CN117535217A - Recombinant bacillus subtilis engineering strain and application thereof in biological preparation of ursodeoxycholic acid - Google Patents
Recombinant bacillus subtilis engineering strain and application thereof in biological preparation of ursodeoxycholic acid Download PDFInfo
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- 244000063299 Bacillus subtilis Species 0.000 title claims abstract description 54
- 235000014469 Bacillus subtilis Nutrition 0.000 title claims abstract description 54
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- Enzymes And Modification Thereof (AREA)
Abstract
The invention provides a recombinant bacillus subtilis engineering strain and application thereof in biological preparation of ursodeoxycholic acid, belonging to the technical field of biological engineering. The invention provides a recombinant bacillus subtilis engineering strain capable of simultaneously and efficiently and stably expressing 7 alpha-hydroxysteroid dehydrogenase (7 alpha-HSDH) and 7 beta-hydroxysteroid dehydrogenase (7 beta-HSDH). In biosynthesis, chenodeoxycholic acid is used as substrate and combined with oxidation under the action of 7alpha-HSDHCoenzyme NADP + The 7 alpha hydroxyl in chenodeoxycholic acid is oxidized into carbonyl, and the generated intermediate 7-keto-lithocholic acid is reduced into ursodeoxycholic acid under the action of 7 beta-HSDH and NADPH. The recombinant bacillus subtilis engineering strain can efficiently and stably biosynthesize ursodeoxycholic acid, greatly reduces the purification work of products in industrial application, and greatly reduces the production cost.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a recombinant bacillus subtilis engineering strain and application thereof in biological preparation of ursodeoxycholic acid.
Background
Ursodeoxycholic acid, alias 3 alpha, 7 beta-dihydroxyl-5 beta-cholestane-24-acid, ursodeoxycholic acid, hyodeoxycholic acid, isodeoxycholic acid, dihydroxyl cholic acid, 3 beta, 6 alpha-dihydroxyl cholanic acid and dihydroxyl cholanic acid, can be used for increasing bile acid secretion, changing bile components, reducing cholesterol and cholesterol fat in bile, being beneficial to gradually dissolving cholesterol in gall-stone, being used for cholesterol-stone which is not suitable for operation treatment, but not dissolving gall-stone, mixed stone and stone which is not transparent to X rays, and also having certain curative effect on cholecystitis, cholangitis and dyspepsia.
There are three main approaches to the traditional sources of ursodeoxycholic acid: natural sources, chemical synthesis and biosynthesis. Natural sources are limited in origin against animal protection laws; although the chemical synthesis is simple to operate and the reagent is cheap, the problems of chemical toxic reagents, chiral difficulties and the like in the process become the bottleneck of industrial technology transformation; the biosynthesis has the advantages of green, simplicity, convenience, high efficiency and the like, but the problems of complex protein extraction and purification work and the like are limited to laboratory operation and are difficult to industrialize due to low enzyme stability.
The prior synthesis method is shown in table 1.
TABLE 1 method for synthesizing ursodeoxycholic acid in the prior art
The industrial synthesis mainly takes simple and easily available animal cholic acid as a raw material, and adopts a chemical synthesis technology method to synthesize UCDA; the method has the advantages of simple operation, low reagent cost and mature industry, but the problems of chemical toxic reagents, chiral difficulties and the like (shown in the table) in the process become the bottleneck of industrial technology transformation. The biosynthesis mode of generating UDCA under the action of biological enzyme catalysis by taking cholic acid and chenodeoxycholic acid as raw materials has the advantages of being green, simple, convenient, efficient and the like, thereby becoming a research hot spot in recent years. In addition, in terms of raw material cost, bile acid used abroad is derived from oxgall, the commercial value of the bile is between dollars 0.1 and 0.4 per liter, and the bile acid accounts for about 0.7 percent (w/w) of the bile. In China, a large number of sources of chicken goose bile make the raw materials not limited by oxgall, and the chicken goose bile has a higher content of chenodeoxycholic acid compared with oxgall. Therefore, the synthesis of UDCA by taking chenodeoxycholic acid as a raw material has considerable prospect in enzyme catalysis.
At present, a biological method is used for synthesizing ursodeoxycholic acid, and a method of regioselective oxidation and subsequent reduction of the ursodeoxycholic acid through 7-OH is received much attention, but in the two-step method, 7-OH in the first step is oxidized into carbonylation, a reaction system is treated to promote the inactivation of 7a-HSDH enzyme, and then intermediate 7-keto-lithocholic acid is used as a substrate to synthesize the ursodeoxycholic acid under the action of 7 beta-HSDH enzyme. Meanwhile, the existing biological method mainly adopts escherichia coli as host cells (CN 109722442B, CN 113061637A) to synthesize UDCA, but the steps of cell wall breaking, protein renaturation and the like involved in the method are complex in operation and high in cost, and the influence of escherichia coli endotoxin brings great difficulty to large-scale industrial production.
