Novel 7beta-hydroxysteroid dehydrogenase gene and application thereof

Abstract

The invention discloses a novel 7 beta-hydroxysteroid dehydrogenase gene and application thereof, and belongs to the technical field of enzyme engineering and biological catalysis. According to the invention, a 7beta-hydroxysteroid dehydrogenase gene with high conversion rate is excavated from a database, the 7beta-hydroxysteroid dehydrogenase gene and a 7alpha-hydroxysteroid dehydrogenase gene are subjected to heterologous expression in different chassis cells, and recombinant bacteria for efficiently preparing ursodesoxycholic acid are constructed. The constructed recombinant strain is used for producing ursodesoxycholic acid through whole-cell catalysis of chenodeoxycholic acid, the feeding amount is 6 g / L, the catalytic reaction time is 24 h, and the conversion rates of recombinant escherichia coli and pichia pastoris are 87.6% and 97.8% respectively.

Description

Technical Field

[0001] The invention relates to a new 7β-hydroxysteroid dehydrogenase gene and application thereof in preparing ursodeoxycholic acid, belonging to the field of genetic engineering and biocatalysis. Background Art

[0002] Ursodeoxycholic acid (UDCA) is the only drug approved by the U.S. Food and Drug Administration (FDA) for the treatment of primary biliary cirrhosis. It can dissolve gallstones, treat cholestasis, biliary pancreatitis, primary cholangitis, primary biliary cirrhosis, drug-induced hepatitis, and improve the effect of liver transplantation. The latest research has found that UDCA also mediates and affects the regulation of carcinogenic signaling pathways in the cancer genome and is used for cancer treatment. In the treatment of progressive neurological diseases, UDCA can improve damaged mitochondrial function and play a neuroprotective role.

[0003] At present, the preparation methods of ursodeoxycholic acid mainly include chemical synthesis and biotransformation. Among them, ursodeoxycholic acid synthesized by chemical synthesis only accounts for 30% of the total synthesis route, which is far from meeting the market demand for the amount and quality of ursodeoxycholic acid. In addition, the chemical synthesis of ursodeoxycholic acid is complicated and involves the use of toxic substances and high temperature and high pressure conditions, which are harmful in terms of production safety and environmental protection. Therefore, new research focuses on obtaining ursodeoxycholic acid by using chenodeoxycholic acid (CDCA) as a precursor and by microbial transformation or bioenzyme catalysis.

[0004] Microbial whole-cell catalysis and enzyme catalysis are green and environmentally friendly, conforming to the general trend of social development and environmental protection, and will be a hot topic for future research. The enzyme catalysis method mainly uses 7α-HSDH and 7β-HSDH from different sources to convert chenodeoxycholic acid into ursodeoxycholic acid through a two-step enzymatic reaction. However, the two-step enzymatic reaction requires the addition of expensive cofactor NAD(P) + , increasing the cost of synthesis. In recent years, good progress has been made in the synthesis of chemical products using common microbial whole-cell catalysis, because it not only has high conversion efficiency and good specificity, but also can utilize the coenzyme regeneration system of the microorganism itself, and avoids the step of protein purification, which lays the foundation for the study of whole-cell catalysis of ursodeoxycholic acid for experimental phase research and industrial production. In addition, factors such as the small number of 7β-HSDH species, low activity and poor stability are also bottlenecks in the current enzymatic synthesis. Therefore, the screening of 7β-HSDH with high catalytic activity and high conversion rate and the optimization of the catalytic process are of great significance for the industrial production of ursodeoxycholic acid. Summary of the invention

[0005] The invention provides a novel 7β-hydroxysteroid dehydrogenase gene, which can be heterologously expressed with a 7α-hydroxysteroid dehydrogenase gene using Escherichia coli and Pichia pastoris as chassis cells, thereby obtaining recombinant strains for different conversion of ursodeoxycholic acid.

