Streptomyces-derived cytochrome P450 enzyme mutant and application thereof in synthesis of ursodesoxycholic acid

Abstract

The invention discloses a streptomyces-derived cytochrome P450 enzyme mutant and an application of the streptomyces-derived cytochrome P450 enzyme mutant in synthesis of ursodesoxycholic acid. The cytochrome P450 enzyme mutant is obtained by carrying out single mutation or multiple mutation on the 181st site, the 192nd site, the 249th site or the 288th site of an amino acid sequence shown in SEQ ID NO.2. The cytochrome P450 enzyme mutant has high activity and regioselectivity, low-cost lithocholic acid is used as a substrate, efficient cytochrome P450 enzyme is used for hydroxylating the 7beta site of the lithocholic acid, ursodesoxycholic acid is synthesized in one step, the industrial production cost is greatly reduced, and the cytochrome P450 enzyme mutant has good application prospects in industrial production of ursodesoxycholic acid.

Description

(I) Technical field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a cytochrome P450 enzyme mutant derived from Streptomyces and application thereof in synthesizing ursodeoxycholic acid. (II) Background technology

[0002] Ursodeoxycholic acid (UDCA) is the only drug approved by the U.S. FDA for the treatment of primary biliary cirrhosis. It has important clinical applications in the treatment of various liver diseases such as gallstones, cholestasis, biliary pancreatitis, and primary cholangitis. In addition, UDCA can also be used for cancer treatment by mediating and affecting the regulation of carcinogenic signaling pathways. Studies have shown that UDCA can specifically regulate the threshold of cell apoptosis, inhibit the growth of cancer cells, and induce cell autophagy and apoptosis. At the same time, UDCA can improve damaged mitochondrial function and play a protective role in neurological diseases. In recent years, the market demand for UDCA has increased year by year, and its market share in the treatment of biliary diseases has reached 82%.

[0003] Due to its broad market prospects, the synthesis method of UDCA has also become a research hotspot. At present, UDCA is mainly synthesized chemically and biologically. Chemical synthesis uses bile acid (CA) or chenodeoxycholic acid (CDCA) from cow bile, chicken, duck and goose bile as substrates, and obtains UDCA after 7 steps of chemical reactions. This route requires the use of toxic reagents such as hydrazine, chromium trioxide and pyridine, and the discharge of three wastes is large. The total yield is only about 30%, which greatly limits industrial production and application. In the biological synthesis route, the preparation of UDCA using expensive CDCA as raw material and enzyme catalysts such as 7α-HSDH and 7β-HSDH is currently a popular route. However, in this method, 7β-HSDH relies on NADPH as a coenzyme, and the cofactor regeneration and reduction systems of the two enzymes will interfere with each other. It can only be divided into two steps, facing the defects of cumbersome steps and high cost. Therefore, the development of an efficient UDCA synthesis method with simple catalytic steps is of great significance for its industrial application.

[0004] Cytochrome P450 (CYP) enzymes have multiple biocatalytic functions such as hydroxylation, dealkylation, deamination, desulfurization, dehalogenation, sulfoxide oxidation, N-oxide reduction, epoxidation and demethylation, and are important tool enzymes for the synthesis of steroid drugs and their drug intermediates. Using cheap lithocholic acid (LCA) as a substrate, UDCA can be synthesized in one step by hydroxylating its 7β position through cytochrome P450 enzymes. Compared with other biosynthetic UDCA routes, it has the advantages of low cost and simple catalytic steps, and has important industrial application potential. However, the cytochrome P450 enzymes that have been found to catalyze LCA have defects such as low catalytic activity and poor selectivity. (III) Summary of the invention

[0005] The present invention aims to provide a cytochrome P450 enzyme mutant derived from Streptomyces antibioticus and its application in the synthesis of ursodeoxycholic acid. The cytochrome P450 enzyme derived from Streptomyces is molecularly modified by semi-rational design to improve its catalytic activity and selectivity, thereby laying a foundation for the industrial enzymatic synthesis of UDCA and solving the problems of low 7β-hydroxylation activity and poor regioselectivity of the existing cytochrome P450 enzyme for lithocholic acid.

[0006] The technical solution adopted by the present invention is:

[0007] The invention provides a cytochrome P450 enzyme mutant derived from Streptomyces, wherein the cytochrome P450 enzyme mutant is obtained by single mutation or multiple mutation of the 181st, 192nd, 249th or 288th position of the amino acid sequence shown in SEQ ID NO.2.