Compared with escherichia coli, the bacillus subtilis not only has the advantages of non-pathogenicity, no endotoxicity, low codon preference, high solubility of expressed protein, good biological activity, high secretion and the like, but also has the advantages that the single-layer membrane structure enables the protein secretion to be easier, and the fusion expression of the protein secretion signal peptide in the bacillus subtilis can guide the target protein to carry out extracellular secretion more smoothly, so that the extracellular secretion of heterologous protein is optimized, the purification work of a required protein product in industrial application is greatly reduced, the operation is simplified, and the generation cost is reduced.
Disclosure of Invention
In view of the above, the invention aims to provide a recombinant bacillus subtilis engineering strain which can be used for efficiently producing ursodeoxycholic acid by a one-pot method by constructing a strain containing coding genes 7 alpha-HSDH and 7 beta-HSDH for synthesizing ursodeoxycholic acid key catalytic enzyme.
The invention provides a recombinant bacillus subtilis engineering strain, which takes bacillus subtilis as host bacteria, and comprises a recombinant vector expressing a 7 alpha-hydroxysteroid dehydrogenase gene with optimized codons and a recombinant vector expressing a 7 beta-hydroxysteroid dehydrogenase gene with optimized codons or a recombinant vector containing a fusion gene formed by the 7 alpha-hydroxysteroid dehydrogenase gene with optimized codons and the 7 beta-hydroxysteroid dehydrogenase gene.
Preferably, the nucleotide sequence of the 7 alpha-hydroxysteroid dehydrogenase gene after codon optimization is shown as SEQ ID NO. 1;
the nucleotide sequence of the 7 beta-hydroxysteroid dehydrogenase gene after codon optimization is shown as SEQ ID NO. 2;
the nucleotide sequence of the fusion gene formed by the codon optimized 7 alpha-hydroxysteroid dehydrogenase gene and the 7 beta-hydroxysteroid dehydrogenase gene is shown as SEQ ID NO. 3.
Preferably, the strain of bacillus subtilis includes WB800N.
The invention provides application of the recombinant bacillus subtilis engineering strain in biological preparation of ursodeoxycholic acid.
The invention provides a method for biologically preparing ursodeoxycholic acid based on the recombinant bacillus subtilis engineering strain, which comprises the following steps:
taking chenodeoxycholic acid as a substrate, and carrying out catalytic reaction on a culture supernatant of the recombinant bacillus subtilis engineering strain under the action of coenzyme NADP to obtain ursodeoxycholic acid.
Preferably, the temperature of the catalytic reaction is 25-37 ℃.
Preferably, the catalytic reaction time is 24-72 hours.
Preferably, the culture method of the culture supernatant of the recombinant bacillus subtilis engineering strain comprises the following steps: inoculating the seed liquid into a liquid LB culture medium, and culturing for 5-6h at 37 ℃ and 200 rpm; when the bacterial cells grow to the logarithmic growth phase, 1mM isopropyl-beta-D-thiopyran galactoside with the final concentration is added, the culture is continued for 18 hours at 25-30 ℃ and 200rpm, and the culture solution is separated, so that the culture supernatant containing 7 alpha-hydroxysteroid dehydrogenase and 7 beta-hydroxysteroid dehydrogenase is obtained.
Preferably, the catalytic reaction system is that 0.25-0.75 g of chenodeoxycholic acid and 2-5 mgNADP are added into each 100ml of culture supernatant of recombinant bacillus subtilis engineering strain;
preferably, 0.5g of chenodeoxycholic acid and 2mgNADP are added to 100ml of the culture supernatant of the recombinant Bacillus subtilis engineering strain.
Preferably, the catalytic reaction further comprises separating the product; the method for separating the product comprises the steps of regulating the pH value of a system after catalytic reaction to 4, separating a ursodeoxycholic acid crude product, mixing and refluxing the ursodeoxycholic acid crude product and ethyl acetate, and filtering to obtain ursodeoxycholic acid with the purity of more than 85%.
