A cytochrome p450 enzyme mutant derived from streptomyces and its application in synthesis of ursodeoxycholic acid

Abstract

The application discloses a cytochrome P450 enzyme mutant derived from Streptomyces and application of the cytochrome P450 enzyme mutant in synthesis of ursodeoxycholic acid, wherein the cytochrome P450 enzyme mutant is obtained by single mutation or multiple mutations of positions 181, 192, 249 or 288 of the amino acid sequence shown in SEQ ID NO. 2. The cytochrome P450 enzyme mutant has higher activity and regional selectivity, and can use cheap lithocholic acid as a substrate, and hydroxylate 7beta of the substrate by using the high-efficiency cytochrome P450 enzyme to synthesize ursodeoxycholic acid in one step, thereby greatly reducing the industrial production cost, and having a good application prospect in industrial production of ursodeoxycholic acid.

Description

(I) TECHNICAL FIELD

[0001] The present application belongs to the field of bioengineering technology, and particularly relates to a cytochrome P450 enzyme mutant derived from Streptomyces and application thereof in synthesis of ursodeoxycholic acid. (II) BACKGROUND

[0002] Ursodeoxycholic acid (UDCA) is the only drug approved by the FDA in the United States for the treatment of primary biliary cirrhosis, and has important clinical applications in the treatment of gallstones, cholestasis, biliary pancreatitis, primary cholangitis and other liver diseases. In addition, UDCA can also be used for cancer treatment by mediating and influencing 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 autophagy and apoptosis. At the same time, UDCA can improve damaged mitochondrial function and play a protective role in nervous system diseases. In recent years, the market demand for UDCA has been increasing year by year, and its market share in the treatment of gallbladder diseases is as high as 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 by chemical and biological methods. Chemical synthesis takes the cholic acid (CA) or chenodeoxycholic acid (CDCA) derived from bovine gall, chicken, duck and goose gall as the substrate, and UDCA is obtained after 7-step chemical reactions. In this route, toxic reagents such as hydrazine, chromium trioxide and pyridine are used, the amount of three wastes is large, and the total yield is only about 30%, which greatly limits the industrial production and application. In the biological synthesis route, UDCA is prepared by using 7α-HSDH and 7β-HSDH enzymes as catalysts with expensive CDCA as raw material, which is the current popular route. However, in this method, 7β-HSDH depends on NADPH as coenzyme, and the coenzyme regeneration reduction system of the two enzymes will interfere with each other, which can only be carried out in two steps, and has the defects of complicated steps and high cost. Therefore, it is of great significance to develop an efficient and simple catalytic step synthesis method of UDCA for its industrial application.

[0004] Cytochrome P450 (CYP) has multiple biological catalytic functions such as hydroxylation, dealkylation, deamination, desulfurization, dehalogenation, sulfoxide oxidation, N-oxide reduction, epoxidation and demethylation, and is an important tool enzyme for the synthesis of steroid drugs and their drug intermediates. By hydroxylating 7β position of lithocholic acid (LCA) through cytochrome P450 enzyme, UDCA can be synthesized in one step, which has the advantages of low cost and simple catalytic step compared with other biological synthesis routes of UDCA, and has important industrial application potential. However, the currently discovered cytochrome P450 enzymes have the defects of low catalytic activity and poor selectivity in catalyzing LCA. (III) SUMMARY

[0005] The application aims to provide a cytochrome P450 enzyme mutant derived from Streptomyces antibioticus and application thereof in synthesis of ursodeoxycholic acid, to improve catalytic activity and selectivity of cytochrome P450 enzyme derived from Streptomyces antibioticus by molecular modification of the cytochrome P450 enzyme, and to lay a foundation for industrial enzymatic synthesis of UDCA, and to solve the problems of low hydroxylation activity at 7beta position of lithocholic acid and poor regioselectivity of existing cytochrome P450 enzyme.

[0006] The technical scheme adopted by the application is:

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

[0008] The amino acid sequence shown in SEQ ID NO. 2 is a cytochrome P450 enzyme mutant Olep / F84Q / S240A / V291G (hereinafter referred to as Olep-3) derived from Streptomyces antibioticus, and the nucleotide sequence is shown in SEQ ID NO. 1, and the cytochrome P450 enzyme mutant with improved catalytic activity and regioselectivity is obtained by site-directed mutagenesis of Olep-3.