[0008] The amino acid sequence shown in SEQ ID NO.2 of the present invention is a cytochrome P450 enzyme mutant Olep / F84Q / S240S / V291G (hereinafter referred to as Olep-3) derived from Streptomyces antibioticus as the parent sequence, and the nucleotide sequence is shown in SEQ ID NO.1. By performing site-directed mutagenesis on Olep-3, a cytochrome P450 enzyme mutant with improved catalytic properties such as catalytic activity and regional selectivity is obtained.

[0009] Preferably, the cytochrome P450 enzyme mutant is a mutant in which the amino acid sequence shown in SEQ ID NO.2 is mutated into one of the following: (1) the serine at position 181 is mutated into threonine (OleP-3-S181T, the amino acid sequence is shown in SEQ ID NO.4, and the nucleotide sequence is shown in SEQ ID NO.3); (2) the serine at position 181 is mutated into threonine, and the serine at position 249 is changed into threonine (OleP-3-S181T / S249T, the amino acid sequence is shown in SEQ ID NO.6, and the nucleotide sequence is shown in SEQ ID NO.5); (3) the serine at position 181 is mutated into threonine, the serine at position 249 is changed into threonine, and the valine at position 192 is mutated into alanine (OleP-3-S181T / S249T / V192A, the amino acid sequence is shown in SEQ ID NO.8, and the nucleotide sequence is shown in SEQ ID NO.7); (4) the serine at position 181 is mutated to threonine, the serine at position 249 is changed to threonine, the valine at position 192 is mutated to alanine, and the threonine at position 288 is changed to isoleucine (OleP-3-S181T / S249T / V192A / T288I, the amino acid sequence is shown in SEQ ID NO.9, and the nucleotide sequence is shown in SEQ ID NO.10).

[0010] Conservative substitution forms of other amino acid positions of the above-mentioned cytochrome P450 enzyme mutants, forms of adding or deleting one or several amino acids, forms of amino-terminal truncation, forms of carboxyl-terminal truncation, these mutant forms are also included in the scope of the present invention.

[0011] The present invention also provides a gene encoding the cytochrome P450 enzyme mutant, and the nucleotide sequence of the gene encoding is shown in SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7 or SEQ ID NO.9.

[0012] The present invention also relates to a recombinant vector containing the coding gene of the cytochrome P450 enzyme mutant and a recombinant genetic engineering bacterium containing the recombinant vector; the original vector of the recombinant vector is pET28b. The recombinant vector is used to transform a host cell to obtain a recombinant genetic engineering bacterium, and the host cell can be any conventional host cell in the art, and preferably the host cell is Escherichia coli BL21.

[0013] The present invention also provides a method for preparing the cytochrome P450 enzyme mutant, which comprises the following steps:

[0014] (1) Designing site-directed mutagenesis primers, using a recombinant plasmid carrying a gene fragment having a nucleotide sequence as shown in SEQ ID NO. 1 as a template, and performing overlap extension PCR to obtain mutant products of 19 amino acids at positions S181, V192, S249, and T288 in the amino acid sequence of the parental cytochrome P450 enzyme, excluding the parent amino acids;

[0015] (2) using the dominant mutant product carrying the OleP-3-S181T gene obtained in step (1) as a template, performing overlap extension PCR to obtain a mutant product in which the S at position 249 is mutated to T;

[0016] (3) using the mutant product carrying the OleP-3-S181T / S249T gene obtained in step (2) as a template, performing overlap extension PCR to obtain a mutant product in which the V at position 192 is mutated to A;

[0017] (4) using the mutant product carrying the OleP-3-S181T / S249T / V192A gene obtained in step (3) as a template, performing overlap extension PCR to obtain a mutant product in which the T at position 288 is mutated to I;

[0018] (5) The mutant products obtained in steps (1), (2), (3) and (4) are inserted into the T7 promoter of pET-28a(+) and transformed into host bacteria respectively, and the cytochrome P450 enzyme mutant expression strain is screened to obtain the mutants OleP-3-S181T, OleP-3-S181T / S249T, OleP-3-S181T / S249T / V192A and OleP-3-S181T / S249T / V192A / T288I are obtained after inducing expression.