The invention simultaneously expresses 7 alpha-hydroxysteroid dehydrogenase gene and 7 beta-hydroxysteroid dehydrogenase gene which are subjected to codon optimization in bacillus subtilis to form fusion genes, thereby obtaining fusion enzyme conjunctThe ursodeoxycholic acid prepared by the one-pot method can fully and effectively utilize the NADP + And the conversion of NADPH saves the energy consumption in cells and improves the yield. The fusion enzyme conjunct one-step synthesis method not only realizes NADP+/NADPH self-circulation, but also has the conversion efficiency of more than 85 percent. The ursodeoxycholoease catalytic process comprises two steps, the step catalyzed by 7α -HSDH requires consumption of nadp+, while the step catalyzed by 7β -HSDH requires consumption of NADPH. Therefore, in order to solve the problems, the invention constructs the 7 alpha/beta-HSDH fusion enzyme conjunct to realize NADP+/NADPH self-circulation and simplify the operation steps by a one-step method, and lays a foundation for realizing one-step synthesis of UDCA and industrialized production thereof.
The invention is realized by utilizing the following technical scheme:
the 7 alpha-HSDH is derived from Bacteroides fragiles, the 7 beta-HSDH is derived from Ruminococcus gnavus, and the nucleotide sequence of fusion of the optimized 7 alpha-hydroxysteroid dehydrogenase gene and the optimized 7 beta-hydroxysteroid dehydrogenase gene is shown as SEQ ID NO. 1.
The invention constructs a bacillus subtilis engineering strain for stably and efficiently expressing 7 alpha/beta-HSDH, and the 7 alpha/beta-HSDH gene with optimized codons can be efficiently expressed in bacillus subtilis WB800N. The bacillus subtilis has more excellent exogenous secretion capacity, can directly secrete the synthesized ursodeoxycholic acid into the culture solution outside the cell, does not need to crush host cells, and effectively solves the problem that intracellular complex components interfere with target components during cell crushing, thereby greatly simplifying the extraction and purification steps. The step of ursodeoxycholoease catalytic process comprising catalysis by 7α -HSDH requires consumption of NADP + Whereas the step catalyzed by 7β -HSDH requires consumption of NADPH. Therefore, in order to solve the problems, the invention constructs the 7 alpha/beta-HSDH fusion enzyme conjunct, realizes NADP+/NADPH self-circulation and one-step simplified operation steps, has the conversion efficiency of more than 85 percent, and lays a foundation for realizing one-step synthesis and industrial production of ursodeoxycholic acid.
Drawings
FIG. 1 is a schematic diagram of the process of synthesizing ursodeoxycholic acid using chenodeoxycholic acid as a substrate;
FIG. 2 is a 7. Alpha. -HSDH vector spectrum;
FIG. 3 is a 7. Beta. -HSDH vector spectrum;
FIG. 4 is a 7. Alpha. Beta. -HSDH vector spectrum;
FIG. 5 is a diagram showing the digestion verification of an alpha-HSDH EcoRI vector, wherein the Marker is 10000bp;
FIG. 6 is a 7. Beta. -HSDH EcoRI cleavage verification chart in which Marker is 10000bp;
FIG. 7 is a 7. Alpha. Beta. -HSDH EcoRI cleavage map in which Marker is 10000bp.
Detailed Description
The invention provides a recombinant bacillus subtilis engineering strain, which takes bacillus subtilis as host bacteria, and comprises a recombinant vector expressing a 7 alpha-hydroxysteroid dehydrogenase gene with optimized codons and a recombinant vector expressing a 7 beta-hydroxysteroid dehydrogenase gene with optimized codons or a recombinant vector containing a fusion gene formed by the 7 alpha-hydroxysteroid dehydrogenase gene with optimized codons and the 7 beta-hydroxysteroid dehydrogenase gene.
