Double-enzyme coupling co-expression engineering bacterium and application thereof in preparation of dehydroepiandrosterone

Abstract

The invention discloses a double-enzyme coupling co-expression engineering bacterium and application thereof in preparation of dehydroepiandrosterone. The co-expression engineering bacterium can simultaneously express short-chain dehydrogenase and alcohol dehydrogenase. Short-chain dehydrogenase for generating dehydroepiandrosterone through high-activity catalysis of 5AD reduction is screened, genetically engineered bacteria for coupling expression of the short-chain dehydrogenase and alcohol dehydrogenase through double recombinant plasmids are constructed, engineering bacteria fermentation liquor is directly used as a catalyst to achieve double-enzyme concerted catalysis, the limitation of traditional enzyme reaction is broken through, and the yield of dehydroepiandrosterone is increased. 5AD is catalyzed by short-chain dehydrogenase to be reduced to generate dehydroepiandrosterone, and NADP < + > is converted by alcohol dehydrogenase to generate NADPH, so that regeneration of coenzyme NADPH is realized, additional addition of coenzyme is avoided, and the cost is saved. According to the method, beta-cyclodextrin is added as a cosolvent, a reaction system is optimized, the efficiency of reducing 5-androstene-3, 17-dione into dehydroepiandrosterone is remarkably improved, and the substrate conversion rate is 90% or above.

Description

(I) Technical field

[0001] The invention belongs to the field of biotechnology and relates to a dual-enzyme coupling co-expression engineering bacterium and an application thereof in preparing dehydroepiandrosterone. (II) Background technology

[0002] Drospirenone is a synthetic progestin that helps control blood pressure, effectively improves symptoms such as acne, hirsutism and seborrheic dermatitis, improves premenstrual syndrome (PMS) and premenstrual dysphoric disorder (PMDD), and prevents osteoporosis. Drospirenone plays an important role in contraception, endocrine disorder treatment and menopausal management due to its multiple pharmacological properties. In industrial production, 5-androsten-3,17-dione (5AD) is usually reduced to prepare dehydroepiandrosterone, which is further prepared to prepare drospirenone. Dehydroepiandrosterone (DHEA) is an important intermediate for the synthesis of drospirenone. Dehydroepiandrosterone is an endogenous steroid hormone synthesized by the adrenal cortex, gonads and brain. It has multiple functions in human health, can regulate gonadal function, improve symptoms caused by sex hormone deficiency, and has immune regulation, treatment of depression and anxiety, and anti-aging effects.

[0003] Both chemical and biological methods can be used to catalyze the reduction of 5AD to produce dehydroepiandrosterone. The chemical reduction process requires the addition of precious metal catalysts, but this method has disadvantages such as expensive catalysts, harsh reaction conditions, harsh reaction equipment requirements, and environmental pollution, so its development is limited. Compared with chemical synthesis, the biological reduction of 5AD to prepare dehydroepiandrosterone has the advantages of mild reaction conditions, simple reaction steps, low environmental pollution, and low cost. It can solve some of the drawbacks of the chemical preparation method and has attracted attention. The key to the biological reduction of 5AD to prepare dehydroepiandrosterone is to find efficient enzymes or microorganisms to realize the reduction reaction process.

[0004] With the rapid development of synthetic biology, the preparation of efficient enzymes through genetically engineered bacteria has significantly promoted the progress of biotransformation technology. This technology can improve the catalytic efficiency and substrate specificity of enzymes, reduce production costs, and overcome the limitations of traditional enzymes in reaction conditions and substrate range, thereby expanding the application of biotransformation in pharmaceutical, chemical and agricultural fields. At the same time, the environmental friendliness and resource conservation characteristics of efficient enzymes meet the needs of green and sustainable development, and help the precise biosynthesis of high value-added compounds. The production of efficient enzymes by genetically engineered bacteria has become an important pillar of modern biomanufacturing, providing strong support for the realization of green and efficient industrial transformation.

[0005] The main problem of biotransformation is the low production efficiency due to the low substrate processing capacity. In order to further improve the substrate processing capacity and production efficiency, multi-enzyme cascade catalytic reactions can be used. Multi-enzyme cascade reactions are processes that use two or more enzymes to jointly catalyze different chemical reactions in a reaction system. Each enzyme catalyzes a separate reaction, and the resulting product is usually used as a substrate for the next enzyme. This reaction path is similar to the multi-step reaction in the metabolic pathway of an organism. Due to its high efficiency and specificity, multi-enzyme cascade reactions have been widely used in biochemical synthesis, drug synthesis, and environmental protection. Multi-enzyme cascade reactions can improve reaction efficiency, selectivity, and environmental protection by connecting multiple enzyme-catalyzed reactions in series. It not only reduces the steps and energy consumption in traditional chemical synthesis, but also provides a greener and more sustainable way to synthesize complex molecules. With the continuous development of biocatalytic technology, the application prospects of multi-enzyme cascade reactions in industrial production, drug synthesis, and environmental protection will be more extensive. (III) Summary of the invention

[0006] The present invention aims to provide a dual-enzyme coupling co-expression engineering bacterium and its application in the preparation of dehydroepiandrosterone, by constructing a genetically engineered bacterium to achieve the coupled expression of short-chain dehydrogenase and alcohol dehydrogenase, by short-chain dehydrogenase catalyzing the reduction of 5AD to generate dehydroepiandrosterone, and by alcohol dehydrogenase converting NADP + Generate NADPH to achieve the regeneration of coenzyme NADPH. Adequate NADPH supply reduction reaction provides a continuous supply of hydrogen donors, thereby improving the bioconversion rate of 5AD.

[0007] The technical solution adopted by the present invention is:

[0008] The invention provides a dual-enzyme coupling co-expression engineering bacterium for efficiently preparing dehydroepiandrosterone, wherein the co-expression engineering bacterium simultaneously expresses a short-chain dehydrogenase and an alcohol dehydrogenase.

[0009] Furthermore, the short-chain dehydrogenase includes the short-chain dehydrogenase BcSDR4 derived from Burkholderia cepacia CGMCCNO.28566, and the amino acid sequence is shown in SEQ ID NO.1.

