Biosynthesis method for producing 3,5-dihydroxy-4-methoxybenzyl alcohol using bacillus subtilis

Abstract

Provided is a biosynthesis method for producing 3,5-dihydroxy-4-methoxybenzyl alcohol using Bacillus subtilis. The method comprises constructing, by means of a genetic engineering, a recombinant plasmid containing genes of key enzymes (carboxylic acid reductase and O-methyltransferase) required for the synthesis of 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA), transforming the recombinant plasmid into Bacillus subtilis, and allowing same to perform biosynthesis in a culture medium containing a shikimic acid to finally obtain DHMBA by means of isolation and purification. The method is characterized by high efficiency, cost-effectiveness, and environmental friendliness, and is applicable to industrial production.

Description

A biosynthesis method for producing 3,5-dihydroxy-4-methoxybenzyl alcohol by using bacillus subtilis TECHNICAL FIELD

[0001] The present application belongs to the field of genetic engineering and biotechnology, and particularly relates to a biosynthesis method for producing 3,5-dihydroxy-4-methoxybenzyl alcohol by using bacillus subtilis. BACKGROUND

[0002] 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA) contains three hydroxyl groups, and has a chemical formula of C8H 10 O4, is a phenolic organic compound extracted from filter-feeding bivalves, and has strong antioxidant effect. DHMBA is a rare antioxidant that has both immediate direct antioxidant effect and sustainable indirect antioxidant effect. Due to its hydrophilic and lipophilic properties, it can penetrate into cell membranes with high oil content and cells with high water content, and active oxygen free radicals in the deep part of the cell can also be fully oxidized and eliminated. Studies have shown that the antioxidant activity of DHMBA is about 2.3 times that of representative antioxidant vitamin C, and 6 times that of chlorogenic acid. At the same time, the safety (IC 50 ) of DHMBA is 5 times that of curcumin and lycopene, and it is a safe and stable super antioxidant.

[0003] In terms of physiological functions, DHMBA is particularly beneficial to liver health, can protect liver cells from oxidative stress-induced damage and apoptosis, and at the same time, DHMBA helps the normal operation of glutamatergic neuronal activity, helps the memory and learning function of the brain, and reduces emotional anxiety; secondly, it helps to reduce body weight and reduce fatty liver; also has the functions of accelerating the penetration of the blood-brain barrier, promoting sleep; improving memory, delaying senile dementia; promoting hair regrowth, preventing hair loss, etc. The related effects are supported by a large amount of clinical data as well as animal model and cell experiment tests.

[0004] In summary, DHMBA is a powerful and safe antioxidant substance, and has great development prospects in the fields of beauty and daily use, pet feed, health products, medicine, etc.

[0005] The Chinese patent "Method for generating 3,5-dihydroxy-4-methoxybenzyl alcohol from oyster meat" with patent application number CN201480081399.9 first mentioned using oyster meat processing to obtain 3,5-dihydroxy-4-methoxybenzyl alcohol, and the method is to heat treat oyster meat directly at a temperature above 98℃, or heat treat after pressurization above 90℃, which belongs to the extraction method of DHMBA. The Chinese patent "Method for fermenting 3,5-dihydroxy-4-methoxybenzyl alcohol and application of the method" with patent application number CN202310206016.7 mentioned using oyster meat for fermentation, and obtained oyster meat powder containing DHMBA. Since DHMBA is found in oysters, this method simulates the natural production of DHMBA, and is not synthesized by genetic engineering. SUMMARY

[0006] In view of the problems existing in the prior art, the purpose of the present application is to provide a biosynthesis method for producing DHMBA by using Bacillus subtilis. The method constructs a recombinant plasmid containing key enzyme genes required for synthesizing DHMBA by genetic engineering means, and transforms it into Bacillus subtilis, so that it can biosynthesize in a medium containing shikimic acid, and finally obtain DHMBA by separation and purification.