In the invention, the nucleotide sequence of the 7 alpha-hydroxysteroid dehydrogenase gene after codon optimization is shown as SEQ ID NO. 1 (gcccaccatcaccatcaccatATGAATCGGTTTGAAAATAAAATCATCATTATTACCGGAGCGGCCGGCGGAATCGGAGCGTCTACCACTCGAAGAATCGTATCTGAAGGCGGCAAGGTAGTTATTGCTGATTATTCAAGAGAAAAAGCAGATCAATTTGCGGCTGAATTGTCAAACAGCGGAGCTGACGTTAGGCCCGTGTATTTCAGCGCGACAGAGTTAAAGTCGTGTAAGGAGCTGATTACGTTCACAATGAAAGAATACGGACAGATTGATGTGCTTGTCAACAATGTCGGCGGTACGAATCCGCGTCGTGATACAAATATTGAAACACTTGATATGGATTACTTTGATGAAGCGTTTCACTTGAATCTGAGCTGTACGATGTATTTGTCCCAGCTGGTCATTCCGATTATGAGTGCTCAAGGCGGAGGCAACATAGTGAATGTAGCGTCCATTTCTGGTATCACTGCAGACAGCAACGGGACCCTCTACGGCGCATCAAAAGCGGGTGTCATCAATTTAACAAAATATATCGCAACACAGACAGGAAAAAAAAATATCCGCTGCAATGCCGTCGCTCCAGGGCTTATACTGACACCGGCTGCATTAAACAACCTGAATGAAGAAGTGAGAAAAATTTTTCTCGGACAATGCGCGACGCCTTATTTAGGTGAGCCTCAGGATGTTGCTGCCACAATAGCATTCCTTGCCTCAGAGGACGCCCGCTATATTACTGGCCAAACGATCGTTGTTGACGGGGGGCTAACCATTCATAACCCGACGATTAACCTTGTGagttagcagcccgcctaatgagcg, wherein lower case letter bases at two ends of the sequence are vector homology arms). The nucleotide sequence of the 7 beta-hydroxysteroid dehydrogenase gene after codon optimization is shown as SEQ ID NO. 2 (gcccaccatcaccatcaccatATGACCCTTCGTGAAAAATACGGTGAATGGGGAATAATTTTGGGGGCAACAGAGGGCGTTGGTAAGGCCTTCTGTGAGCGGCTTGCAAAAGAAGGCATGAATGTCGTCATGGTTGGGAGAAGGGAAGAGAAGTTAAAAGAGCTTGGAGAAGAGTTGAAAAATACATATGAAATCGATTATAAAGTTGTGAAAGCGGATTTTTCACTTCCTGACGCGACAGATAAAATATTCGCCGCAACGGAAAACCTGGACATGGGCTTTATGGCGTATGTAGCCTGTTTACATTCTTTCGGAAAGATTCAAGACACTCCATGGGAAAAGCATGAAGCGATGATTAACGTGAATGTGGTCACGTTTATGAAATGCTTTTATCATTATATGAAAATCTTTGCGGCGCAGGACAGAGGCGCTGTTATCAATGTAAGCAGCATGACAGGCATTTCATCGTCCCCGTGGAATGGTCAATATGGAGCAGGGAAAGCATTTATCTTGAAAATGACTGAAGCAGTTGCTTGCGAAACAGAGAAAACAAACGTCGATGTTGAAGTCATTACGCTGGGAACGGTGTTAACACCGAGTCTGCTCTCCAATCTACCCGGCGGCCCTCAGGGAGAGGCCGTGATGAAGACCGCTCAGACCCCGGAAGAAGTAGTAGATGAAGCCTTTGAAAAGCTCGGAAAAGAGCTGTCAGTGATTAGTGGTGAGCGCAACAAAGCTTCTGTTCACGATTGGAAAGCTAACCACACTGAAGATGATTACATCCGCTACATGGGGAGCTTCTATCAAGAAagttagcagcccgcctaatgagcggg, wherein lower case letter bases at two ends of the sequence are carrier homology arms). The nucleotide sequence of the fusion gene formed by the codon optimized 7 alpha-hydroxysteroid dehydrogenase gene and the 7 beta-hydroxysteroid dehydrogenase gene is shown as SEQ ID NO. 3 (gcccaccatcaccatcaccatATGAATCGGTTTGAAAATAAAATCATCATTATTACCGGAGCGGCCGGCGGAATCGGAGCGTCTACCACTCGAAGAATCGTATCTGAAGGCGGCAAGGTAGTTATTGCTGATTATTCAAGAGAAAAAGCAGATCAATTTGCGGCTGAATTGTCAAACAGCGGAGCTGACGTTAGGCCCGTGTATTTCAGCGCGACAGAGTTAAAGTCGTGTAAGGAGCTGATTACGTTCACAATGAAAGAATACGGACAGATTGATGTGCTTGTCAACAATGTCGGCGGTACGAATCCGCGTCGTGATACAAATATTGAAACACTTGATATGGATTACTTTGATGAAGCGTTTCACTTGAATCTGAGCTGTACGATGTATTTGTCCCAGCTGGTCATTCCGATTATGAGTGCTCAAGGCGGAGGCAACATAGTGAATGTAGCGTCCATTTCTGGTATCACTGCAGACAGCAACGGGACCCTCTACGGCGCATCAAAAGCGGGTGTCATCAATTTAACAAAATATATCGCAACACAGACAGGAAAAAAAAATATCCGCTGCAATGCCGTCGCTCCAGGGCTTATACTGACACCGGCTGCATTAAACAACCTGAATGAAGAAGTGAGAAAAATTTTTCTCGGACAATGCGCGACGCCTTATTTAGGTGAGCCTCAGGATGTTGCTGCCACAATAGCATTCCTTGCCTCAGAGGACGCCCGCTATATTACTGGCCAAACGATCGTTGTTGACGGGGGGCTAACCATTCATAACCCGACGATTAACCTTGTGggttctggatccggttctggaagtggatccATGACCCTTCGTGAAAAATACGGTGAATGGGGAATAATTTTGGGGGCAACAGAGGGCGTTGGTAAGGCCTTCTGTGAGCGGCTTGCAAAAGAAGGCATGAATGTCGTCATGGTTGGGAGAAGGGAAGAGAAGTTAAAAGAGCTTGGAGAAGAGTTGAAAAATACATATGAAATCGATTATAAAGTTGTGAAAGCGGATTTTTCACTTCCTGACGCGACAGATAAAATATTCGCCGCAACGGAAAACCTGGACATGGGCTTTATGGCGTATGTAGCCTGTTTACATTCTTTCGGAAAGATTCAAGACACTCCATGGGAAAAGCATGAAGCGATGATTAACGTGAATGTGGTCACGTTTATGAAATGCTTTTATCATTATATGAAAATCTTTGCGGCGCAGGACAGAGGCGCTGTTATCAATGTAAGCAGCATGACAGGCATTTCATCGTCCCCGTGGAATGGTCAATATGGAGCAGGGAAAGCATTTATCTTGAAAATGACTGAAGCAGTTGCTTGCGAAACAGAGAAAACAAACGTCGATGTTGAAGTCATTACGCTGGGAACGGTGTTAACACCGAGTCTGCTCTCCAATCTACCCGGCGGCCCTCAGGGAGAGGCCGTGATGAAGACCGCTCAGACCCCGGAAGAAGTAGTAGATGAAGCCTTTGAAAAGCTCGGAAAAGAGCTGTCAGTGATTAGTGGTGAGCGCAACAAAGCTTCTGTTCACGATTGGAAAGCTAACCACACTGAAGATGATTACATCCGCTACATGGGGAGCTTCTATCAAGAAgcagcccgcctaatgagcg, wherein lower case bases at two ends of the sequence are carrier homology arms, and lower case bases in the middle are connecting arms). The backbone vector of the recombinant vector is preferably a T50 expression vector. The fusion gene is preferably inserted into the XbaI multiple cloning site of the backbone vector. The strain of bacillus subtilis includes WB800N.
In the invention, the construction method of the recombinant bacillus subtilis engineering strain preferably comprises the following steps:
constructing a recombinant vector for expressing the 7 alpha-hydroxysteroid dehydrogenase gene with optimized codons and a recombinant vector for expressing the 7 beta-hydroxysteroid dehydrogenase gene with optimized codons or a recombinant vector for expressing the 7 alpha-hydroxysteroid dehydrogenase gene with optimized codons and the 7 beta-hydroxysteroid dehydrogenase gene;
the recombinant vector expressing the 7 alpha-hydroxysteroid dehydrogenase gene with optimized codons and the recombinant vector expressing the 7 beta-hydroxysteroid dehydrogenase gene with optimized codons or the recombinant vector containing the 7 alpha-hydroxysteroid dehydrogenase gene with optimized codons and the 7 beta-hydroxysteroid dehydrogenase gene are transformed into bacillus subtilis, and the recombinant bacillus subtilis engineering strain is obtained through screening.
In the method of constructing a recombinant vector comprising a fusion gene formed by expressing the codon-optimized 7α -hydroxysteroid dehydrogenase gene and 7β -hydroxysteroid dehydrogenase gene, preferably, the fusion gene formed by expressing the codon-optimized 7α -hydroxysteroid dehydrogenase gene and 7β -hydroxysteroid dehydrogenase gene is cloned into a backbone vector to obtain the recombinant vector.
The method of cloning is not particularly limited in the present invention, and may be accomplished by cloning methods well known in the art, such as homologous recombination methods.
The method of transformation is not particularly limited in the present invention, and can be accomplished by transformation methods well known in the art, for example, by the Spizizen transformation method, specifically, see the following documents (Anagnostopoulos C, spizizen J (1961) Requirements for transformation in Bacillus, bacteria 81:741-746; li Ruifang, xue, yellow light et al, preparation of competent cells of Bacillus subtilis, plasmid transformation methods research [ J ]. Biotechnology report, 2011 (05): 227-230.DOI:10.13560/j.cnki. Biotech. Bull.1985.2011.05.035; li Chunyan, feng Fengzhao, feng Lou, cheng Yi, cheng Xiaosong. Spizizen transformation method optimization of wild type Bacillus subtilis N4 [ J ]. University of northeast agriculture, 2015,46 (02): 78-82+108.)
