.

[0028] 2. The present invention also knocks out genes of the starting bacteria on the basis of preparing recombinant Bacillus subtilis producing ene reductase and alcohol dehydrogenase. The recombinant Bacillus subtilis producing ene reductase and alcohol dehydrogenase prepared by the starting bacteria after knocking out the lytC, pcfA and cwlC genes can increase the yield by about two times when catalyzing compound II to produce compound I. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematic diagram for the construction of recombinant plasmids;

[0030] Figure 2 SDS-PAGE analysis of the fermentation broth of the recombinant strain;

[0031] Figure 3 HPLC analysis of compound I standard (A), compound II standard (B) and catalytic product (C);

[0032] Figure 4 Schematic diagram for CRISPR Cas9 editing plasmid construction. DETAILED DESCRIPTION

[0033] The present invention is described in detail below in conjunction with the accompanying drawings and embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] Example 1: Construction of strains expressing olefin reductase and alcohol dehydrogenase

[0035] A1. Construction of recombinant plasmid:

[0036] (1) Using the genome of Escherichia coli BL21 (DE3) as a template, primer pairs curA-F1 / curA-R1, curA-F1 / curA-R2, and curA-F2 / curA-R1 were used to amplify the olefin reductase curA gene fragments containing different homology arms, respectively. The amino acid sequence encoded by the gene is shown in SEQ ID No. 1;

[0037] (2) Using an artificially synthesized alcohol dehydrogenase gene fragment as a template, the amino acid sequence encoded by the gene is shown in SEQ ID No. 2, and the DNA sequence is shown in SEQ ID No. 3, primer pairs adh-F1 / adh-R1, adh-F1 / adh-R2, and adh-F2 / adh-R1 are used to amplify alcohol dehydrogenase adh gene fragments containing different homology arms, respectively; wherein the alcohol dehydrogenase amino acid sequence is derived from Levilactobacillus brevis ATCC 14869, and SEQ ID No. 3 is an optimized artificial DNA synthesized based on the amino acids from the source.

[0038] (3) Using the pP43NMK plasmid as a template, the primer pair P43-F / P43-R was used to amplify the P 43 Promoter plasmid backbone.

[0039] The above fragments were amplified by PCR using a high-fidelity enzyme, and the primer sequences are shown in Table 1.

[0040] Table 1 Primer sequences

[0041]

[0042]

[0043] (4) Detect the size of PCR product bands by agarose gel electrophoresis. After verifying the correct bands, recover the bands and construct the recombinant plasmids by Gibson assembly. The schematic diagram of plasmid construction is shown in the attached figure. Figure 1As shown, the recombinant plasmid expressing olefin reductase curA alone was named pP43curA, the recombinant plasmid expressing alcohol dehydrogenase adh alone was named pP43adh, the recombinant plasmid expressing adh-curA fusion protein (adh at the N-terminus and curA at the C-terminus) through a flexible connecting peptide was named pP43adh-curA, and the recombinant plasmid expressing curA-adh fusion protein (curA at the N-terminus and adh at the C-terminus) through a flexible connecting peptide was named pP43curA-adh.

[0044] (5) The recombinant plasmid was transformed into E. coli DH5α competent cells by the heat shock method, and a single clone was selected for sequencing verification. The correct recombinant plasmid was stored at -20°C for future use.

[0045] A2. Bacillus subtilis electrotransformation:

[0046] (1) Take a glycerol strain of Bacillus subtilis 168, streak it on a non-antibiotic LB solid medium, and invert it at 37°C for 12 hours. Pick a fresh single colony and place it in 5 mL LB medium, and culture it at 37°C, 200 rpm overnight; transfer it to 100 mL GM medium (LB medium supplemented with 0.5 M sorbitol) at a volume ratio of 3%, and culture it at 37°C, 200 rpm for 3-4 hours until the OD reaches 0. 600 It is about 1.0.

[0047] (2) After the cultured bacterial solution was ice-bathed for 10 min, it was centrifuged at 4°C, 5000 g for 10 min to collect the bacterial cells, and 40 mL of ice-cold ETM solution (0.5 M sorbitol, 0.5 M mannitol, 10% glycerol) was used to resuspend the bacterial cells, and the cells were centrifuged at 4°C, 5000 g for 5 min. The washing was repeated three times; the washed bacterial cells were resuspended with EMT at a volume ratio of 1:150 to a cell density of 10 10 / mL, 60μL per tube was dispensed into 1.5mL centrifuge tubes and stored at -80℃ for later use. This is the prepared competent state.