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

[0010] Other conservative substitution forms, forms with one or more amino acids added or deleted, forms with amino-terminal truncation, and forms with carboxyl-terminal truncation of the above-mentioned cytochrome P450 enzyme mutants are also included in the scope of the present application.

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

[0012] The present application also relates to a recombinant vector containing the coding gene of the cytochrome P450 enzyme mutant, and a recombinant genetically engineered 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 genetically engineered bacterium, and the host cell can be various conventional host cells in the art, and preferably the host cell is E. coli BL21.

[0013] The present application also provides a preparation method of the cytochrome P450 enzyme mutant, and the preparation method comprises the following steps:

[0014] (1) Design site-directed mutation primers, take the recombinant plasmid carrying the gene fragment with nucleotide sequence as shown in SEQ ID NO. 1 as a template, perform overlap extension PCR to obtain 19 amino acids of each mutation product of positions S181, V192, S249 and T288 of the parent cytochrome P450 enzyme amino acid sequence except the parent amino acid;

[0015] (2) Take the dominant mutation product carrying the OleP-3-S181T gene obtained in step (1) as a template, perform overlap extension PCR to obtain a mutation product with S mutated to T at position 249;

[0016] (3) Take the mutation product carrying the OleP-3-S181T / S249T gene obtained in step (2) as a template, perform overlap extension PCR to obtain a mutation product with V mutated to A at position 192;

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

[0018] (5) Insert the mutation products obtained in steps (1), (2), (3) and (4) into the T7 promoter of pET-28a(+) respectively, transform into host bacteria, screen to obtain cytochrome P450 enzyme mutant expression strains, induce expression to obtain mutants OleP-3-S181T, OleP-3-S181T / S249T, OleP-3-S181T / S249T / V192A and OleP-3-S181T / S249T / V192A / T288I.

[0019] The application also provides the application of the cytochrome P450 enzyme mutant in catalyzing lithocholic acid (LCA) to prepare ursodeoxycholic acid (UDCA), and the method of the application is as follows: taking the wet bacteria obtained by fermenting and centrifuging the engineering bacteria co-expressing the cytochrome P450 enzyme mutant encoding gene and the oxidoreductase gene as a catalyst, taking lithocholic acid (LCA) as a substrate, adding glucose, NaCl, dimethyl sulfoxide (DMSO) and glycerol, taking a buffer with a pH of 5-10 as a reaction medium to form a reaction system, and then performing water bath reaction under the conditions of 20-55 DEG C and 100-400 rpm (preferably 28 DEG C and 120 rpm), and then extracting the reaction solution with ethyl acetate, taking the upper organic phase, and then performing volatilization treatment on the ethyl acetate with a vacuum concentration instrument to obtain the product ursodeoxycholic acid (UDCA); the oxidoreductase gene is a combination of the genes encoding putidaredoxin reductase Pdr and putidaredoxin Pdx from Pseudomonas putida.

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

[0021] Preferably, the engineering bacteria co-expressing the cytochrome P450 enzyme mutant encoding gene and the oxidoreductase gene are constructed according to the following steps: inserting the cytochrome P450 enzyme mutant into the T7 promoter of pET-28a (+), and then co-transferring the recombinant plasmid pACYCDuet-Pdr-Pdx expressing putidaredoxin reductase Pdr and putidaredoxin Pdx into the host bacteria to obtain the co-expression engineering bacteria.

[0022] The engineering bacteria co-expressing the cytochrome P450 enzyme mutant and the oxidoreductase gene can be used in the form of whole cells of the engineering bacteria, or in the form of crude enzymes without purification, or in the form of partially purified or completely purified enzymes. The cytochrome P450 enzyme mutant and the oxidoreductase of the application can also be made into immobilized enzymes or immobilized cells in the form of a biological catalyst by using immobilization techniques known in the art.

[0023] Preferably, the substrate is added at a concentration of 1-5 g / L (preferably 4 g / L), the catalyst is used at a concentration of 50-150 g / L (preferably 80 g / L) based on the weight of the wet bacteria, 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%).