In the present invention, the screening method is preferably to pick single colony in 4ml liquid LB medium (4. Mu.l 20mg/ml chloramphenicol has been added), put in a shaking table at 37℃and 200rpm for overnight culture; and taking out the bacterial liquid cultured overnight, performing microscopic examination, quickly extracting genome, and verifying target genes by PCR.
The invention provides application of the recombinant bacillus subtilis engineering strain in biological preparation of ursodeoxycholic acid.
The invention provides a method for biologically preparing ursodeoxycholic acid based on the recombinant bacillus subtilis engineering strain, which comprises the following steps:
chenodeoxycholic acid is used as a substrate, and catalytic reaction is carried out under the action of culture supernatant fluid of recombinant bacillus subtilis engineering strain and NADP, so as to obtain ursodeoxycholic acid.
In the invention, the culture method of the culture supernatant of the recombinant bacillus subtilis engineering strain comprises the following steps: inoculating the seed liquid into a liquid LB culture medium, and culturing for 5-6h at 37 ℃ and 200 rpm; when the cell amount is up to the logarithmic growth phase, 1mM isopropyl-beta-D-thiopyran galactoside with the final concentration is added, the culture is continued for 18 hours at 25-30 ℃ and 200rpm, and the obtained bacterial liquid is centrifuged and the sediment is removed, so that the culture supernatant containing 7 alpha-hydroxysteroid dehydrogenase and 7 beta-hydroxysteroid dehydrogenase is obtained.
In the invention, the preparation method of the seed liquid is preferably to inoculate recombinant bacillus subtilis engineering strains into LB culture medium, and culture the recombinant bacillus subtilis engineering strains to a logarithmic phase at 25-30 ℃ to obtain the seed liquid.
In the present invention, the catalytic reaction system is preferably such that 0.25 to 0.75g of chenodeoxycholic acid (CDCA) is added per 100ml of the culture supernatant of the recombinant Bacillus subtilis engineering strain, 2 to 5mg of NADP is added, more preferably 0.5g of chenodeoxycholic acid (CDCA) per 100ml of the culture supernatant of the recombinant Bacillus subtilis engineering strain, 2mg of NADP is added. Separation of the products is also preferably included after the catalytic reaction has ended. The method for separating the product is preferably to add hydrochloric acid into a catalytic reaction system to adjust the product to be acidic (pH=4), promote the crystallization of ursodeoxycholic acid (UDCA) product, centrifuge the product at 10000rmp for 20min to remove protein and solution, distill to remove residual liquid to obtain a crude UDCA product, add the obtained crude UDCA product and ethyl acetate into a reaction vessel, heat and reflux for 1 hour, cool to normal temperature, and filter to obtain the UDCA with purity of more than 85%. The temperature of the catalytic reaction is preferably 25 to 37 ℃, more preferably 30 ℃. The time of the catalytic reaction is preferably 24 to 72 hours, more preferably 36 to 48 hours, and most preferably 42 hours.
In the invention, the recombinant bacillus subtilis engineering strain can stably and efficiently express 7 alpha-hydroxysteroid dehydrogenase (7 alpha-HSDH) and 7 beta-hydroxysteroid dehydrogenase (7 beta-HSDH), and during biosynthesis, chenodeoxycholic acid (CDCA) is taken as an initial substrate, and under the action of the 7 alpha-hydroxysteroid dehydrogenase (7 alpha-HSDH), oxidized coenzyme NADP is combined + Oxidizing the hydroxyl group at position 7α in chenodeoxycholic acid to carbonyl group, thereby producing intermediate 7-keto-lithocholic acid (7-K-LCA); 7-keto-lithocholic acid as a substrate for 7β -hydroxysteroid dehydrogenase (7β -HSDH) is reduced to ursodeoxycholic acid by the action of 7β -HSDH in combination with reduced coenzyme NADPH. Therefore, the 7 alpha-HSDH and 7 beta-HSDH prepared by the recombinant bacillus subtilis engineering strain provided by the invention have high biological activityThe ursodeoxycholic acid can be efficiently and stably biosynthesized, and the bacillus subtilis is a nonpathogenic microorganism, so that the self-metabolite is low in toxicity and harmless, the purification work of the product in industrial application is greatly reduced, the product separation step is simplified, the operation complexity is reduced, and the production cost is greatly reduced.
The following examples further illustrate the invention but are not to be construed as limiting the invention.