[0048] (3) Thaw the competent cell on ice, add 5 μL of the recombinant plasmid, mix well and let stand on ice for 5 min, then transfer to a pre-cooled 1 mm electroporation cup, set the electroporation instrument parameters to 25 μF, 200 Ω, 1700 V, and shock once. The shock time is about 4.5 ms to 5.0 ms. Immediately after electroporation, add 1 mL of pre-cooled RM medium (LB supplemented with 0.5 M sorbitol and 0.38 M mannitol), and then culture at 37 °C, 200 rpm for 1 h.

[0049] (4) After the culture was completed, 100 μL of the bacterial solution was spread on a LB solid medium containing 50 μg / mL kanamycin and cultured overnight at 37°C. The recombinant Bacillus subtilis containing the recombinant plasmids pP43curA, pP43adh, pP43adh-curA, and pP43curA-adh were named BS01, BS02, BS03, and BS04, respectively.

[0050] Example 2: Fermentation expression and detection of olefin reductase and alcohol dehydrogenase

[0051] B1. Prepare culture medium:

[0052] (1) The fermentation medium composition is as follows: 20 g / L glycerol, 10 g / L yeast extract, 5 g / L disodium hydrogen phosphate dodecahydrate, 3 g / L potassium dihydrogen phosphate, 1 g / L ammonium chloride, 0.5 g / L sodium chloride, 0.5 g / L magnesium sulfate heptahydrate, 0.5 g / L tryptophan, and 0.01 g / L calcium chloride.

[0053] (2) The feed medium composition is: 400 g / L glycerol, 40 g / L yeast extract, and 5 g / L tryptophan. Except for tryptophan, which is sterilized by wet heat sterilization, the remaining components are mixed evenly and sterilized by high pressure wet heat sterilization at 121°C for 30 min.

[0054] B2. Bacterial culture and detection:

[0055] (1) The recombinant Bacillus subtilis glycerol bacteria prepared in Example 1 were inoculated into 50 mL of LB medium containing 1% glucose and cultured at 37°C and 200 rpm for 12 h. Then, the inoculum was transferred to a 5 L fermenter containing 2 L of fermentation medium. The temperature was maintained at 37°C and the pH was 6.8. 25% ammonia water was used to automatically adjust the pH. The dissolved oxygen (DO) was maintained at more than 20% by controlling the rotation speed and ventilation volume. The foam generation was controlled by adding a defoamer. After about 9 h of culture, the DO suddenly increased, and the feed medium was added at a rate of 6-10 g / L / h. After 24 h of culture, the fermentation was stopped and the bacteria were collected by centrifugation at 7000 g.

[0056] (2) Resuspend the collected cells in deionized water and control the OD 600 For 4, SDS-PAGE protein electrophoresis analysis was performed, 5× loading buffer was added to the sample and the sample was treated in a boiling water bath for 10 min. The concentration of the concentrated gel used was 5%, the concentration of the separating gel was 12%, and the sample volume was 10 μL.

[0057] (3) SDS-PAGE results are as follows Figure 2As shown, compared with the control, the figure shows that strain BS01 has an obvious overexpression band, the size of which is consistent with curA, indicating that curA is successfully expressed in this strain; strain BS02 has no obvious overexpression band, indicating that the expression level of adh protein in this strain is extremely low or not expressed; strain BS03 shows a weaker overexpression band, the size of which is consistent with the size of adh-curA fusion protein, indicating that the fusion protein is successfully expressed in this strain; strain BS04 shows the most obvious overexpression band, the size of which is consistent with the size of adh-curA fusion protein, indicating that the expression level of the fusion protein in this strain is high.