[0010] Furthermore, the alcohol dehydrogenase includes alcohol dehydrogenase CpSADH derived from Candida parapsilosis CCTCC M 20221544, and the amino acid sequence is shown in SEQ ID NO.2.

[0011] Furthermore, the expression of the short-chain dehydrogenase is carried out using plasmid pET28a(+) as a vector; the expression of the alcohol dehydrogenase is carried out using plasmid pETDuet1 as a vector, and both use E. coli BL21(DE3) as a host bacteria.

[0012] Furthermore, the co-expression engineering bacteria are constructed as follows:

[0013] (1) The genome of Burkholderia cepacia CGMCC No. 28566 was used as a template to obtain the target gene fragment of BcSDR4 by PCR amplification, and then ligated with the pET28a(+) vector that had also been digested with restriction endonucleases NcoI and NdeI to construct the recombinant expression plasmid pET28a(+)-BcSDR4;

[0014] F: 5'-AAGAAGGAGATATACCATGGATGATTTCATTCAACGGCAAGACC-3';

[0015] R: 5'-CAGTCATGCTAGCCATATGTCAGAAATTGAAGCCGTTGCCC-3';

[0016] (2) Using the genome of Candida parapsilosis CCTCC M 20221544 as a template, the CpSADH target gene fragment was obtained by PCR amplification and ligated with the pETDuet1 vector that had also been digested with restriction endonucleases BamHI and Takara to construct the recombinant expression plasmid pETDuet1-CpSADH;

[0017] Primer F: 5'-CGCGGATCCATGTCAATTCCATC-3',

[0018] Primer R: 5′-CCGCTCGAGGGATTCTATGGATT-3′;

[0019] (3) Introduce pET28a(+)-BcSDR4 and pETDuet1-CpSADH in the above step into the host bacteria to obtain the co-expression engineered bacteria E. coli BL21(DE3)-(pET28a(+)-BcSDR4 / pETDuet1-CpSADH).

[0020] The present invention also provides an application of the dual-enzyme coupling co-expression engineering bacteria in the efficient preparation of dehydroepiandrosterone, and the application method is: adding 5-androsten-3,17-dione (5AD) and β-cyclodextrin (β-CD) to the fermentation liquid obtained by fermentation culture of the dual-enzyme coupling co-expression engineering bacteria, carrying out a biotransformation reaction for 24 to 120 hours (preferably 96 hours) at 30 to 40° C. (preferably 38° C.) and 400 to 900 rpm (preferably 500 rpm), and controlling the pH of the biotransformation system to 7.0-9.0 (preferably pH 8.0); after the conversion is completed, extracting the biotransformation liquid with ethyl acetate, and removing the ethyl acetate by rotary evaporation of the ethyl acetate extract to obtain dehydroepiandrosterone.

[0021] Furthermore, the bacterial concentration of the fermentation broth is 100-200 g / L (preferably 140 g / L); the concentration of 5-androstene-3,17-dione added to the fermentation broth is 50-200 g / L, and the concentration of β-cyclodextrin added to the fermentation broth is 1-20 g / L (preferably 10 g / L).

[0022] Further, the fermentation broth is prepared in a fermenter according to the following steps:

[0023] (1) inoculating the dual-enzyme coupling co-expression engineering bacteria into LB liquid culture medium, culturing for 12 hours at 30-40° C. (preferably 37° C.) and 120-180 rpm (preferably 160 rpm) to obtain a primary seed solution;

[0024] (2) Inoculate the primary seed solution into the seed culture medium at a volume concentration of 6-8%, and culture at 30-40°C (preferably 37°C) and 120-180 rpm (preferably 160 rpm) for 4-8 h until the OD 600 is 1.0-2.0 (preferably 1.5) to obtain a secondary seed solution;

[0025] (3) The secondary seed liquid is inoculated into a fermentation tank filled with fermentation medium at a volume concentration of 6-8%, and the fermentation culture is carried out under the conditions of ventilation 2VVM, dissolved oxygen 1.5mg / L, 30-40°C (preferably 37°C), 120-180rpm (preferably 160rpm), and constant dissolved oxygen is controlled by feeding the medium, and the culture is carried out until OD 600 When the pH value reaches 0.6-0.8, add IPTG with a final concentration of 0.1-1 mM (preferably 0.6 mM), and induce culture for 4-18 h (preferably 16 h) at 20-25° C. (preferably 23° C.) and 400-900 rpm (preferably 700 rpm). During the induction process, the constant dissolved oxygen is controlled by feeding the medium. When the bacterial concentration reaches 100-200 g / L (preferably 140 g / L), the fermentation is terminated to obtain the fermentation liquid.

[0026] Furthermore, the seed culture medium consists of 5 g / L glycerol, 25 g / L yeast extract powder, 4 g / L K2HPO4, 2 g / L NaH2PO4, 2 g / L NaCl, 2 g / L (NH4)2SO4, 2 g / L glucose, and 0.5 g / L MgSO4, the solvent is water, and the pH is 7.0.

[0027] Furthermore, the fermentation medium composition is: 8 g / L glycerol, 10 g / L yeast extract powder, 4 g / L K2HPO4, 2 g / L NaH2PO4, 2 g / L NaCl, 2 g / L (NH4)2SO4, 2 g / L glucose, 0.5 g / L MgSO4, the solvent is water, and the pH value is 7.0; the feed medium composition is: 800 g / L glycerol, 100 g / L yeast extract powder, the solvent is water, and the pH value is 7.0.

[0028] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0029] (1) The present invention screens a short-chain dehydrogenase that has high activity and catalyzes the reduction of 5AD to generate dehydroepiandrosterone, and constructs a genetically engineered bacterium that expresses a short-chain dehydrogenase and an alcohol dehydrogenase by coupling a double recombinant plasmid. The fermentation liquid of the engineered bacterium is directly used as a catalyst to achieve dual enzyme synergistic catalysis, thereby breaking through the limitations of traditional enzyme reactions. The short-chain dehydrogenase catalyzes the reduction of 5AD to generate dehydroepiandrosterone, and the alcohol dehydrogenase converts NADP + Generate NADPH to achieve the regeneration of coenzyme NADPH, avoid additional addition of coenzyme and save costs.