[0019] The present invention also provides the use of the cytochrome P450 enzyme mutant in catalyzing lithocholic acid (LCA) to prepare ursodeoxycholic acid (UDCA). The method of the use is: using wet bacteria obtained by fermentation and centrifugation of engineered bacteria co-expressing cytochrome P450 enzyme mutant encoding genes and redox chaperone genes as catalysts, using lithocholic acid (LCA) as substrate, adding glucose, NaCl, dimethyl sulfoxide (DMSO) and glycerol, and using a buffer solution with a pH of 5 to 10 as a reaction medium to form a reaction system, after the water bath reaction is completed at 20 to 55° C. and 100 to 400 rpm (preferably 28° C. and 120 rpm), extracting the reaction solution with ethyl acetate, taking the upper organic phase, and volatilizing the ethyl acetate with a vacuum concentrator to obtain the product ursodeoxycholic acid (UDCA); the redox chaperone gene is a combination of genes encoding ferredoxin reductase Pdr (putidaredoxin reductase) and ferredoxin Pdx (putidaredoxin) from Pseudomonas putida.

[0020] Preferably, the amino acid sequence of the ferredoxin reductase Pdr is shown in SEQ ID NO:11, and the nucleotide sequence is shown in SEQ ID NO:12; the amino acid sequence of the ferredoxin Pdx is shown in SEQ ID NO:13, and the nucleotide sequence is shown in SEQ ID NO:14.

[0021] Preferably, the engineered bacteria that co-express the cytochrome P450 enzyme mutant encoding gene and the redox chaperone gene are constructed according to the following steps: the cytochrome P450 enzyme mutant is inserted into the T7 promoter of pET-28a(+), and is co-transformed into the host bacteria together with the recombinant plasmid pACYCDuet-Pdr-Pdx expressing ferredoxin reductase Pdr and ferredoxin Pdx to obtain a co-expression engineered bacteria.

[0022] The engineered bacteria co-expressing the cytochrome P450 enzyme mutant and the redox partner gene of the present invention can be used in the form of engineered bacteria whole cells, or in the form of unpurified crude enzymes, or in the form of partially purified or completely purified enzymes. The cytochrome P450 enzyme mutant and the redox partner of the present invention can also be made into immobilized enzymes or engineered bacteria into biocatalysts in the form of immobilized cells using immobilization techniques known in the art.

[0023] Preferably, the substrate is added to the reaction system at a concentration of 1-5 g / L (preferably 4 g / L), the catalyst is used in an amount of 50-150 g / L (preferably 80 g / L) based on the weight of the wet cells, NaCl is added at a concentration of 10-30 mM (preferably 20 mM), glucose is added at a concentration of 50-150 g / L (preferably 100 g / L), glycerol is added at a concentration of 1-5 g / L (preferably 4 g / L), and dimethyl sulfoxide is added at a concentration of 1-10% (preferably 4-8%) by volume.

[0024] Preferably, the reaction medium is a 200 mM KH2PO4 / K2HPO4 buffer solution at pH 7.4.

[0025] The culture medium used for the engineered bacteria of the present invention can be a culture medium in the art that can grow the engineered bacteria and produce the cytochrome P450 enzyme of the present invention, preferably TB culture medium: 12g / L peptone, 5g / L glycerol, 24g / L yeast extract, 17mM potassium dihydrogen phosphate, 72mM potassium dihydrogen phosphate, the solvent is water, and sterilized at 121°C for 15min. There are no special requirements for the culture method and culture conditions, as long as the engineered bacteria can grow and produce cytochrome P450 enzymes.

[0026] Preferably, the wet cells are prepared as follows:

[0027] The engineered bacteria co-expressing the cytochrome P450 enzyme mutant encoding gene and the redox chaperone gene are inoculated into a TB culture medium containing 50 μg / mL kanamycin and 25 μg / mL chloramphenicol, and cultured at 37°C for 6-8 hours to obtain a seed solution; the seed solution is then inoculated into a fresh TB liquid culture medium containing a final concentration of 50 μg / mL kanamycin and 25 μg / mL chloramphenicol at a volume concentration of 2% (v / v), and cultured at 37°C and 180-200rpm until the bacterial concentration OD600 reaches 0.3-0.45, and then 0.1-0.5mM isopropyl-β-D-thiogalactopyranoside (IPTG) is added to the culture solution, and the induction culture is carried out at 28°C and 180-200rpm for 10-12 hours, and the wet bacteria are collected by centrifugation at 4°C and 8000-12000rpm for 10 minutes.