[0058] Example 3: Preparation of Compound I by Catalysis of Crude Enzyme Liquid of Recombinant Strain

[0059] S1. Catalytic process:

[0060] (1) Prepare different catalytic systems according to the following ratios

[0061] Table 2 Content of each component in the catalytic system

[0062] Reaction ① Reaction ② Reaction ③ Bacteria BS01 20g / L, BS02 20g / L BS03 20g / L BS04 20g / L Compound II 10g / L 10g / L 10g / L Isopropyl alcohol 50g / L 50g / L 50g / L <![CDATA[NADP + ]]> 1mM 1mM 1mM

[0063] The bacteria were cultured using the method of step B2-(1) of Example 2, and were added in the form of crude enzyme solution after being crushed by high-pressure homogenization. The pH was adjusted to 6.5 using hydrochloric acid or NaOH solution.

[0064] (2) Take 50 mL of the prepared catalytic system and stir catalyze it in a round-bottom flask under water. Control the temperature at 30±5°C and catalyze for 24 hours to obtain a catalytic solution.

[0065] S2. Test results:

[0066] (1) Take 200 μL of the catalyst solution, add 1 mL of ethyl acetate for extraction, vortex and oscillate for 1 min, and then centrifuge at 8000 g for 5 min. After centrifugation, the organic phase and the aqueous phase are separated, and the organic phase is separated and the ethyl acetate is removed by nitrogen blowing. Dissolve the precipitate with 1 mL of chromatographic methanol, dilute it a certain number of times, and filter it with a 0.22 μm organic filter membrane. Add the filtrate to a liquid sample bottle and wait for high performance liquid chromatography (HPLC) detection and analysis.

[0067] (2) Accurately weigh 100.0 mg of the standard sample of Compound I, place it in a 100 mL volumetric flask, add an appropriate amount of chromatographically pure acetonitrile to dissolve, and dilute to the mark with acetonitrile to prepare a standard stock solution with a concentration of 1.00 mg / mL. Accurately draw a certain volume of the standard stock solution, dilute it step by step with acetonitrile, and prepare standard working solutions with concentrations of 0.80 mg / mL, 0.60 mg / mL, 0.40 mg / mL, 0.20 mg / mL, and 0.10 mg / mL. Filter through a 0.22 μm microporous membrane for HPLC determination.

[0068] (3) Adjust the HPLC instrument according to the chromatographic conditions described in Table 3 below, and take 10 μL of the standard working solution of the compound of appropriate concentration and the sample for liquid chromatography determination, and obtain the peak area of ​​the standard working solution and the peak area of ​​the test solution, respectively. If the peak area in the test sample is significantly different from the peak area in the standard working solution, adjust the dilution factor of the test solution and then perform the determination.

[0069] Table 3 Chromatographic conditions

[0070]

[0071]

[0072] (4) Test results are as follows Figure 3 Under the above chromatographic conditions, compound I exists in two forms, the retention time of the keto form is about 5 minutes, the retention time of the enol form is 9 minutes, and the retention time of compound II is about 12 minutes.

[0073] Let the concentration of the standard working solution be X, and the total peak area of ​​the keto and enol structures of compound I measured by the UV detector be Y. Calculate the linear regression equation and correlation coefficient R 2 When R 2 When ≥0.999, the standard curve is available. The yield of compound I is calculated by comparing the mass of compound I calculated in the reaction solution with the mass of compound II added initially.

[0074] The yield of compound I was 11.4% when the crude enzyme solution of strain BS01 and BS02 was used for catalysis together; the yield of compound I was 23.6% when the crude enzyme solution of strain BS03 was used for catalysis alone; the yield of compound I was 46.4% when the crude enzyme solution of strain BS04 was used for catalysis alone. This shows that the total activity of olefin reductase curA and alcohol dehydrogenase adh is low when they are expressed separately. The total activity is higher than that of individual expression when fusion expression is used, and the total activity is highest when olefin reductase curA is located at the N-terminus of the fusion protein and alcohol dehydrogenase adh is located at the C-terminus of the fusion protein.

[0075] Example 4: Gene knockout improves the catalytic activity of recombinant Bacillus subtilis crude enzyme solution

[0076] D1. Gene knockout method:

[0077] CRISPR Cas9 gene editing technology was used to knock out the genes of the starting strain B. subtilis 168. The pJOE8999 plasmid was used as the knockout plasmid, and the sgRNA sequence and repair template were simultaneously integrated into the plasmid. The corresponding sgRNA sequence and upstream and downstream homology arms were amplified using the primer pair pJOE-F / pJOE-R, and pJOE8999 was amplified to obtain a linearized plasmid. The corresponding recombinant editing plasmid was then constructed by Gibson assembly. The schematic diagram of plasmid construction is shown in Figure 4 , the primer sequences are shown in Table 4 below.