[0030] (2) Based on the construction of a dual-enzyme co-expression engineered bacterium, the present invention optimizes the reaction system by adding β-cyclodextrin as a cosolvent, thereby ensuring a high bacterial concentration (140 g / L) and a high conversion rate, and significantly improving the efficiency of reducing 5-androsten-3,17-dione (5AD) to dehydroepiandrosterone, with a substrate conversion rate of more than 90%.

[0031] (3) The method of the present invention has the advantages of being green, environmentally friendly, low cost, and simple process, and is suitable for large-scale industrial production, providing a new solution for the green and efficient synthesis of dehydroepiandrosterone. (IV) Description of the drawings

[0032] Figure 1 It is a process in which 5AD is converted into dehydroepiandrosterone by dual enzyme coupling catalysis. (V) Specific implementation methods

[0033] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0034] The Burkholderia cepacia CGMCC NO.28566 used to amplify the short-chain dehydrogenase (BcSDR4) gene in the embodiment of the present invention has been disclosed in patent application 202311851393.2. Candida parapsilosis CCTCC NO:M 20221544 used to amplify alcohol dehydrogenase (CpSADH) has been disclosed in patent application 202211632852.3. In the present invention, the short-chain dehydrogenase gene 8G9M (amino acid sequence as shown in SEQ ID NO.3) from Acinetobacter_baumannii, the short-chain dehydrogenase gene Py2 (amino acid sequence as shown in SEQ ID NO.4) from Xanthobacter autotrophicus, the short-chain dehydrogenase gene 4H16 (amino acid sequence as shown in SEQ ID NO.5) from Sinorhizobium meliloti 1021, the short-chain dehydrogenase gene 5EL0 (amino acid sequence as shown in SEQ ID NO.6) from Brucella ovis, and the short-chain dehydrogenase gene 7ULH (amino acid sequence as shown in SEQ ID NO.7) from Mycobacterium avium 104 are all synthesized by Beijing Qingke Company.

[0035] The various culture media used in the present invention are composed as follows:

[0036] The solid culture medium for strain preservation consists of 25 g / L glucose, 3 g / L yeast extract, 5 g / L ammonium sulfate, 0.25 g / L magnesium sulfate, 1.5 g / L potassium hydrogen phosphate trihydrate, 1.5 g / L potassium dihydrogen phosphate, 16 g / L agar powder, and the solvent is water.

[0037] LB liquid culture medium composition: NaCl 10g / L, peptone 10g / L, yeast extract 5g / L, solvent is water, pH 7.0.

[0038] LB solid medium is prepared by adding agar to LB liquid medium at a final concentration of 20 g / L.

[0039] Seed culture medium composition: glycerol 5g / L, yeast extract powder 25g / L, K2HPO4 4g / L, NaH2PO4 2g / L, NaCl2g / L, (NH4)2SO4 2g / L, glucose 2g / L, MgSO4 0.5g / L, solvent is water, pH 7.0.

[0040] Fermentation medium composition: glycerol 8g / L, yeast extract powder 10g / L, K2HPO4 4g / L, NaH2PO4 2g / L, NaCl2g / L, (NH4)2SO4 2g / L, glucose 2g / L, MgSO4 0.5g / L, solvent is water, pH 7.0.

[0041] The composition of the feed medium: 800 g / L glycerol, 100 g / L yeast extract powder, solvent is water, pH 7.0.

[0042] Example 1: Construction of dual enzyme co-expression engineering bacteria

[0043] 1. Construction of recombinant expression plasmid pET-28a(+)-BcSDR4

[0044] (1) Primer design and synthesis for amplification of short-chain dehydrogenase BcSDR4

[0045] Primer F1 and primer R1 were designed based on the short-chain dehydrogenase BcSDR4 gene (GeneID: 56664023) from Burkholderia cepacia in the NCBI database and synthesized by Beijing Qingke Biotechnology Co., Ltd. Hangzhou Branch.

[0046] Primer F1:

[0047] 5'-AAGAAGGAGATATAAAGAAGGAGATATACCATGGATGATTTCATTCAACGGCAAGACC-3'

[0048] Primer R1: 5′-CAGTCATGCTAGCCATATGTCAGAAATTGAAGCCGTTGCCC-3′

[0049] (2) Extraction, PCR amplification, verification and purification of B. cepacia CGMCC NO:28566 genomic DNA

[0050] A single colony of B.cepacia CGMCC NO:28566 was inoculated into LB liquid medium and cultured overnight at 37°C and 180rpm to obtain the bacteria. According to the instructions of the fungal genome extraction kit provided by Yisheng Biotechnology Co., Ltd., the genomic DNA of B.cepacia CGMCC NO:28566 was extracted. The quality of the extracted genomic DNA was verified by agarose gel electrophoresis. Using the extracted genomic DNA as a template, primers F1 and R1 were used to PCR amplify the BcSDR4 gene fragment.

[0051] The PCR reaction system was as follows: total reaction volume 50 μL, including genomic DNA 2 μL, primer F1 2 μL, primer R12 μL, 2×Hieff PCR Master Mix 25 μL, and ddH2O 19 μL.

[0052] The PCR amplification program was as follows: 94°C for 5 min (pre-denaturation); 94°C for 30 s (denaturation); 55°C for 30 s (annealing); 72°C for 1 min (extension); 72°C for 10 min (terminal extension); and 35 cycles.

[0053] The PCR amplification results were verified by agarose gel electrophoresis. The target fragment BcSDR4 was recovered and purified using a column DNA gel recovery and extraction kit from Sangon. The purified BcSDR4 fragment was sent to Beijing Qingke Biotechnology Co., Ltd. Hangzhou Branch for sequencing verification. The nucleotide sequence is shown in SEQ ID NO.2, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.1.

[0054] (3) Double restriction digestion of pET-28a(+) vector

[0055] The pET-28a(+) vector was double-digested with Takara endonucleases NcoI and NdeI. The double-digestion reaction system of the vector was configured as follows: NcoI 2μL, NdeI 2μL, 10×K buffer 10μL, BSA 10μL, pET-28a(+) 70μL, ddH2O 6μL. The reaction was carried out at 37°C for 3h, and the digestion results were verified by agarose gel electrophoresis. Subsequently, the pET-28a(+) vector fragment after digestion was purified using the Shanghai Biotech Column DNA Gel Recovery Kit.