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

[0029] The present invention improves the activity and regional selectivity of cytochrome P450 enzymes through semi-rational design, and constructs cytochrome P450 enzyme mutants. Compared with the parent cytochrome P450 enzyme Olep-3, the activity of the mutant OleP-3-S181T is increased by 2.2 times, the activity of the mutant OleP-3-S181T / S249T is increased by 3.12 times, the activity of the mutant OleP-3-S181T / S249T / V192A is increased by 4.83 times, the content of the byproduct murideoxycholic acid (MDCA) is reduced by 61.44% compared with the wild type, the activity of the mutant OleP-3-S181T / S249T / V192A / T288I is increased by 4.85 times, and the content of the byproduct MDCA is reduced by 62.71% compared with the wild type.

[0030] The cytochrome P450 enzyme mutant of the invention has high activity and regional selectivity, takes cheap lithocholic acid as a substrate, utilizes efficient cytochrome P450 enzyme to hydroxylate its 7β position, synthesizes ursodeoxycholic acid in one step, greatly reduces the industrial production cost, and has good application prospect in the industrial production of ursodeoxycholic acid. (IV) Description of the drawings

[0031] Figure 1 Schematic diagram of the construction of genetically engineered bacteria for co-expression of P450 enzyme mutants.

[0032] Figure 2 This is the gel electrophoresis diagram of the genetically engineered bacteria co-expressing P450 enzyme mutants and the wet bacteria with reduced chaperone proteins.

[0033] Figure 3 This is a reaction process diagram for the production of UDCA catalyzed by genetically engineered bacteria co-expressing P450 enzyme mutants.

[0034] Figure 4 Schematic diagram of the reaction for producing ursodeoxycholic acid by co-expressing recombinant Escherichia coli containing cytochrome P450 enzymes. (V) Specific implementation methods

[0035] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0036] The molecular biology experiments in the embodiments, including plasmid construction, enzyme digestion, ligation, competent cell preparation, transformation, culture medium preparation, etc., are mainly carried out with reference to "Molecular Cloning Experiment Guide" (3rd edition, edited by J. Sambrook and DW Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002).

[0037] TB medium: 12 g / L peptone, 5 g / L glycerol, 24 g / L yeast extract, 17 mM potassium dihydrogen phosphate, 72 mM potassium hydrogen phosphate, solvent is water, sterilized at 121°C for 15 min.

[0038] Example 1: Construction of parent cytochrome P450 enzyme genetically engineered bacteria

[0039] The cytochrome P450 enzyme mutant Olep / F84Q / S240S / V291G (hereinafter referred to as Olep-3, the amino acid sequence is shown in SEQ ID NO.2) from Streptomyces antibioticus was selected for codon optimization. The optimized gene sequence is shown in SEQ ID NO:1, which was synthesized by Beijing Qingke Biotechnology Co., Ltd. (Beijing, China) and inserted into the T7 promoter of pET-28a(+) to obtain the recombinant expression vector pET28a-OleP-3. The plasmid map is shown in Figure 1 The recombinant expression vector pET28a-OleP-3 was heat-shock transformed into the host Escherichia coli BL21 (DE3) to obtain a recombinant Escherichia coli expressing the parent cytochrome P450 enzyme.

[0040] BL21(DE3)-pET28a-OleP-3 (denoted as OleP-3).

[0041]

[0042]

[0043] Example 2: Construction of a cytochrome P450 enzyme co-expression strain

[0044] Since the CYP107 family is a typical three-component P450 enzyme, a reducing chaperone is required to transfer electrons from the electron donor NAD(P)H to the P450 enzyme active center. Therefore, the ferredoxin reductase Pdr (amino acid sequence as shown in SEQ ID NO.12) and ferredoxin Pdx (amino acid sequence as shown in SEQ ID NO.14) from Pseudomonas putida were selected as reducing chaperones. The ferredoxin reductase gene Pdr and ferredoxin gene Pdx gene (nucleotide sequence as shown in SEQ ID NO.11 and SEQ ID NO.13) after codon optimization were synthesized, and the Pdr and Pdx genes were subcloned into the plasmid pACYCDuet to obtain the recombinant plasmid pACYCDuet-Pdr-Pdx. The above recombinant plasmids pET28a-oleP-3 and pACYCDuet-Pdr-Pdx were transformed into the Escherichia coli expression host BL21 (DE3) to obtain a recombinant strain E. coli ET28a-oleP-3 / pACYCDuet-Pdr-Pdx (denoted as oleP-Pdr / Pdx). Figure 1 At the same time, the recombinant strain E. coli pACYCDuet-Pdr-Pdx (denoted as Pdr / Pdx) was constructed.