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

[0025] The culture medium used for the engineered bacteria of the present application can be any culture medium known in the art that allows the engineered bacteria to grow and produce the cytochrome P450 enzyme of the present application, preferably TB medium: 12 g / L peptone, 5 g / L glycerol, 24 g / L yeast extract, 17 mM potassium dihydrogen phosphate, 72 mM dipotassium hydrogen phosphate, solvent water, sterilized at 121°C for 15 min. The culture method and culture conditions are not particularly limited, as long as the engineered bacteria can grow and produce the cytochrome P450 enzyme.

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

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

[0028] Compared with the prior art, the present application has the following advantages:

[0029] The present invention constructs cytochrome P450 enzyme mutants by improving cytochrome P450 enzyme activity and regioselectivity through semi-rational design. Compared with the parent cytochrome P450 enzyme Olep-3, the enzyme 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, and 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 present invention has high activity and regioselectivity, uses cheap lithocholic acid as a substrate, utilizes efficient cytochrome P450 enzyme to hydroxylate its 7β position, and synthesizes ursodeoxycholic acid in one step, greatly reducing the cost of industrial production, and has good application prospects in the industrial production of ursodeoxycholic acid. (IV) Description of the accompanying 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 with reference to specific embodiments, but the protection scope of the present invention is not limited thereto:

[0036] The molecular biology experiments in the examples, including plasmid construction, enzyme digestion, ligation, competent cell preparation, transformation, culture medium preparation, etc., were mainly carried out with reference to Molecular Cloning Laboratory Manual (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 phosphate dibasic, 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 / S240A / V291G (hereinafter referred to as Olep-3, the amino acid sequence is shown as SEQ ID NO. 2) derived from Streptomyces antibioticus was codon-optimized, the optimized gene sequence is shown as SEQ ID NO: 1, synthesized by Beijing Genki Biological Company (Beijing, China), and inserted into the T7 promoter of pET-28a(+), to obtain the recombinant expression vector pET28a-OleP-3, and the plasmid map is shown as Figure 1 The recombinant expression vector pET28a-OleP-3 was transformed into the host Escherichia coli BL21 (DE3) by heat shock, to obtain the recombinant Escherichia coli BL21 (DE3) expressing the parent cytochrome P450 enzyme

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

[0041] SEQ ID NO. 2

[0042]

[0043]

[0044] Example 2, Construction of cytochrome P450 enzyme co-expression strain

[0045] Since CYP107 family is a typical three-component P450 enzyme, which needs a reductase to transfer electron from electron donor NAD(P)H to the active center of P450 enzyme, 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 reductase. Codon-optimized ferredoxin reductase gene Pdr and ferredoxin gene Pdx (nucleotide sequences as shown in SEQ ID NO. 11 and SEQ ID NO. 13) were synthesized and subcloned into plasmid pACYCDuet to obtain recombinant plasmid pACYCDuet-Pdr-Pdx. The above recombinant plasmids pET28a-oleP-3 and pACYCDuet-Pdr-Pdx were transformed into E. coli expression host BL21 (DE3) to obtain a recombinant strain E. coli pET28a-oleP-3 / pACYCDuet-Pdr-Pdx (denoted as oleP-Pdr / Pdx), as shown in Figure 1 Meanwhile, a recombinant strain E. coli pACYCDuet-Pdr-Pdx (denoted as Pdr / Pdx) was constructed.

[0046] Example 3, induced expression of cytochrome P450 enzyme

[0047] The glycerol tube of the engineered bacteria oleP-Pdr / Pdx and Pdr-Pdx prepared by the method of Example 2 was inoculated into 10 mL of liquid TB medium (containing 50 μg / mL kanamycin and 25 μg / mL chloramphenicol), and cultured at 37°C, 200 rpm overnight. The culture was inoculated into 100 mL of fresh TB medium (containing 50 μg / mL kanamycin and 25 μg / mL chloramphenicol) at a volume concentration of 2%, and the culture was continued until the OD 600 was 0.4. IPTG was added at a final concentration of 0.5 mM, and the culture was induced at 28°C, 180 rpm for 12 h. After the culture was completed, the bacteria were collected by centrifugation at 8000 rpm for 10 min at 4°C, and washed twice with 0.9% physiological saline to obtain wet bacterial cells. The gel electrophoresis diagram is shown in Figure 2 .