[0056] (4) Preparation of recombinant expression plasmid pET-28a(+)-BcSDR4

[0057] Under the conditions of the corresponding enzyme ligation system, the BcSDR4 gene was ligated with the pET-28a(+) vector fragment after double enzyme digestion. The reaction was carried out at 25°C for 30 minutes, and the recombinant expression plasmid pET-28a(+)-BcSDR4 was successfully obtained.

[0058] The seamless cloning reaction system was configured as follows: 2× Seamless Cloning Mix 5 μL, pET-28a(+) 2 μL, BcSDR4 2 μL, ddH2O 1 μL.

[0059] The same method was used to construct plasmids pET28a(+)-8G9M, pET28a(+)-Py2, pET28a(+)-4H16, pET28a(+)-5EL0 and pET28a(+)-7ULH of the short-chain dehydrogenase gene 8G9M of Acinetobacter_baumannii, the short-chain dehydrogenase gene Py2 of Xanthobacter autotrophicus, the short-chain dehydrogenase gene 4H16 of Sinorhizobium meliloti 1021, the short-chain dehydrogenase gene 5EL0 of Brucella ovis and the short-chain dehydrogenase gene 7ULH of Mycobacterium avium 104, respectively.

[0060] 2. Construction of recombinant expression plasmid pETDuet1-CpSADH

[0061] (1) Primer design and synthesis for CpSADH amplification

[0062] Primer F2 and primer R2 were designed based on the alcohol dehydrogenase CpSADH gene from C. parapsilosis in the NCBI database (GenBank: AB010636.1) and synthesized by Beijing Qingke Biotechnology Co., Ltd. Hangzhou Branch.

[0063] Primer F2: 5′-CGCGGATCCATGTCAATTCCATC-3′

[0064] Primer R2: 5′-CCGCTCGAGGGATTCTATGGATT-3′

[0065] (2) Extraction, PCR amplification, verification and purification of C. parapsilosis CCTCC NO:M 20221544 genomic DNA

[0066] A single colony of C. parapsilosis CCTCC NO: M 20221544 was picked and inoculated into LB liquid medium, and cultured overnight at 37 ° C and 180 rpm. Subsequently, the fungal genome extraction kit provided by Yisheng Biotechnology Co., Ltd. was used to extract the genomic DNA of C. parapsilosis according to the instructions, and the extraction effect was verified by agarose gel electrophoresis. The extracted genomic DNA was used as a template, and primers F2 and R2 were used to perform PCR amplification of the target fragment (CpSADH). The amplified product was verified by agarose gel electrophoresis and sent to the Hangzhou Branch of Beijing Qingke Biotechnology Co., Ltd. for sequencing confirmation.

[0067] The PCR amplification system was configured as follows: total reaction volume 50 μL, including genomic DNA template 2 μL, primer F2 2 μL, primer R2 2 μL, 2×Hieff PCR Master Mix 25 μL, and ddH2O 19 μL.

[0068] The PCR amplification program was as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, terminal extension at 72°C for 10 min, and the steps from denaturation to terminal extension were cycled 35 times.

[0069] The PCR amplification results were verified by agarose gel electrophoresis. Finally, the target fragment (CpSADH) in the PCR product was purified using a Sangon column DNA gel recovery and extraction kit, and the purified CpSADH was sent to Beijing Qingke Biotechnology Co., Ltd. Hangzhou Branch for sequencing confirmation. The nucleotide sequence is shown in SEQ ID NO.4, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.3.

[0070] (3) Double restriction enzyme digestion of pETDuet1 vector

[0071] The pETDuet1 vector was double-digested with Takara endonucleases BamHI and XhoI, and the digestion reaction was carried out at 37°C for 6 hours. The double digestion reaction system of the pETDuet1 vector was: BamHI 10μL, XhoI 10μL, 10×K buffer 20μL, pETDuet1 70μL, ddH2O 2μL. After the reaction, the target fragment and vector after digestion were recovered and purified using the Bio-Tech Column DNA Gel Recovery Kit.

[0072] (4) Preparation of recombinant expression plasmid pETDuet1-CpSADH

[0073] Under the conditions of the corresponding enzyme ligation system, the CpSADH gene was ligated with the pETDuet1 vector fragment after double enzyme digestion. The reaction was carried out at 25°C for 30 minutes, and the recombinant expression plasmid pETDuet1-CpSADH was successfully obtained.

[0074] The seamless cloning reaction system was configured as follows: 2×Seamless Cloning Mix 5 μL, pETDuet1 2 μL, CpSADH 2 μL, and ddH2O 1 μL.

[0075] 3. Construction of co-expression engineered bacteria E. coli BL21(DE3)-(pET28a(+)-BcSDR4 / pETDuet1-CpSADH)

[0076] The recombinant expression plasmids pET-28a(+)-BcSDR4 and pETDuet1-CpSADH were transformed into E.coli DH5α competent cells (purchased from Beijing Qingke Biotechnology Co., Ltd.). The operation steps are as follows: First, add 100μL of E.coli DH5α competent cells to two centrifuge tubes, add 10μL of pET-28a(+)-BcSDR4 and pETDuet1-CpSADH recombinant plasmids respectively, mix them by gently blowing, and incubate them on ice for 20 minutes. Then perform a heat shock treatment at 42℃ for 90s and ice bath again for 2min. Subsequently, add 0.9mL of anti-antibody LB medium and recover at 37℃ and 180rpm for 1 hour. After centrifugation of the recovered bacterial solution, discard 900μL of supernatant, retain 100μL, and mix evenly with the precipitate. The mixture of pET-28a(+)-BcSDR4 and E.coli DH5α was spread on LB solid medium containing 100 μg / mL kanamycin and cultured overnight at 37°C; the mixture of pETDuet1-CpSADH and E.coli DH5α was spread on LB solid medium containing 100 μg / mL ampicillin and cultured overnight at 37°C. Through this process, the recombinant plasmid was successfully transformed into E.coli DH5α, and the engineered strains E.coli DH5α-pET-28a(+)-BcSDR4 and E.coli DH5α-pETDuet1-CpSADH were obtained.