[0045] Example 3: Inducible expression of cytochrome P450 enzymes

[0046] The glycerol tubes of the engineered bacteria oleP-Pdr / Pdx and Pdr-Pdx prepared by the method of Example 2 were inoculated into 10 mL of liquid TB medium (containing 50 μg / mL kanamycin and 25 μg / mL chloramphenicol), cultured overnight at 37°C, 200 rpm, and transferred to 100 mL of fresh TB medium (containing 50 μg / mL kanamycin and 25 μg / mL chloramphenicol) at a volume concentration of 2%, and continued to be cultured until OD 600 The final concentration of IPTG was 0.4, and IPTG was added at a final concentration of 0.5 mM. The cells were induced and cultured at 28°C and 180 rpm for 12 h. After the culture was completed, the cells were collected by centrifugation at 8000 rpm for 10 min at 4°C, and washed twice with 0.9% saline to obtain wet bacterial cells. The gel electrophoresis diagram is shown in the figure below. Figure 2 shown.

[0047] Example 4: Construction of OleP-3 mutant library

[0048] Homology modeling of OleP-3 was performed using AlphaFold2 (AlphaFold2TIB Server (biodesign.ac.cn)), and further computational aids such as molecular docking and kinetic simulation were used to screen the key amino acids Ser181, Val192, Ser249 and Thr288 in the catalytic pocket that affect the stereoselective recognition of substrates based on steric hindrance and interaction forces. In order to saturate the Ser (S) at position 181, the Val (V) at position 192, the Ser (S) at position 249 and the Thr (T) at position 288 in the parental amino acid sequence, the corresponding primers were designed, as shown in Table 1.

[0049] Table 1: Primer design table

[0050]

[0051] Note: N=A / G / C / T, K=G / T, M=A / C.

[0052] The recombinant plasmid pET28a-OleP-3 containing the target gene fragment was used as a template, and the whole plasmid of the template was amplified according to the overlap extension PCR method.

[0053] The PCR amplification system was (50 μL): template DNA 0.1 ng-1 ng, 2×Phanta Max Buffer 25 μL, dNTPs (10 mM each) 1 μL, upstream and downstream mutation primers 1 μL each, Phanta Max Super-Fidelity DNA Polymerase 1 U, and the rest ddH2O was added to the total volume.

[0054] PCR reaction parameters: (1) pre-denaturation at 95°C for 30 s; (2) denaturation at 95°C for 15 s; (3) annealing at 63°C for 4 min 30 s; (4) extension at 72°C for 6 min, and steps (2) to (4) were cycled 30 times; (5) complete extension at 72°C for 5 min, and stored at 4°C.

[0055] After the PCR product was positive after 0.9% agarose gel electrophoresis analysis, 20 μL of the PCR reaction solution was taken, and 1 μL of endonuclease Dpn I was added to remove the template plasmid DNA at 37°C for 3h, and inactivated at 65°C for 10min. Heat shock transformation was performed into E. coli BL21 (DE3) competent cells, and after recovery, it was spread on LB plates containing 50 mg / L kanamycin and cultured at 37°C overnight. A single colony was picked and cultured in LB liquid medium containing kanamycin resistance (final concentration of 50 mg / L), and the plasmid was extracted for sequencing.

[0056] Finally, engineered strains of OleP-3 mutants at sites 181, 192, 249 and 288 were obtained.

[0057] Example 5: Screening of optimal single-point mutants

[0058] All mutant plasmids obtained in Example 4 were transformed into the expression host Escherichia coli BL21 (DE3) together with the pACYCDuet-Pdr-Pdx plasmid according to the method of Example 2 to obtain a co-expression recombinant strain.

[0059] The co-expression recombinant strain was inoculated into TB medium containing kanamycin (final concentration of 50 mg / L) and chloramphenicol (final concentration of 25 mg / L), cultured at 37°C, 180 rpm for 6-8 h, and then transferred to fresh TB liquid medium containing kanamycin (final concentration of 50 mg / L) and chloramphenicol (final concentration of 25 mg / L) at a 2% (v / v) inoculum, and cultured at 37°C, 180 rpm until the bacterial OD 600 When the pH value was 0.3-0.45, IPTG (final concentration was 0.5 mM) was added to the above TB liquid culture medium, and the culture was induced at 28°C and 180 rpm for 10-12 h. The bacterial cells were collected by centrifugation at 4°C and 8000 rpm for 10 min.