[0006] In order to solve the problems of low catalytic activity, low conversion rate and immature catalytic process of 7β-hydroxysteroid dehydrogenase, the present invention screened out a 7β-hydroxysteroid dehydrogenase derived from Clostridium innocuum by comparing and searching the NCBI database. The 7β-hydroxysteroid dehydrogenase has better catalytic potential in participating in the catalytic production process of ursodeoxycholic acid, provides a new selection direction for the process of whole-cell catalytic production of ursodeoxycholic acid, and selects different chassis cells as a platform for efficient production of ursodeoxycholic acid.

[0007] The invention provides a recombinant expression plasmid for catalyzing chenodeoxycholic acid to synthesize ursodeoxycholic acid. The recombinant expression vector is based on the pETDuet-1 double promoter plasmid of Escherichia coli and pPIC3.5K and pPICZX of Pichia pastoris, and is connected with two dehydrogenase genes, E7α-HSDH and Cin7β-HSDH.

[0008] The present invention provides a method for constructing the recombinant expression plasmid described in the above technical solution, comprising the following steps:

[0009] PCR was used to amplify the E7αHSDH and Cin7β-HSDH genes, and the dual promoter vector was digested. 2μl of the digested product and 2μl of the PCR product were taken for agarose gel electrophoresis to verify the band size. After digestion and purification of the PCR product, the target fragment and the backbone were homologously recombined, and the recombinant system was transformed into the competent Escherichia coli JM109. The transformant was picked to extract the plasmid, and the sequencing was verified to be correct, and the recombinant plasmid was obtained. Other recombinant plasmids were constructed by the above method.

[0010] The present invention also provides a recombinant engineering strain for catalyzing chenodeoxycholic acid to synthesize ursodeoxycholic acid. The recombinant engineering strain is obtained by respectively transforming a recombinant expression plasmid into Escherichia coli and integrating it into Pichia pastoris through electroporation.

[0011] The present invention also provides a method for synthesizing ursodeoxycholic acid by whole-cell catalysis of chenodeoxycholic acid, comprising the following steps:

[0012] The recombinant Escherichia coli was inoculated into TB liquid medium for fermentation. 600 When it reached 0.8, 2 mM IPTG was added for induction at a temperature of 25°C. The fermentation was continued for 18 h, and then 6 g / L chenodeoxycholic acid was added as a substrate for continued fermentation and conversion to obtain ursodeoxycholic acid.

[0013] The recombinant Pichia pastoris is inoculated into a YPG liquid medium for fermentation culture, and an appropriate amount of bacterial liquid is transferred to BMGY for continued culture, so that when the bacterial liquid density value reaches 7-8, the bacterial bodies are collected, the supernatant is discarded, and the precipitate is taken. The bacterial sludge is transferred to a BMMY medium, and the fermentation culture liquid is mixed with a substrate chenodeoxycholic acid and an inducer methanol; the amount of chenodeoxycholic acid added is 6 g / L, and the volume percentage of methanol added is 3.3%; the fermentation conversion culture condition is 28-30°C for fermentation conversion culture, and ursodeoxycholic acid is obtained.

[0014] Finally, the fermentation products were extracted from the fermentation broth using ethyl acetate, and the conversion of the fermentation products was analyzed by TLC and HPLC.

[0015] The novel 7β-hydroxysteroid dehydrogenase gene obtained by screening and the whole-cell catalytic method of the present invention can be used to produce ursodeoxycholic acid after high-efficiency expression in recombinant Escherichia coli and Pichia pastoris. Under the condition of 6 g / L feed amount, the recombinant Escherichia coli catalyzes the reaction for 24 hours, and the conversion rate of Pichia pastoris can be as high as 97.8%.

[0016] Through mining and analysis of the database, the present invention provides a novel 7β-hydroxysteroid dehydrogenase gene and its application, which can promote its application in the fields of medicine and the like; in addition, the culture medium used in the fermentation of the present invention is simple, does not require the addition of expensive cofactors, has low economic cost, and has great reference significance for future industrial applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Diagram of the cascade reaction catalyzed by E7α-HSDH and Cin7β-HSDH dual enzymes in whole cells.

[0018] Figure 2 Schematic diagram of the expression plasmid pETDuet-1-Cin7β-HSDH of recombinant Escherichia coli.

[0019] Figure 3 Schematic diagram of the recombinant Pichia pastoris expression vector pPIC3.5K-E7α-HSDH.