[0077] Randomly select 10 E.coli DH5α-pET-28a(+)-BcSDR4 and 10 E.coli DH5α-pETDuet1-CpSADH single colonies from the overnight culture plates and dissolve them in 20μL ddH2O of the corresponding number. Take 10μL of each colony, boil for 5 minutes and use it as a template for colony PCR, and verify the results by agarose gel electrophoresis. The remaining 10μL was transferred to LB medium containing kanamycin or ampicillin, and cultured overnight at 37°C and 180rpm. Finally, the culture solution was sent to the Hangzhou Branch of Beijing Qingke Biotechnology Co., Ltd. for sequencing verification.

[0078] The correct E. coli DH5α-pET-28a(+)-BcSDR4 and E. coli DH5α-pETDuet1-CpSADH recombinant bacteria verified by sequencing were used to extract pET-28a(+)-BcSDR4 and pETDuet1-CpSADH recombinant plasmids respectively, and after confirmation by agarose gel electrophoresis, the two recombinant plasmids were simultaneously transformed into E. coli BL21(DE3) competent cells to obtain E. coli BL21-(pET-28a(+)-BcSDR4 / pETDuet1-CpSADH) co-expression engineering bacteria. The specific operation steps are as follows: 100 μL of E. coli BL21 (DE3) competent cells were mixed with 5 μL of pET-28a (+) -BcSDR4 and 5 μL of pETDuet1-CpSADH recombinant plasmids, and then heat-shocked at 42°C for 90 seconds after ice bathing for 20 minutes, and then ice bathed for 2 minutes. Then, 0.9 mL of LB antibiotic-free medium was added and the cells were revived at 37°C and 180 rpm for 1 hour. The revived bacterial solution was centrifuged, 900 μL of supernatant was discarded, 100 μL was retained and mixed evenly with the precipitate, and then spread on LB solid culture medium containing 100 μg / mL kanamycin and 100 μg / mL ampicillin, and cultured overnight at 37°C to obtain the short-chain dehydrogenase and alcohol dehydrogenase dual enzyme coupling co-expression engineering bacteria E. coli BL21-(pET-28a(+)-BcSDR4 / pETDuet1-CpSADH), which was inoculated into the solid culture medium for strain preservation and stored at 4°C.

[0079] 4. Construction of five other co-expression engineered bacteria

[0080] The following co-expression engineering bacteria were constructed using the method of step 3:

[0081] pET28a(+)-8G9M and pETDuet1-CpSADH were introduced into the host bacteria to obtain the co-expression engineered bacteria E. coli BL21(DE3)-(pET28a(+)-8G9M / pETDuet1-CpSADH).

[0082] pET28a(+)-Py2 and pETDuet1-CpSADH were introduced into the host bacteria to obtain the co-expression engineered bacteria E. coli BL21(DE3)-(pET28a(+)-Py2 / pETDuet1-CpSADH).

[0083] pET28a(+)-4H16 and pETDuet1-CpSADH were introduced into the host bacteria to obtain the co-expression engineered bacteria E. coli BL21(DE3)-(pET28a(+)-4H16 / pETDuet1-CpSADH).

[0084] pET28a(+)-5EL0 and pETDuet1-CpSADH were introduced into the host bacteria to obtain the co-expression engineered bacteria E. coli BL21(DE3)-(pET28a(+)-5EL0 / pETDuet1-CpSADH).

[0085] pET28a(+)-7ULH and pETDuet1-CpSADH were introduced into the host bacteria to obtain the co-expression engineered bacteria E. coli BL21(DE3)-(pET28a(+)-7ULH / pETDuet1-CpSADH). A total of 5 dual enzyme co-expression engineered bacteria were obtained. The construction process was the same as the construction method of E. coli BL21-(pET-28a(+)-BcSDR4 / pETDuet1-CpSADH) in step 3.

[0086] Example 2: Preparation of fermentation broth of engineered bacteria expressing dual enzymes

[0087] (1) Primary seed solution

[0088] 10 μL of the glycerol bacteria of the co-expression engineered bacteria constructed in Example 1 was inoculated into 10 mL of LB liquid culture medium and cultured at 37° C. and 160 rpm for 12 h to obtain a primary seed solution.

[0089] (2) Secondary seed solution

[0090] Inoculate 10 ml of the first-level seed solution into 150 mL of seed culture medium and culture at 37°C and 160 rpm for 4 to 8 h to obtain the second-level seed solution OD 600 is 1.5, and the secondary seed solution is obtained.

[0091] (3) Fermentation broth

[0092] 150 mL of secondary seed liquid was inoculated into a 5 L fermenter containing 2.5 L of fermentation medium, with aeration of 2 VVM, dissolved oxygen of 1.5 mg / L, cultured at 37 °C and 160 rpm, and fed with a constant dissolved oxygen condition controlled by a feeding medium until OD 600 At 0.6-0.8, IPTG was added with a final concentration of 0.6 mM, and the culture was induced at 23°C and 700 rpm for 16-20 h. During the induction process, the feeding medium was used to control the constant dissolved oxygen conditions for feeding. When the bacterial concentration reached 140 g / L, the fermentation was terminated to obtain 3.5 L of fermentation liquid.

[0093] Example 3: Screening of engineered bacteria with dual enzyme co-expression and high transformation efficiency

[0094] Under aseptic operation conditions, 3.5 L of the six fermentation broths obtained by the method in Example 2 were respectively added with a substrate 5AD having a final concentration of 120 g / L, and then β-CD having a final concentration of 10 g / L was respectively added, and the biotransformation was carried out at 38° C. and 500 rpm for 96 h, and the pH of the biotransformation system was controlled to be 8.0. After the transformation was completed, the biotransformation liquid was extracted with ethyl acetate, the ethyl acetate extract was rotary evaporated to remove the ethyl acetate, the remaining sample was dissolved with acetonitrile, and the content of the substrate and the product was detected by high performance liquid chromatography to determine the conversion rate. The results are shown in Table 1.