[0060] The final concentration of the enzyme activity detection reaction system (10mL) is as follows: 20mM NaCl, 100g / L glucose, 4g / L glycerol, 0.8g wet bacteria, 1g / L substrate lithocholic acid (LCA), 2% DMSO (dimethyl sulfoxide) by volume, pH7.4, 200mM KH2PO4 / K2HPO4 buffer solution constitutes a 10mL reaction system. After the reaction solution is preheated at 28°C for 2min, it is placed on a water bath shaker at 28°C and 180rpm for 1h. The reaction solution is extracted twice with an equal volume of ethyl acetate, the upper organic phase is taken, and the ethyl acetate is volatilized with a vacuum concentrator. The precipitated solid is re-dissolved with 400μL of methanol, and then the substrate and product are detected by high performance liquid chromatography-differential refractive index detector, and the enzyme activity and the relative reduction of the byproduct MDCA are calculated. The results are shown in Table 2.

[0061] Definition of enzyme activity unit: Under the above conditions, the amount of enzyme required to convert 1 μmol of substrate in 1 minute is 1 enzyme activity unit (U).

[0062] Specific activity: the enzyme activity per milligram of enzyme protein, unit U / mg.

[0063] Table 2. Enzyme activities of dominant single-site mutants

[0064]

[0065] The HPLC model is UlitMate 3000 (Thermo Fisher Scientific), and the chromatographic column model is Omega PSC18. Chromatographic conditions were as follows: mobile phase was acetonitrile / water = 1 / 1 (v / v) + 0.1% trifluoroacetic acid, flow rate was 1 mL / min, injection volume was 30.0 μL, injection port and detector temperature were both 40°C, column temperature was 40°C, and retention time was 20 min.

[0066] The results showed that the engineered bacteria E. coli BL21 (DE3) / pET28b-OleP-3-S181T / pACYCDuet-Pdr-Pdx (denoted as OleP-3-S181T) had the highest hydrolysis activity (8.80 U / g), which was 2.2 times that of the wild type.

[0067] Example 6, iterative mutation of OleP-3

[0068] 1. Double mutation

[0069] Based on the mutant strain S181T with the most improved activity obtained in Example 5, the optimal single-point mutations of the three sites S249, V192, and T288 were further superimposed in the form of permutations and combinations: S249T, V192A, and T288I. Using the pET28b-OleP-3-S181T plasmid as a mutation template, the mutation primers of S249T (Table 3) were designed for full plasmid PCR amplification.

[0070] Table 3. Primer design table for mutations at S249T, V192A, and T288I sites

[0071]

[0072] PCR conditions: (1) pre-denaturation at 95°C for 5 min; (2) denaturation at 95°C for 15 s, annealing at 60°C for 5 s, extension at 72°C for 3.5 min, step (2) for 35 cycles; (3) final extension at 72°C for 5 min, and storage at 4°C.

[0073] The amplified PCR product was digested with endonuclease Dpn I at 37°C for 3h and inactivated at 65°C for 10min. According to the method in Implementation 2, it was co-transformed into the expression host Escherichia coli BL21 (DE3) with the pACYCDuet-Pdr-Pdx plasmid to obtain a co-expression recombinant strain. It was spread on an LB plate containing kanamycin (50μg / mL) and chloramphenicol (25μg / mL) resistance and cultured overnight at 37°C. The most active engineering bacteria E.coli BL21 (DE3) / pET28b-OleP-3-S181T / S249T / pACYCDuet-Pdr-Pdx (denoted as OleP-3-S181T / S249T) were screened using the method in Example 5, and the enzyme activity reached 12.46U / g, as shown in Table 4.

[0074] 2. Triple mutation

[0075] Using pET28b-OleP-3-S181T / S249T plasmid as mutation template, V192A mutation primers (Table 3) were designed for whole plasmid PCR amplification.

[0076] PCR conditions: (1) pre-denaturation at 95°C for 5 min; (2) denaturation at 95°C for 15 s, annealing at 60°C for 5 s, extension at 72°C for 3.5 min, step (2) for 35 cycles; (3) final extension at 72°C for 5 min, and storage at 4°C.