[0007] To achieve the above-mentioned purpose of the application, the present application provides the following technical scheme:

[0008] A biosynthesis method for producing DHMBA by using Bacillus subtilis, comprising the following steps:

[0009] (1) Constructing a recombinant plasmid containing carboxylate reductase and O-methyltransferase genes;

[0010] (2) Transforming the recombinant plasmid into Bacillus subtilis to obtain a recombinant Bacillus subtilis;

[0011] (3) Culturing the recombinant Bacillus subtilis in a medium containing shikimic acid;

[0012] (4) Separating and purifying the obtained DHMBA from the medium.

[0013] The carboxylate reductase is derived from Streptomyces, and its coding gene is carA gene, and the nucleotide sequence is shown in SEQ ID NO. 1.

[0014] The O-methyltransferase is derived from Streptomyces, and its coding gene is omt gene, and the nucleotide sequence is shown in SEQ ID NO. 2.

[0015] In the above-mentioned biosynthesis method, the specific metabolic pathway is as follows:

[0016] The first step is to convert shikimic acid into 3,4,5-trihydroxy-1-cyclohexene-1-carboxaldehyde (DHBA) under the action of carboxylic acid reductase.

[0017] The second step is to methylate 3,4,5-trihydroxy-1-cyclohexene-1-carboxaldehyde (DHBA) into DHMBA under the action of O-methyltransferase.

[0018] In step (3), the culture conditions are as follows: pH value is 6.5-7.5, temperature is 32-40℃, and time is 24-72h.

[0019] Preferably, the culture conditions are as follows: pH value is 6.5-7.5, temperature is 34-36℃, and time is 48-60h.

[0020] In step (3), the culture medium is composed of 20-30g / L of glucose, 5g / L of yeast extract, and 10g / L of sodium chloride, and the solvent is water.

[0021] In step (3), the concentration of shikimic acid in the culture medium is 1-10g / L.

[0022] Preferably, the concentration of shikimic acid in the culture medium is 6-8g / L.

[0023] In step (4), the separation and purification are performed by high performance liquid chromatography.

[0024] The application also provides the use of the above-mentioned biosynthesis method for producing DHMBA from Bacillus subtilis in the preparation of DHMBA.

[0025] The application first proposes a biosynthesis method for DHMBA. The method constructs a recombinant plasmid containing the key enzyme genes required for synthesizing DHMBA by genetic engineering means, and transforms it into Bacillus subtilis, so that it can be biosynthesized in a culture medium containing shikimic acid, and finally DHMBA is obtained through separation and purification. The method has the characteristics of high efficiency, economy and environmental protection, and is suitable for industrial production. BRIEF DESCRIPTION OF DRAWINGS

[0026] Fig. 1 is the influence of different temperatures on the expression amount of DHMBA.

[0027] Fig. 2 is the influence of different glucose concentrations on the expression amount of DHMBA.

[0028] Fig. 3 is the influence of different shikimic acid concentrations on the expression amount of DHMBA.

[0029] Fig. 4 is the influence of different fermentation times on the expression amount of DHMBA. DETAILED DESCRIPTION

[0030] The following examples are further illustrations of the application and are not intended to limit the same.

[0031] Example 1

[0032] The method for preparing the recombinant plasmid and strain required for synthesizing DHMBA is as follows:

[0033] (1) Gene acquisition:

[0034] A. The carboxylate reductase (carA gene) and O-methyltransferase (omt gene) gene sequences were amplified from Streptomyces by PCR technology, and the DNA fragments of the target genes were obtained by PCR amplification using specific primers.

[0035] The carA gene sequence is shown in SEQ ID NO. 1:

[0036] The omt gene sequence is shown in SEQ ID NO. 2:

[0037] B. The quality of the PCR amplification product was checked, and the presence of the target gene was confirmed using agarose gel electrophoresis.

[0038] (2) Selection and preparation of vector

[0039] A. Select the plasmid vector pHT01 suitable for expression in Bacillus subtilis.

[0040] B. Use restriction enzymes (EcoRI and BamHI) to treat the vector with enzymes, producing sticky ends or blunt ends compatible with the ends of the target gene.

[0041] C. The enzyme-treated vector was dephosphorylated to prevent self-circularization.

[0042] (3) Cloning and linking of target genes

[0043] A. The PCR-amplified carA and omt genes were treated with the same restriction enzymes as the vector, producing ends compatible with the vector.