[0095] The HPLC detection conditions are as follows: chromatographic column Agilent ZORBAX SB-C18 3.5μm, 150mm×4.6mm, mobile phase: mobile phase is water / acetonitrile, gradient elution is adopted, elution conditions are 0-25min water / acetonitrile ratio is 4:36, 25-55min water / acetonitrile ratio is 25:75, 55-75min water / acetonitrile ratio is 64:36. UV detection wavelength is 210nm, and mobile phase flow rate is 1.0ml / min.

[0096] Table 1 Screening of dual enzyme co-expressing engineering bacteria with high transformation efficiency

[0097]

[0098] The results in Table 1 show that E. coli BL21-(pET-28a(+)-BcSDR4 / pETDuet1-CpSADH) has the best biotransformation effect. Under the above transformation conditions, the conversion rate reaches 99%. Further research will be carried out using the co-expression strain E. coli BL21-(pET-28a(+)-BcSDR4 / pETDuet1-CpSADH) as the catalytic strain.

[0099] Example 4, screening of cosolvent

[0100] Under aseptic conditions, 5AD was added to 3.5 L of the fermentation broth of the co-expression strain E. coli BL21-(pET-28a(+)-BcSDR4 / pETDuet1-CpSADH) obtained by the method of Example 2, and α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, methyl-β-cyclodextrin, hydroxyethyl-β-cyclodextrin and hydroxypropyl-β-cyclodextrin were added as cosolvents at a final concentration of 10 g / L, respectively, and biotransformed for 96 h at 38 ° C and 500 rpm, and the pH of the biotransformation system was controlled to 8.0. After the transformation was completed, the biotransformation liquid was extracted with ethyl acetate, the ethyl acetate extract was rotary evaporated to remove ethyl acetate, and the remaining sample was dissolved with acetonitrile. The content of the substrate and the product was detected by high performance liquid chromatography according to Example 3 to determine the conversion rate. The results are shown in Table 2.

[0101] Table 2 Selection of cyclodextrin cosolvents

[0102]

[0103] The results showed that the addition of six cyclodextrin solvents helped to improve the conversion rate, among which β-cyclodextrin had the best effect as an auxiliary agent, with a conversion rate of 99.0%. Therefore, β-cyclodextrin was added as an auxiliary agent in subsequent experiments.

[0104] Example 5: Effect of the Feed Amount of Substrate 5AD on the Conversion Reaction

[0105] Under aseptic conditions, the substrate 5AD was added to 3.5 L of the fermentation broth of the co-expression strain E. coli BL21-(pET-28a(+)-BcSDR4 / pETDuet1-CpSADH) obtained by the method of Example 2, respectively, so that the feeding concentration of the substrate 5AD was 50, 100, 120, 150 and 200 g / L, respectively, and then β-CD was added at a final concentration of 10 g / L, and the biotransformation was carried out at 38°C and 500 rpm for 96 hours, and the pH of the biotransformation system was controlled to 8.0. After the transformation was completed, the biotransformation liquid was extracted with ethyl acetate, the ethyl acetate extract was rotary evaporated to remove ethyl acetate, and the remaining sample was dissolved with acetonitrile. The content of the substrate and the product was detected by high performance liquid chromatography according to Example 3 to determine the conversion rate. The results are shown in Table 3.

[0106] Table 3 Effect of feed concentration of substrate 5AD on conversion rate of growing cell conversion reaction

[0107]

[0108] In the biotransformation system, the effect of substrate concentration on conversion rate shows a typical three-stage change. When the substrate concentration is low, the conversion rate is proportional to the substrate concentration, and the reaction rate increases linearly with the increase of substrate concentration, because the active sites of the enzyme or microorganism are not yet saturated. As the substrate concentration increases, the reaction rate gradually tends to be flat. At this time, the active sites of the enzyme begin to be limited by the substrate concentration, and the conversion rate is no longer proportional to the concentration. Finally, after entering the high concentration zone, the reaction rate tends to the maximum value and reaches saturation. Further increasing the substrate concentration no longer significantly increases the conversion rate, and may even reduce the reaction efficiency due to the substrate inhibition effect. The reason for this phenomenon is the saturation state of the enzyme. Excessive substrate concentration may cause the active sites of the enzyme to be occupied, and the binding mode of the substrate and the enzyme will change, which will inhibit the reaction. At the same time, high concentration substrates may cause toxic effects on microorganisms or enzymes, affecting their growth and catalytic activity, or lead to insufficient supply of nutrients such as oxygen, further limiting the conversion process. Therefore, the optimization of substrate concentration is crucial to improving the efficiency of biotransformation.

[0109] The results in Table 3 show that when the concentration of substrate 5AD is in the range of 50-120 g / L, the conversion rate remains at a high level. When the concentration of substrate 5AD is in the range of 120-200 g / L, the conversion rate decreases rapidly as the substrate concentration increases. The reason may be that the substrate concentration is too high and a longer conversion time is required to achieve a higher conversion rate, or that the substrate concentration is too high and has a toxic effect on the cells, further affecting the catalytic activity of the enzyme. The optimal feed amount of substrate 5AD is determined to be 120 g / L.

[0110] Example 6: Effect of the amount of β-CD added on the conversion reaction

[0111] The feed concentration of substrate 5AD in Example 5 was fixed at 120 g / L, and the final concentrations of β-CD were changed to 1, 3, 6, 10, 15, and 20 g / L, respectively. Other operations were the same. The results are shown in Table 4.

[0112] Table 4 Effect of the amount of β-CD added on the conversion rate of the growing cell transformation reaction

[0113]

[0114] The results in Table 4 show that the conversion rate shows an upward trend with the increase of the amount of β-CD added. When the amount of β-CD added is 10 g / L, the bioconversion rate is 99.0%. The optimal amount of β-CD added is determined to be 10 g / L.

[0115] In the process of steroid biotransformation, β-CD plays an important role as an auxiliary agent. It increases the solubility of steroid compounds in water by forming inclusion complexes, thereby increasing the bioavailability and conversion rate of the reactants. In addition, after β-CD binds to steroid molecules, it may change their spatial configuration, making the substrate more susceptible to reacting with specific enzymes, thereby enhancing the selectivity and catalytic efficiency of the enzyme. β-CD can also reduce the nonspecific interaction between substrates and solvents or other chemicals, thereby reducing the occurrence of side reactions and increasing the purity and yield of the target product. By regulating the microenvironment of the reaction system, β-CD helps to optimize the reaction conditions and further improve the conversion effect. Therefore, β-CD has multiple roles in the process of steroid biotransformation and can significantly improve the selectivity and efficiency of the reaction.