[0020] Figure 4 Schematic diagram of the recombinant Pichia pastoris expression vector pPICZX-Cin7β-HSDH.

[0021] Figure 5 This is the conversion rate diagram of ursodeoxycholic acid prepared by recombinant Escherichia coli at a feed amount of 6g / L chenodeoxycholic acid.

[0022] Figure 6 This is the conversion rate diagram of ursodeoxycholic acid prepared by recombinant Pichia pastoris at a feed amount of 6 g / L chenodeoxycholic acid.

[0023] Figure 7 This is the HPLC analysis result of ursodeoxycholic acid synthesized by recombinant Pichia pastoris. DETAILED DESCRIPTION

[0024] The method of the present invention is described below by specific embodiments. The experimental methods in the following examples are conventional methods unless otherwise specified. The embodiments should be understood to be illustrative rather than limiting the scope of the present invention, and the essence and scope of the present invention are limited only by the claims. The test materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0025] The present invention involves strains: Escherichia coli JM109, Escherichia coli BL21 (DE3), Pichia pastoris GS115, Tianjin University of Science and Technology Microbial Culture Collection Center.

[0026] Example 1

[0027] (1) Construction of recombinant plasmid and recombinant Escherichia coli

[0028] The 7β-HSDH from Clostridium innocuum (amino acid sequence as shown in SEQ ID NO. 1) was retrieved from the NCBI database, and the 7β-hydroxysteroid dehydrogenase gene was synthesized to construct the expression vector pETDuet-1-Cin7β-HSDH (construction schematic diagram as shown in Figure 2 ), 7α-HSDH was derived from E. coli BL21 (amino acid sequences are shown in SEQ ID NO. 2). The expression vector carrying pETDuet-1-Cin7β-HSDH was transformed into the E. coli host cell BL21, and the verified correct strain was named EE7α-Cin7β-HSDH.

[0029] Table 1 Primer sequences

[0030] serial number Primers Sequence (5′-3′) SEQ.NO.3 7β-HSDH-F CATATGACAATGACTGAC SEQ.NO.4 7β-HSDH-R GACTCGAGTTATTCGTAG

[0031] Cin7β-HSDH PCR verification reaction system (50μL):

[0032]

[0033] PCR procedure:

[0034]

[0035] (2) Synthesis of ursodeoxycholic acid catalyzed by recombinant Escherichia coli whole cells

[0036] The recombinant strain expressing EE7α-Cin7β-HSDH was inoculated into TB liquid medium for fermentation, and the components were 11.8 g / L tryptone, 23.6 g / L yeast extract, 2.2 g / L KH 2 PO4, 9.4g / LK 2 HPO4, 0.4% glycerol. The fermentation conditions were: culture temperature at 3°C, rotation speed at 200 rpm, and when the bacterial concentration OD 600 When the concentration reached 0.8, 2 mM IPTG was added for induction at 25 °C. The fermentation was continued for 18 h. CDCA was then added to continue the fermentation and conversion for 24 h. The supernatant was extracted with ethyl acetate after sampling. The conversion was shown in the figure. Figure 5 shown.

[0037] Embodiment 2:

[0038] (1) Construction of recombinant yeast GS115-pPIC3.5K-E7α-HSDH

[0039] Using the E.coli BL21 genome as a template, PCR amplified the target gene E7α-HSDH. The pPIC3.5K plasmid was digested with BamH I to prepare a homologous recombination backbone, and primers with homology arms were designed using the E7α-HSDH target gene as a template. 2 μL of the digested product and 2 μL of the PCR product were taken for agarose gel electrophoresis to verify the band size. After digestion and purification of the PCR product, the target fragment and the backbone were homologously recombined, and the recombination system was transformed into the competent E. coli JM109. The transformant was picked to extract the plasmid, and sequencing was used to verify that it was correct, thus obtaining the pPIC3.5K-E7α-HSDH recombinant plasmid (construction schematic diagram as shown in the figure). Figure 3 ). The pPIC3.5K-E7α-HSDH plasmid successfully verified by sequencing was linearized by single enzyme digestion with Sal I, and electroporated into the genome of Pichia pastoris to obtain Pichia pastoris GS115-pPIC3.5K-E7α-HSDH containing the pPIC3.5K-E7α-HSDH recombinant plasmid. Afterwards, high-copy transformants were further screened on solid YPD plates with 2 mg / mL and 4 mg / mL G418 resistance, and the successfully verified positive transformants were made into GS115-pPIC3.5K-E7α-HSDH competent cells for use.