[0044] B. Mix the enzyme-treated target genes and vector in the appropriate ratio, add T4 DNA ligase, and perform a ligation reaction to form a recombinant plasmid.

[0045] (4) Transformation and selection of recombinant plasmid

[0046] A. Recombinant plasmid is introduced into competent cells of B. subtilis by electroporation.

[0047] B. The transformed B. subtilis is plated on selective medium plates containing appropriate antibiotic (chloramphenicol) and incubated for 24-48 hours to select antibiotic resistant strains containing the recombinant plasmid.

[0048] C. Several antibiotic resistant strains are selected and the presence of the target gene in the recombinant plasmid is verified by plasmid extraction and PCR. The correctness of the recombinant plasmid is further confirmed by sequencing.

[0049] (5) Verification of the function of the recombinant plasmid

[0050] A. B. subtilis containing the recombinant plasmid is inoculated into appropriate medium to induce gene expression.

[0051] B. The expression of carboxylate reductase and O-methyltransferase is detected using techniques such as SDS-PAGE and Western Blot.

[0052] C. The crude enzyme solution in the engineered bacteria is extracted and in vitro enzyme activity assay is performed to confirm the function of carboxylate reductase and O-methyltransferase.

[0053] The culture medium and culture method required for the synthesis of DHMBA are as follows:

[0054] Prepare the basic culture medium as follows, adjust the pH to 6.5-7.5:

[0055] Prepare different concentrations of shikimic acid solution, dissolve shikimic acid with sterile water, and sterilize by filtering through a 0.22 μm filter membrane after dissolution. Store the shikimic acid solution in a 4°C refrigerator for standby use.

[0056] Sterilize the basic culture medium and cool it to below 50°C, add shikimic acid solution to a final concentration of 1-10 g / L. Add kanamycin solution to a final concentration of 50 μg / mL. Mix well and then dispense into sterile culture bottles.

[0057] Take an appropriate amount of overnight culture broth and inoculate into the culture medium containing shikimic acid at a volume ratio of 1:100 (for example: inoculate 1 mL of overnight culture broth into 100 mL of culture medium). Incubate in a shaking incubator at different temperatures (200 rpm). Incubation temperature: 32-40°C. Incubation time: 24-72 hours.

[0058] Take an appropriate amount of culture broth and centrifuge (6000 x g, 10 minutes) to collect the supernatant. Use high performance liquid chromatography (HPLC) to detect DHMBA in the supernatant. Calculate the concentration of DHMBA according to the standard curve drawn by the standard.

[0059] The DHMBA in the fermentation broth was extracted with ethyl acetate. The organic phase was collected and the solvent was evaporated to obtain the crude product. The crude product was purified using column chromatography method.

[0060] Example 2

[0061] Effect of different fermentation temperatures on the expression of DHMBA:

[0062] The fermentation culture was carried out using the following conditions to discuss the effect of fermentation temperature:

[0063] Glucose concentration: 20 g / L;

[0064] Shikimic acid concentration: 5 g / L;

[0065] Fermentation time: 48 hours;

[0066] Fermentation temperature: 32℃, 34℃, 36℃, 38℃, 40℃;

[0067] After fermentation, the concentration of DHMBA in the fermentation supernatant was tested. The test results are shown in Figure 1. Different temperatures have a great influence on the expression of DHMBA, and the optimal fermentation temperature is between 34-36℃.

[0068] Example 3

[0069] Effect of different glucose concentrations on the expression of DHMBA:

[0070] The fermentation culture was carried out using the following conditions to discuss the effect of glucose concentration:

[0071] Fermentation temperature: 36℃;

[0072] Shikimic acid concentration: 5 g / L;

[0073] Fermentation time: 48 hours;

[0074] Glucose concentration: 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L;

[0075] After fermentation, the concentration of DHMBA in the fermentation supernatant was tested. The test results are shown in Figure 2. Different glucose concentrations have a great influence on the expression of DHMBA, and the optimal glucose concentration is between 20-30 g / L.