[0077] The PCR product obtained by amplification was digested with endonuclease Dpn I at 37°C for 3h and inactivated at 65°C for 10min. According to the method in Implementation 2, it was co-transformed into the Escherichia coli expression host BL21 (DE3) with the pACYCDuet-Pdr-Pdx plasmid to obtain a co-expression recombinant strain. It was coated on an LB plate containing kanamycin (50μg / mL) and chloramphenicol (25μg / mL) resistance and cultured overnight at 37°C. The engineering bacteria E.coli BL21 (DE3) / pET28b-OleP-3-S181T / S249T / V192A / pACYCDuet-Pdr-Pdx (denoted as OleP-3-S181T / S249T / V192A) with the highest activity was screened using the method in Example 5. The enzyme activity reached 19.31U / g, and the byproduct MDCA content was reduced by 61.44%, as shown in Table 4.

[0078] 3. Four mutations

[0079] Using pET28b-OleP-3-S181T / S249T / V192A plasmid as a mutation template, T288I mutation primers (Table 3) were designed for full plasmid PCR amplification.

[0080] PCR conditions: (1) pre-denaturation at 95°C for 5 min; (2) denaturation at 95°C for 15 s, annealing at 60°C for 5 s, extension at 72°C for 3.5 min, step (2) for 35 cycles; (3) final extension at 72°C for 5 min, and storage at 4°C.

[0081] The amplified PCR products were digested with endonuclease Dpn I at 37°C for 3h, inactivated at 65°C for 10min, and transformed into the expression host Escherichia coli BL21 (DE3) with the pACYCDuet-Pdr-Pdx plasmid according to the method in Example 2 to obtain a co-expression recombinant strain. The strains were spread on LB plates containing kanamycin (50 μg / mL) and chloramphenicol (25 μg / mL) resistance and cultured at 37°C overnight. The method of Example 5 was used to screen the engineered bacteria E. coli BL21 (DE3) / pET28b-OleP-3-S181T / S249T / V192A / T288I / pACYCDuet-Pdr-Pdx (denoted as OleP-3-S181T / S249T / V192A / T288I) with the highest activity. The enzyme activity reached 19.36 U / g, which was 4.85 times higher than that of the wild type. In addition, the content of the byproduct MDCA was reduced by 62.71% relative to the wild type, as shown in Table 4.

[0082] Table 4. Comparison of the activity of various dominant mutants of OleP-3

[0083]

[0084] Example 7: Application of co-expressed recombinant Escherichia coli containing cytochrome P450 enzymes in the preparation of ursodeoxycholic acid

[0085] The wet bacterial cells of E.coliBL21(DE3)-OleP-3 / pACYCDuet-Pdr-Pdx obtained in Example 2, E.coliBL21(DE3)-OleP-3-S181T / pACYCDuet-Pdr-Pdx obtained by the method of Example 5, E.coliBL21(DE3)-OleP-3-S181T / S249T / pACYCDuet-Pdr-Pdx, E.coliBL21(DE3)-OleP-3-S181T / S249T / V192A / pACYCDuet-Pdr-Pdx, and E.coliBL21(DE3)-OleP-3-S181T / S249T / V192A / T288I / pACYCDuet-Pdr-Pdx were used as catalysts.

[0086] The transformation system was added with the final concentration of: 20 mM NaCl, 100 g / L glucose, 4 g / L glycerol, 0.8 g wet bacteria, 4 g / L lithocholic acid (LCA) substrate, 8% DMSO, pH 7.4, 200 mM KH2PO4 / K2HPO4 buffer to form a 10 mL reaction system, and reacted at 28° C. and 120 rpm. Samples were taken every 2-4 hours, and the reaction progress was detected using the high performance liquid chromatography-differential refractive index detector described in Example 5.

[0087] Results Figure 3 After 24 hours of reaction, the concentration of OleP-3 product was 0.3 g / L, the concentration of the optimal single mutant OleP-3-S181T product was 0.37 g / L, which was 1.2 times that of the wild type, the concentration of the optimal double mutant OleP-3-S181T / S249T product was 0.51 g / L, which was 1.7 times that of the wild type, the concentration of the optimal triple mutant OleP-3-S181T / S249T / V192A product was 1.28 g / L, which was 4.26 times that of the wild type, and the concentration of the optimal quadruple mutant OleP-3-S181T / S249T / V192A / T288I product was 1.49 g / L, which was 4.97 times that of the wild type.

[0088] The present invention is not limited by the above specific textual description. The present invention can make various changes within the scope summarized by the claims, and these changes are all within the scope of the present invention.