Example 1
Synthesis of 7α -hydroxysteroid dehydrogenase (7α -HSDH) and/or 7β -hydroxysteroid dehydrogenase (7β -HSDH) and construction of recombinant vector therefor
Genes for 7α -HSDH (derived from Bacteroides fragiles, genBank: OGX 95366.1) and 7β -HSDH (derived from Ruminococcus gnavus, genBank: R9UAM 1) were codon-optimized according to the codon preference of Bacillus subtilis. Digging into 7 alpha-HSDH sequence and 7 beta-HSDH sequence by utilizing a gene digging method, carrying out codon optimization synthesis genes (SEQ ID NO:1 and SEQ ID NO: 2) according to protein sequences, respectively constructing into T50 expression vectors, wherein a gene insertion site is XbaI, simultaneously fusing the two genes to synthesize a fusion gene (SEQ ID NO: 3), inserting into the T50 expression vectors, and carrying out spectrograms of the recombinant expression vectors with the XbaI gene insertion site as shown in figures 2-4.
The artificially synthesized gene fragments are respectively used as templates, and the primer pairs 7 alpha-50 for/7 alpha-50 rev in the table 1 are used for amplifying 7 alpha-HSDH gene fragments, 7 beta-50 for/7 beta-50 rev are used for amplifying 7 beta-HSDH gene fragments and 7 alpha beta-50 for/7 alpha beta-50 rev are used for amplifying fusion fragments formed by 7 alpha-HSDH and 7 beta-HSDH. The amplification reaction system is shown in Table 2, and the reaction procedure is shown in Table 3.
TABLE 1 primer sequence information
TABLE 2 reaction system
Table 3 reaction procedure
And (3) obtaining an amplified target fragment, connecting the amplified target fragment with an expression vector T50 by a Gibson method, transferring the obtained recombinant vector into an escherichia coli clone strain, selecting a single colony culture plasmid, and verifying the recombinant plasmid by using EcoRI double enzyme digestion. The results are shown in FIGS. 5-7, which demonstrate the success of recombinant plasmid construction.
Example 2
Construction of engineering strain for expressing fusion gene 7 alpha beta-HSDH
The recombinant plasmid obtained in example 1 was transformed into bacillus subtilis competent cell WB800N by Spizizen transformation method, spread on solid LB medium containing ampicillin for culturing, single clone fungus was picked up and dropped in liquid medium for culturing, the recombinant fungus after screening was subjected to microscopic examination, genome was quickly extracted, taQ enzyme was confirmed to recombinant plasmid, and preliminary protein induction expression was carried out, and analysis and identification were carried out on three enzymes 7α -HSDH (32 kDa), 7β -HSDH (33.7 kDa) and 7αβ -HSDH (64.3 kDa) by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The results all demonstrated that the gene was correctly incorporated into the expression vector.
500. Mu.l of a Bacillus subtilis WB800N strain containing T50-7α/7β -HSDH was added to 50ml of LB liquid medium, while 12.5. Mu.l of 20mg/ml chloramphenicol was added thereto, shaken well, and shake-cultured overnight at 200rpm at 37℃as a seed solution. Taking out the seed bacterial liquid cultured overnight, performing microscopic examination, quickly extracting genome, performing PCR verification, adding the seed bacterial liquid cultured overnight into 500ml LB fermentation medium containing 125 μl of 20mg/ml chloramphenicol at 1% inoculation amount, culturing in a shaker at 37deg.C and 200rpm for 5-6 hr, and culturing when the bacterial body amount is up to logarithmic phase (OD 600 When=0.6-1), 1mM IPTG was added and induced in a shaker at 30 ℃/25℃and 200rpm for 18 hours, the induced bacterial liquid was centrifuged at 4000rpm for 30 minutes, the bacterial cells were discarded to leave a supernatant, and a supernatant containing 7α/7β -HSDH was obtained.
Example 3
Method for converting ursodeoxycholic acid by recombinant bacterium fermentation broth
100ml of the supernatant containing 7α/7β -HSDH prepared in example 2 was taken, the pH was adjusted to 8.5 with NaOH, and 0.5g of chenodeoxycholic acid and 2mg of NADP were taken + After dissolving in 0.1mol/LPBS (pH=8.0) solution, the solution was added dropwise to the supernatant to maintain pH>7.5, detecting the reaction progress by thin layer chromatography, detecting after 20 hours, completely reacting the substrate, adjusting the reaction pH to 6.0, and filtering and collecting the precipitate to obtain 0.45g ursodeoxycholic acid.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. A recombinant bacillus subtilis engineering strain is characterized by taking bacillus subtilis as a host bacterium, and comprises a recombinant vector for expressing a 7 alpha-hydroxysteroid dehydrogenase gene with optimized codons and a recombinant vector for expressing a 7 beta-hydroxysteroid dehydrogenase gene with optimized codons or comprises a recombinant vector for expressing a fusion gene formed by the 7 alpha-hydroxysteroid dehydrogenase gene with optimized codons and the 7 beta-hydroxysteroid dehydrogenase gene.