[0076] Example 4

[0077] Effect of different concentrations of shikimic acid on the expression of DHMBA:

[0078] The fermentation culture was carried out using the following conditions to discuss the effect of shikimic acid concentration:

[0079] Fermentation temperature: 36℃;

[0080] Glucose concentration: 25 g / L;

[0081] Fermentation duration: 48 hours;

[0082] Shikimic acid concentration: 1 g / L, 2 g / L, 4 g / L, 6 g / L, 8 g / L, 10 g / L;

[0083] After fermentation, the concentration of DHMBA in the fermentation supernatant was tested. The test results are shown in Figure 3. Different shikimic acid concentrations have a great influence on the expression amount of DHMBA, and low shikimic acid concentration makes little synthesis of DHMBA. The optimized shikimic acid concentration is 6-8 g / L.

[0084] Example 5

[0085] Influence of different fermentation durations on the expression amount of DHMBA:

[0086] Fermentation culture was carried out using the following conditions, and the influence of fermentation duration was discussed:

[0087] Fermentation temperature: 36℃;

[0088] Glucose concentration: 25 g / L;

[0089] Fermentation duration: 24 hours, 36 hours, 48 hours, 60 hours, 72 hours;

[0090] Shikimic acid concentration: 6 g / L;

[0091] After fermentation, the concentration of DHMBA in the fermentation supernatant was tested. The test results are shown in Figure 4. Different fermentation durations have a great influence on the expression amount of DHMBA, and with the increase of fermentation time, DHMBA gradually accumulates, and the growth slows down after 48 hours of fermentation, and the optimal fermentation duration is between 48-60 hours.

[0092] The above embodiments are only some preferred embodiments of the present application, and cannot be used to limit the scope of protection of the present application. Any non-essential changes and substitutions made by those skilled in the art on the basis of the present application are within the scope of the present application.

Claims

1. A biosynthetic method for producing 3,5-dihydroxy-4-methoxybenzyl alcohol using Bacillus subtilis, characterized by, The method comprises the following steps: (1) constructing a recombinant plasmid containing carboxylate reductase and O-methyltransferase genes; (2) transforming the recombinant plasmid into Bacillus subtilis to obtain a recombinant Bacillus subtilis; (3) culturing the recombinant Bacillus subtilis in a culture medium containing shikimic acid; (4) isolating and purifying 3,5-dihydroxy-4-methoxybenzyl alcohol obtained from the culture medium.

2. The biosynthetic method according to claim 1, characterized in that, The nucleotide sequence of the carboxylate reductase is shown as SEQ ID NO. 1, and the nucleotide sequence of the O-methyltransferase is shown as SEQ ID NO.

2.

3. The biosynthetic method of claim 1, wherein, The specific metabolic pathway is as follows: In the first step, shikimic acid is converted into 3,4,5-trihydroxy-1-cyclohexene-1-carboxaldehyde under the action of carboxylate reductase; In the second step, 3,4,5-trihydroxy-1-cyclohexene-1-carboxaldehyde is methylated into 3,5-dihydroxy-4-methoxybenzyl alcohol under the action of O-methyltransferase.

4. The biosynthetic method of claim 1, wherein, The culture conditions are as follows: pH value is 6.5-7.5, temperature is 32-40℃, and time is 24-72h.

5. The biosynthetic method according to claim 4, wherein, The culture conditions are as follows: pH value is 6.5-7.5, temperature is 34-36℃, and time is 48-60h.

6. The biosynthetic method of claim 1, wherein, The culture medium comprises 20-30g / L of glucose, 5g / L of yeast extract, and 10g / L of sodium chloride, and the solvent is water.

7. The biosynthetic method of claim 1, wherein, The concentration of shikimic acid in the culture medium is 1-10g / L.

8. The biosynthetic method of claim 7, wherein, The concentration of shikimic acid in the culture medium is 6-8g / L.

9. The biosynthetic method of claim 1, wherein, The isolation and purification are performed by high performance liquid chromatography.

10. Use of the biosynthesis method for producing 3,5-dihydroxy-4-methoxybenzyl alcohol by Bacillus subtilis according to any one of claims 1-9 in the preparation of 3,5-dihydroxy-4-methoxybenzyl alcohol.

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