[0078] Table 4 Primer sequences

[0079]

[0080]

[0081] D2. Strain culture method:

[0082] According to the electroporation transformation method in Example 1, the editing plasmid was electroporated into B. subtilis 168 competent cells, the recovery solution was spread on a plate containing kanamycin, and cultured at 30°C. The correct transformants were selected and transferred to LB liquid culture medium containing 0.5% mannose and kanamycin for culture at 30°C and 200 rpm for 12 h, and then streaked onto antibiotic-free LB solid culture medium to remove the editing plasmid containing the temperature-sensitive replicon.

[0083] D3. Preparation of recombinant strains after gene knockout:

[0084] Primers were designed based on the genomic DNA sequence of B. subtilis 168 to verify the gene knockout, and the knockout strain with the correct verification was prepared as a competent state for the next round of gene knockout. After knocking out the lytC, pcfA, and cwlC genes in sequence, the competent state was prepared again and transferred into the recombinant plasmid pP43curA-adh to obtain a recombinant strain with the genotype of ΔlytCΔpcfAΔcwlC OEcurA-adh, which was named BS05.

[0085] D4, the recombinant strain after gene knockout catalyzes the preparation of compound Ⅰ:

[0086] The strain BS05 was fermented and catalyzed, and the addition amount of compound II was set to 10g / L, 20g / L, and 40g / L, respectively, and the other conditions were the same as in Example 2 and Example 3. After 24 hours of catalysis, when the addition concentration of compound II was 10g / L, the yield of compound I was 89.1%; when the addition concentration of compound II was 20g / L, the yield of compound I was 82.3%; when the addition concentration of compound II was 40g / L, the yield of compound I was 71.4%. This shows that knocking out the lytC, pcfA, and cwlC genes increases the total activity of ene reductase and alcohol dehydrogenase in recombinant Bacillus subtilis.

[0087] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and implementation modes, and can be fully applicable to various fields suitable for the present invention. For those familiar with the art, for those of ordinary skill in the art, various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principle and spirit of the present invention. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.

Claims

1. A biocatalytic method for preparing compound I from compound II, characterized in that: The catalytic method uses olefin reductase and alcohol dehydrogenase to catalyze compound II to prepare compound I; The chemical formula of the compound I is shown in Formula 1 below: (Formula 1); The chemical formula of the compound II is shown in Formula 2 below: (Formula 2); The dosage of compound II is 10-50 g / L; The ene reductase and alcohol dehydrogenase are obtained by inoculating recombinant Bacillus subtilis into a fermentation medium for fermentation; The recombinant Bacillus subtilis is a DNA encoding an alkene reductase and an alcohol dehydrogenase expressed in Bacillus subtilis; The olefin reductase and alcohol dehydrogenase are expressed in the form of fusion proteins, using pP43NMK plasmid as template. 43 The alkene reductase is located at the N-terminus of the fusion protein, and the alcohol dehydrogenase is located at the C-terminus of the fusion protein, and the two are connected by a flexible connecting peptide; The amino acid sequence encoded by the olefin reductase DNA is shown in SEQ ID No. 1; The amino acid sequence encoded by the alcohol dehydrogenase DNA is shown in SEQ ID No. 2; The recombinant Bacillus subtilis Bacillus subtilis 168 is the starting strain; knockout strain Bacillus subtilis Genes in 168 LYT , pcfA and C .

2. The biocatalytic method according to claim 1, characterized in that The fermentation temperature is 20-45°C, and the fermentation time is 12-60 hours.

3. The biocatalytic method according to claim 1, characterized in that: The catalytic method comprises the following raw materials: Broken or unbroken recombinant Bacillus subtilis: dosage is 5-50 g / L wet bacteria; Isopropyl alcohol: dosage is 10-70 g / L; NADP + :The dosage is 0.01-1 mM; The solution pH of the catalytic system is 5.5-7.5, the temperature of the catalytic reaction is 25-45° C., and the time of the catalytic reaction is 1-72 h.

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