[0040] Table 2 Primer sequences

[0041]

[0042] E7α-HSDH PCR reaction system (Total 50 μL):

[0043]

[0044] PCR reaction conditions:

[0045]

[0046] Enzyme digestion reaction system (TotaL 50μL):

[0047]

[0048] Recombination reaction system (Total 10μL)

[0049]

[0050] (2) Construction of a dual enzyme co-expression strain

[0051] Using the pETDuet-1-Cin7β-HSDH plasmid as a template, PCR amplified the target gene Cin7β-HSDH. The pPICZX plasmid was digested with EcoR I to prepare a homologous recombination backbone, and primers with homology arms were designed using the Cin7β-HSDH target gene as a template. 2μL of the digested product and 2μL of the PCR product were taken for agarose gel electrophoresis to verify the band size. After digestion and purification of the PCR product, the target fragment and the backbone were homologously recombined, and the recombination system was transformed into the competent Escherichia coli JM109. The transformant was picked to extract the plasmid, and the sequencing was verified to be correct, thus obtaining the pPICZX-Cin7β-HSDH recombinant plasmid (construction schematic diagram as shown in the figure). Figure 4 The pPICZX-Cin7β-HSDH plasmid successfully verified by sequencing was linearized by single enzyme digestion with SacI, and then transformed into the Pichia pastoris GS115-pPIC3.5K-E7α-HSDH competent medium prepared in step (1) of Example 2 by electroporation, and high-copy transformants were screened on solid YPD plates with 200 μg / mL and 400 μg / mL bleomycin resistance, and PCR verification was performed using its genomic DNA as a template. If the verification was successful, a dual-enzyme co-expression strain containing the pPIC3.5K-E7α-HSDH and pPICZX-Cin7β-HSDH recombinant plasmids, namely PE7α-Cin7β-HSDH, was obtained.

[0052] Table 3 Primer sequences

[0053]

[0054] Cin7β-HSDH PCR reaction system (Total 50μL):

[0055]

[0056] PCR procedure:

[0057]

[0058] Enzyme digestion reaction system (TotaL 50μL):

[0059]

[0060] Recombination reaction system (Total 10μL):

[0061]

[0062] (3) Recombinant Pichia pastoris for the synthesis of ursodeoxycholic acid

[0063] Pick a single colony from the Pichia recombinant bacteria plate and place it in YPG medium, and culture it in a shaker at 3°C ​​and 200 r / min for 24 h. Transfer an appropriate amount of bacterial culture from the YPG culture medium to BMGY medium, and add 10×YNB, phosphate buffer, and trace biotin to make the initial OD 600 The concentration of chenodeoxycholic acid is 0.2. After about 12-14 hours of culture, when the density of the bacterial liquid reaches 7-8, the bacteria are collected, the supernatant is discarded, and the precipitate is taken. The bacterial sludge is transferred to BMMY culture medium, and then 10×YNB, phosphate buffer and biotin are added. The fermentation culture liquid is mixed with the substrate chenodeoxycholic acid and the inducer methanol; the dosage of chenodeoxycholic acid is 6g / L, and the volume percentage of methanol added is 3.3%; the fermentation conversion culture conditions are 28-30℃ for fermentation conversion culture. Samples are taken 24 hours after conversion, and the supernatant is extracted with ethyl acetate. The conversion status is as follows: Figure 6 The extracted product was then re-dissolved in methanol and placed in a liquid phase vial for quantitative analysis of the synthesized product by HPLC-ELSD. The results are shown in Figure 5 shown.