2. The recombinant bacillus subtilis engineering strain according to claim 1, wherein the nucleotide sequence of the 7 alpha-hydroxysteroid dehydrogenase gene after codon optimization is shown in SEQ ID NO. 1;
the nucleotide sequence of the 7 beta-hydroxysteroid dehydrogenase gene after codon optimization is shown as SEQ ID NO. 2;
the nucleotide sequence of the fusion gene formed by the 7 alpha-hydroxysteroid dehydrogenase gene and the 7 beta-hydroxysteroid dehydrogenase gene with optimized codons is shown in SEQ ID NO. 3.
3. The recombinant bacillus subtilis engineering strain according to claim 1, wherein the strain of bacillus subtilis comprises WB800N.
4. Use of the recombinant bacillus subtilis engineering strain according to any one of claims 1-3 in biological preparation of ursodeoxycholic acid.
5. A method for biologically preparing ursodeoxycholic acid based on the recombinant bacillus subtilis engineering strain according to any one of claims 1 to 3, comprising the steps of:
taking chenodeoxycholic acid as a substrate, and carrying out catalytic reaction on a culture supernatant of the recombinant bacillus subtilis engineering strain under the action of coenzyme NADP to obtain ursodeoxycholic acid.
6. The method according to claim 5, wherein the temperature of the catalytic reaction is 25 to 37 ℃.
7. The method according to claim 5, wherein the catalytic reaction is carried out for 24 to 72 hours.
8. The method according to claim 5, wherein the preparation method of the culture supernatant of the recombinant bacillus subtilis engineering strain comprises the following steps: inoculating the seed liquid into a liquid LB culture medium, and culturing for 5-6h at 37 ℃ and 200 rpm; when the bacterial cells grow to the logarithmic growth phase, 1mM isopropyl-beta-D-thiopyran galactoside with the final concentration is added, the culture is continued for 18 hours at 25-30 ℃ and 200rpm, and the culture solution is separated, so that the culture supernatant containing 7 alpha-hydroxysteroid dehydrogenase and 7 beta-hydroxysteroid dehydrogenase is obtained.
9. The method according to claim 5, wherein the catalytic reaction system comprises adding 0.25-0.75 g chenodeoxycholic acid and 2-5 mg NADP per 100ml of culture supernatant of recombinant bacillus subtilis engineering strain;
preferably, 0.5g of chenodeoxycholic acid and 2mg of NADP are added to 100ml of culture supernatant.
10. The method according to any one of claims 5 to 9, further comprising separating the product after the catalytic reaction; the method for separating the product comprises the steps of regulating the pH value of a system after catalytic reaction to 4, separating a ursodeoxycholic acid crude product, mixing and refluxing the ursodeoxycholic acid crude product and ethyl acetate, and filtering to obtain ursodeoxycholic acid with the purity of more than 85%.
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| CN102994604A (en) * | 2012-11-21 | 2013-03-27 | 上海凯宝药业股份有限公司 | Method for preparing binding-form ursodesoxycholic acid by two-step enzymatic method |
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| CN113227364A (en) * | 2018-10-09 | 2021-08-06 | 伊莱思拓遗传学公司 | Cells and methods for producing ursodeoxycholic acid and its precursors |
| CN115992085A (en) * | 2022-10-27 | 2023-04-21 | 聊城大学 | Method for synthesizing ursodeoxycholic acid by double-strain whole-cell catalysis one-step method |
| US20230287333A1 (en) * | 2022-03-11 | 2023-09-14 | Xi'an Yueda Biotechnology Co., Ltd. | Production method of recombinant escherichia coli and high-purity ursodeoxycholic acid |
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| CN102994604A (en) * | 2012-11-21 | 2013-03-27 | 上海凯宝药业股份有限公司 | Method for preparing binding-form ursodesoxycholic acid by two-step enzymatic method |
| CN113227364A (en) * | 2018-10-09 | 2021-08-06 | 伊莱思拓遗传学公司 | Cells and methods for producing ursodeoxycholic acid and its precursors |
| WO2020108327A1 (en) * | 2018-11-29 | 2020-06-04 | 江苏邦泽生物医药技术股份有限公司 | Method of preparing tauroursodeoxycholic acid by biotransformation and application thereof |
| US20230287333A1 (en) * | 2022-03-11 | 2023-09-14 | Xi'an Yueda Biotechnology Co., Ltd. | Production method of recombinant escherichia coli and high-purity ursodeoxycholic acid |
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