[0116] Example 7: Effect of temperature on conversion reaction

[0117] The feed concentration of substrate 5AD in Example 5 was fixed at 120 g / L, the reaction temperature was changed to 30, 32, 34, 36, 38 and 40°C, and other operations were the same. The results are shown in Table 5.

[0118] Table 5 Effect of temperature on the conversion rate of growing cell transformation reaction

[0119]

[0120] In the process of bioconversion, temperature has a significant effect on conversion rate. Lower temperatures reduce the activity of enzymes and microorganisms and slow down the reaction rate because low temperatures reduce the frequency of collisions between molecules and inhibit enzyme catalysis and the metabolic rate of microorganisms. As the temperature rises, the activity of enzymes and microorganisms gradually increases, the conversion rate increases, and reaches a maximum value within a certain optimal temperature range. Usually, the optimal temperature range is 35°C to 45°C, but there are exceptions. Some thermophilic microorganisms may show optimal activity at higher temperatures. However, after exceeding the optimal temperature, the enzyme may denature or inactivate, and high temperature may also have a toxic effect on microorganisms, inhibiting their growth and metabolism, resulting in a decrease in conversion rate. Therefore, controlling the temperature within an appropriate range is the key to optimizing the bioconversion process and improving conversion efficiency.

[0121] The results in Table 5 show that the above reactions can achieve good conversion rates in the range of 30-40°C. Between 30-38°C, the conversion rate increases with increasing temperature. Above 38°C, the conversion rate decreases. The optimal bioconversion temperature is 38°C, with a conversion rate of 99%.

[0122] Example 8: Effect of stirring blade speed on conversion reaction

[0123] The feed concentration of substrate 5AD in Example 5 was fixed at 120 g / L, the stirring blade speeds were 400, 500, 600, 700 and 800 rpm respectively, and other operations were the same. The results are shown in Table 6.

[0124] Table 6 Effect of stirring blade speed on the conversion rate of growing cell transformation reaction

[0125]

[0126] In the bioconversion process, the impeller speed has an important influence on the conversion rate. A lower impeller speed will lead to uneven mixing of the reaction system, low contact efficiency between the substrate and the enzyme or microorganism, and thus reduce the conversion rate. As the speed increases, the mixing effect improves, the contact efficiency of the substrate, enzyme and microorganism increases, and the conversion rate gradually increases. Usually, the best conversion rate can be obtained within a certain speed range. However, when the impeller speed is too high, although the mixing effect is further enhanced, excessive shear force and bubbles may have an adverse effect on the microorganisms or enzymes, resulting in cell damage, reduced enzyme activity, and even foaming problems, which in turn reduces the conversion efficiency. Therefore, choosing the right impeller speed is crucial to optimizing the bioconversion process and improving the conversion rate.

[0127] The results in Table 6 show that in the range of 400-800 rpm, as the speed of the stirring blade increases, the conversion rate remains at a high level, and when the stirring blade speed is higher than 500 rpm, the conversion rate can reach 99%. Considering energy saving and cost saving, the optimal stirring blade speed is determined to be 500 rpm, and the conversion rate reaches 99%.

[0128] Example 9: Effect of conversion time on conversion reaction

[0129] The feed concentration of substrate 5AD in Example 5 was fixed at 120 g / L, and the bioconversion time was changed to 24, 48, 72, 96 and 120 h. Other operations were the same. The results are shown in Table 7.

[0130] Table 7 Effect of transformation time on transformation efficiency of growing cell transformation reaction

[0131]

[0132] In the process of biological transformation, the transformation time has an important influence on the transformation rate, which usually shows a trend of increasing first and then decreasing. In the early stage of the transformation process, the activity of the enzyme or microorganism is high, the substrate is quickly transformed, and the transformation rate increases rapidly. As time goes on, the substrate is gradually consumed, the transformation rate slows down, and enters a stable stage. Finally, in the later stage of the transformation process, the substrate concentration decreases significantly and the transformation rate decreases. The reaction may be further inhibited by factors such as excessive product concentration or enzyme inactivation and microbial metabolic fatigue. Therefore, a long transformation time may lead to a decrease in the transformation rate. The optimal transformation time is usually when the transformation rate is the highest, which needs to be determined by optimizing the experimental conditions.

[0133] The results in Table 7 show that as the biotransformation time increases, the conversion rate increases. When the conversion time reaches 96h, the conversion rate reaches 99%. When the reaction time exceeds 96h, the conversion rate does not increase much. Considering energy saving and cost, the optimal reaction time is determined to be 96h.

[0134] Example 10: Effect of pH value of bioconversion system on conversion reaction

[0135] The feed concentration of substrate 5AD in Example 5 was fixed at 120 g / L, the bioconversion system was controlled at pH 7.0, 7.5, 8.0, 8.5 and 9.0, and other operations were the same. The results are shown in Table 8.

[0136] Table 8 Effect of pH value of biotransformation system on conversion rate of conversion reaction

[0137]

[0138] In biotransformation systems, pH has a significant effect on conversion rates because enzyme activity and microbial growth and metabolism are directly regulated by changes in pH. Each enzyme or microorganism has an optimal pH range, within which the catalytic activity of the enzyme or the growth of the microorganism is most vigorous, and the conversion rate usually reaches a maximum. However, when the pH value deviates from the optimal range, the structure of the enzyme may change, resulting in a decrease in catalytic activity or even inactivation, and the metabolism of the microorganism may also be inhibited, thereby reducing the conversion rate. Under extreme pH conditions (overacidic or overalkaline), the activity of enzymes and microorganisms is usually significantly reduced, or even completely inactivated or dead, resulting in a significant decrease in conversion rate. Therefore, maintaining a suitable pH value is crucial to optimizing the biotransformation process and improving conversion efficiency. It is usually necessary to adjust the pH according to the characteristics of the enzyme or microorganism to ensure that the reaction is carried out under optimal conditions.