Claims

1. A cytochrome P450 enzyme mutant derived from Streptomyces, characterized in that: The cytochrome P450 enzyme mutant is obtained by performing single mutation or multiple mutation on the 181st, 192nd, 249th or 288th position of the amino acid sequence shown in SEQ ID NO.

2.

2. The cytochrome P450 enzyme mutant according to claim 1, characterized in that The cytochrome P450 enzyme mutant is a cytochrome P450 enzyme mutant in which the amino acid sequence shown in SEQ ID NO.2 is mutated into one of the following: (1) the serine at position 181 is mutated into threonine; (2) the serine at position 181 is mutated into threonine, and the serine at position 249 is changed into threonine; (3) the serine at position 181 is mutated into threonine, the serine at position 249 is changed into threonine, and the valine at position 192 is mutated into alanine; (4) the serine at position 181 is mutated into threonine, the serine at position 249 is changed into threonine, the valine at position 192 is mutated into alanine, and the threonine at position 288 is changed into isoleucine.

3. A recombinant genetically engineered bacterium comprising a gene encoding the cytochrome P450 enzyme mutant according to claim 1.

4. Use of the cytochrome P450 enzyme mutant according to claim 1 in catalyzing the preparation of ursodeoxycholic acid from lithocholic acid.

5. The use according to claim 4, characterized in that The application method comprises the following steps: using wet bacteria obtained by fermentation and centrifugation of engineered bacteria co-expressing a cytochrome P450 enzyme mutant encoding gene and a redox chaperone gene as a catalyst, using lithocholic acid as a substrate, adding glucose, NaCl, dimethyl sulfoxide and glycerol, and using a buffer solution with a pH of 5 to 10 as a reaction medium to form a reaction system; after a water bath reaction is completed at 20 to 55° C. and 100 to 400 rpm, extracting the reaction solution with ethyl acetate, taking an upper organic phase, and volatilizing the ethyl acetate with a vacuum concentrator to obtain a product, ursodeoxycholic acid; the redox chaperone gene is a combination of genes encoding a ferredoxin reductase Pdr (putidaredoxinreductase) and a ferredoxin Pdx (putidaredoxin) from Pseudomonas putida.

6. The use according to claim 5, characterized in that The nucleotide sequence of the ferredoxin reductase Pdr is shown in SEQ ID NO:12; the nucleotide sequence of the ferredoxin Pdx is shown in SEQ ID NO:

14.

7. The use according to claim 5, characterized in that The engineering bacteria for co-expressing the cytochrome P450 enzyme mutant encoding gene and the redox chaperone gene are constructed according to the following steps: the cytochrome P450 enzyme mutant is inserted into the T7 promoter of pET-28a(+), and is co-transfected into the host bacteria together with the recombinant plasmid pACYCDuet-Pdr-Pdx expressing ferredoxin reductase Pdr and ferredoxin Pdx to obtain the co-expressing engineering bacteria.

8. The use according to claim 5, characterized in that The substrate is added to the reaction system at a concentration of 1-5 g / L, the catalyst is used in an amount of 50-150 g / L based on the weight of wet cells, NaCl is added at a concentration of 10-30 mM, glucose is added at a concentration of 50-150 g / L, glycerol is added at a concentration of 1-5 g / L, and dimethyl sulfoxide is added at a concentration of 1-10% by volume.

9. The use according to claim 5, characterized in that The reaction medium is a 200 mM potassium phosphate buffer with a pH value of 7.

4.

10. The use according to claim 5, characterized in that The wet cells are prepared as follows: The engineered bacteria co-expressing the cytochrome P450 enzyme mutant encoding gene and the redox chaperone gene are inoculated into a TB culture medium containing 50 μg / mL kanamycin and 25 μg / mL chloramphenicol, and cultured at 37°C for 6-8 hours to obtain a seed solution; the seed solution is then inoculated into a fresh TB liquid culture medium containing a final concentration of 50 μg / mL kanamycin and 25 μg / mL chloramphenicol at a volume concentration of 2%, and cultured at 37°C and 180-200rpm until the bacterial concentration OD600 reaches 0.3-0.45, and then 0.1-0.5mM isopropyl-β-D-thiogalactopyranoside is added to the culture solution, and the induction culture is carried out at 28°C and 180-200rpm for 10-12 hours, and the wet bacteria are collected by centrifugation at 4°C and 8000-12000rpm for 10 minutes.

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