[0064] Example 3: Analysis of fermentation conversion products

[0065] Accurately weigh 10 mg of chenodeoxycholic acid (95%), ursodeoxycholic acid (99%) and 7-ketolithocholic acid (98%) standards into a 10 mL volumetric flask, dissolve them in methanol by ultrasonication and dilute to the scale, filter them with a 0.22 μm filter membrane and set aside, resuspend and re-dissolve them in 100 μL of methanol, and then pass them through a 0.22 μm organic membrane to prepare the sample.

[0066] HPLC determination conditions are as follows: chromatographic column: C18 column (4.6 mm DL×250 L mm, 5 μm); mobile phase: methanol: water 0.1% phosphoric acid (80:20); injection volume: 10 μL; column temperature: 30°C; flow rate: 1 mL·min -1 .

[0067] The above embodiments only express several implementation methods of the present invention, but they are not intended to limit the present invention. It should be noted that any person skilled in the art can make several improvements to the above implementation methods without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the content defined in the claims.

Claims

1. A novel 7β-hydroxysteroid dehydrogenase gene, characterized in that: The nucleotide sequence of the 7β-hydroxysteroid dehydrogenase gene is shown in SEQ ID NO. 1, and a recombinant bacterium capable of efficiently preparing ursodeoxycholic acid is constructed.

2. The novel 7β-hydroxysteroid dehydrogenase gene according to claim 1, characterized in that: 7α-hydroxysteroid dehydrogenase and 7β-hydroxysteroid dehydrogenase were expressed simultaneously, and recombinant Escherichia coli BL21 (DE3) and recombinant Pichia pastoris GS115 were constructed for the efficient conversion of chenodeoxycholic acid to ursodeoxycholic acid.

3. The novel 7β-hydroxysteroid dehydrogenase gene according to claim 2, characterized in that: The recombinant E. coli BL21 for efficiently converting chenodeoxycholic acid into ursodeoxycholic acid comprises the following steps: (1) The 7α-hydroxysteroid dehydrogenase gene is derived from Escherichia coli BL21, and the nucleotide sequence of the 7α-hydroxysteroid dehydrogenase gene is shown in SEQ ID NO.

2. (2) The 7β-hydroxysteroid dehydrogenase gene from Clostridium innocuum was synthesized and the pETDuet-1-Cin7β-HSDH plasmid was constructed. Its characteristics are that the pETDuet-1 vector is a dual-promoter plasmid; (3) The constructed expression plasmid was transformed into Escherichia coli BL21, and the recombinant bacteria was named EE7α-Cin7β-HSDH.

4. The novel 7β-hydroxysteroid dehydrogenase gene according to claim 2, characterized in that: The recombinant Pichia pastoris GS115 for efficiently converting chenodeoxycholic acid into ursodeoxycholic acid comprises the following steps: (1) Construction of recombinant expression plasmids containing 7α-HSDH gene and 7β-HSDH gene: inserting 7α-HSDH gene and 7β-HSDH gene into expression vectors pPIC3.5K and pPICZX, respectively, to obtain recombinant expression plasmids pPIC3.5K-E7α-HSDH and pPICZX-Cin7β-HSDH; (2) The recombinant vectors pPIC3.5K-PE7α-HSDH and pPICZX-Cin7β-HSDH were sequentially electroporated into Pichia pastoris GS115. The recombinant bacteria were named PE7α-Cin7β-HSDH.

5. Application of a novel 7β-hydroxysteroid dehydrogenase gene, characterized in that: The application of the novel 7β-hydroxysteroid dehydrogenase gene in the preparation of ursodeoxycholic acid comprises the following steps: (1) The recombinant Escherichia coli EE7α-Cin7β-HSDH according to claim 3 is inoculated into a liquid culture medium for fermentation culture. When the bacterial cell concentration OD600 is 0.8, IPTG is added for induction. After the induction is completed, it is mixed with chenodeoxycholic acid for fermentation and conversion culture to obtain ursodeoxycholic acid. (2) The recombinant Pichia pastoris PE7α-Cin7β-HSDH of claim 4 is inoculated into a liquid culture medium for fermentation to obtain a fermentation culture solution, and the fermentation culture solution is mixed with chenodeoxycholic acid and methanol inducer for fermentation conversion culture to obtain ursodeoxycholic acid.

6. Ursodeoxycholic acid obtained by the use as claimed in claim 5.

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