[0139] The results in Table 8 show that a good conversion rate can be obtained when the pH of the biotransformation system is between 7.0 and 9.0. When the pH of the biotransformation system is 8.0, the conversion rate reaches the highest value of 99.0%. Therefore, the optimal pH value of the biotransformation system is determined to be 8.0.

[0140] Example 11: Effect of Quiescent Cell Transformation on Transformation Rate

[0141] 3.5 L of the fermentation liquid obtained by the method of Example 3 was centrifuged at 8000 rpm for 20 min, and the supernatant was discarded. The cell precipitate was dispersed in 0.2 mM sodium phosphate buffer (pH 8.0), fully shaken and washed, and then centrifuged to obtain a cell precipitate. The washing was repeated three times. The obtained cell precipitate was dispersed in 3.5 L of 0.2 mM phosphate buffer (pH 8.0) to obtain 3.5 L of bacterial suspension with a bacterial concentration of 140 g / L.

[0142] Sterilized substrate 5AD was added to 3.5 L of bacterial suspension to a concentration of 120 g / L. 10 g / L of β-CD was added. The conversion reaction was carried out at 38°C and 500 rpm. The pH of the conversion system was controlled at 8.0. Samples were taken after 24, 48, 72, 96 and 120 h of reaction. The conversion rate was detected by the method of Example 3. The results are shown in Table 9.

[0143] Table 9 Effect of biotransformation time on conversion rate of resting cell conversion reaction

[0144]

[0145] The results in Table 9 show that as the biotransformation time increases, the conversion rate shows an upward trend. When the conversion time reaches 96h, the conversion rate reaches 95.2%. The results of the resting cell biotransformation are directly compared with the results of the growing cell biotransformation in the fermentation broth in Examples 5-10, and the growing cell conversion effect is better. The reason may be that strict aseptic operation cannot be performed during the process of obtaining resting cells and the resting cell biotransformation process, the reaction time is long, and it is easy to cause contamination by miscellaneous bacteria. Considering simplifying the production process, it is determined that the growing cell conversion 5AD has a better effect.

Claims

1. A dual-enzyme coupling co-expression engineering bacterium for efficiently preparing dehydroepiandrosterone, characterized in that: The co-expression engineering bacteria simultaneously express short-chain dehydrogenase and alcohol dehydrogenase.

2. The dual enzyme coupling co-expression engineering bacteria according to claim 1, characterized in that: The amino acid sequence of the short-chain dehydrogenase is shown in SEQ ID NO.

1.

3. The dual enzyme coupling co-expression engineering bacteria according to claim 1, characterized in that: The alcohol dehydrogenase amino acid sequence is shown in SEQ ID NO.

3.

4. The dual enzyme coupling co-expression engineering bacteria according to claim 1, characterized in that: The expression of the short-chain dehydrogenase uses plasmid pET28a(+) as a vector; the expression of the alcohol dehydrogenase uses plasmid pETDuet1 as a vector, and both use E.coli BL21(DE3) as a host bacterium.

5. Use of the dual-enzyme coupling co-expression engineering bacteria according to claim 1 in the efficient preparation of dehydroepiandrosterone.

6. The use according to claim 5, characterized in that The application method comprises the following steps: adding 5-androsten-3,17-dione and beta-cyclodextrin to the fermentation liquid obtained by fermentation culture of the dual-enzyme coupling co-expression engineering bacteria, carrying out a biotransformation reaction at 30-40°C and 400-900rpm for 24-120h, and controlling the pH value of the biotransformation system to be 7.0-9.0; after the transformation is completed, extracting the biotransformation liquid with ethyl acetate, and removing the ethyl acetate by rotary evaporation of the ethyl acetate extract to obtain dehydroepiandrosterone.

7. The use according to claim 6, characterized in that The bacterial concentration of the fermentation liquid is 100-200 g / L; the concentration of 5-androstene-3,17-dione added to the fermentation liquid is 50-200 g / L, and the concentration of β-cyclodextrin added to the fermentation liquid is 1-20 g / L.

8. The use according to claim 6, characterized in that The fermentation broth is prepared in a fermenter according to the following steps: (1) inoculating the dual-enzyme coupling co-expression engineering bacteria into LB liquid culture medium, culturing at 30-40° C. and 120-180 rpm for 12 h to obtain a primary seed solution; (2) Inoculate the primary seed solution into the seed culture medium at a volume concentration of 6-8%, and culture at 30-40°C and 120-180 rpm for 4-8 h until the OD 600 The pH value of the seed culture medium is 1.0-2.0, and a secondary seed solution is obtained; the seed culture medium comprises: 5 g / L glycerol, 25 g / L yeast extract, 4 g / L K2HPO4, 2 g / L NaH2PO4, 2 g / L NaCl, 2 g / L (NH4)2SO4, 2 g / L glucose, 0.5 g / L MgSO4, the solvent is water, and the pH value is 7.0; (3) The secondary seed liquid was inoculated into a fermentation tank containing fermentation medium at a volume concentration of 6-8%, and the fermentation culture was carried out under the conditions of ventilation of 2VVM, dissolved oxygen of 1.5mg / L, 30-40°C, 120-180rpm, and constant dissolved oxygen was controlled by feeding medium. The culture was continued until OD 600 When the pH value reaches 0.6-0.8, IPTG with a final concentration of 0.1-1 mM is added, and the induction culture is carried out at 20-25° C. and 400-900 rpm for 4-18 hours. During the induction process, a feed medium is used to control the constant dissolved oxygen. When the bacterial concentration reaches 100-200 g / L, the fermentation is terminated to obtain a fermentation liquid. The fermentation medium is composed of: 8 g / L glycerol, 10 g / L yeast extract, 4 g / L K2HPO4, 2 g / L NaH2PO4, 2 g / L NaCl, 2 g / L (NH4)2SO4, 2 g / L glucose, 0.5 g / L MgSO4, the solvent is water, and the pH value is 7.0; the feed medium is composed of: 800 g / L glycerol, 100 g / L yeast extract, the solvent is water, and the pH value is 7.0.

Images