[0048] Example 4, construction of OleP-3 mutant library

[0049] OleP-3 was homologous modeled by AlphaFold2 (AlphaFold2 TIB Server (biodesign.ac.cn)), and further screened the key amino acids Ser181, Val192, Ser249 and Thr288 in the catalytic pocket affecting the stereoselective recognition of the substrate by molecular docking, dynamics simulation and other computational aids according to steric hindrance, interaction force and the like. In order to saturate mutate Ser (S) at the 181st site, Val (V) at the 192nd site, Ser (S) at the 249th site and Thr (T) at the 288th site in the parent amino acid sequence, the corresponding primers were designed, as shown in Table 1.

[0050] Table 1: Primer design table

[0051]

[0052] Note: N = A / G / C / T, K = G / T, M = A / C.

[0053] The recombinant plasmid pET28a-OleP-3 containing the target gene fragment was used as a template, and the template was amplified by overlap extension PCR.

[0054] The PCR amplification system was (50 μL): 0.1 ng-1 ng of template DNA, 25 μL of 2x Phanta Max Buffer, 1 μL of dNTPs (10 mM each), 1 μL of mutation primer upstream and downstream, 1 U of Phanta Max Super-Fidelity DNA Polymerase, and the rest was supplemented with ddH2O to a total volume.

[0055] The PCR reaction parameters were as follows: (1) 95℃ pre-denaturation for 30s; (2) 95℃ denaturation for 15s; (3) 63℃ annealing for 4min30s; (4) 72℃ extension for 6min, steps (2)-(4) were cycled for 30 times; (5) 72℃ thorough extension for 5min, 4℃ storage.

[0056] After the PCR product was analyzed by 0.9% agarose gel electrophoresis and was positive, 20 μL of the PCR reaction solution was added with 1 μL of endonuclease Dpn I, and was incubated at 37℃ for 3h to remove the template plasmid DNA, and was inactivated at 65℃ for 10min. The heat shock was transformed into E. coli BL21 (DE3) competent cells, and after recovery, was plated on LB plates containing 50 mg / L kanamycin and was cultured at 37℃ overnight. Single colonies were picked and cultured in LB liquid medium containing kanamycin resistance (final concentration of 50 mg / L), and the plasmid was extracted and sequenced.

[0057] Finally, 181-site, 192-site, 249-site and 288-site OleP-3 mutant engineering strains were obtained.

[0058] Example 5, screening of optimal single-point mutants

[0059] All mutant plasmids obtained in Example 4 were co-transformed with pACYCDuet-Pdr-Pdx plasmid into expression host E. coli BL21 (DE3) according to the method of Example 2 to obtain co-expression recombinant strains.

[0060] The co-expression recombinant strains were inoculated into TB medium containing kanamycin (final concentration 50 mg / L) and chloramphenicol (final concentration 25 mg / L) and cultured at 37°C, 180 rpm for 6-8 h, then transferred to fresh TB liquid medium containing kanamycin (final concentration 50 mg / L) and chloramphenicol (final concentration 25 mg / L) at a 2% (v / v) inoculation amount, and cultured at 37°C, 180 rpm until the OD 600 When the OD was 0.3-0.45, IPTG (final concentration 0.5 mM) was added to the above-mentioned TB liquid medium, and the culture was induced at 28°C, 180 rpm for 10-12 h, then centrifuged at 4°C, 8000 rpm for 10 min to collect the bacterial cells.

[0061] The enzyme activity detection reaction system (10 mL) had the following final concentrations: 20 mM NaCl, 100 g / L glucose, 4 g / L glycerol, 0.8 g of wet bacterial cells, 1 g / L of substrate lithocholic acid (LCA), 2% (v / v) DMSO (dimethyl sulfoxide), 10 mL of reaction system composed of 200 mM KH2PO4 / K2HPO4 buffer at pH 7.4. After preheating the reaction solution at 28°C for 2 min, it was placed in a water bath shaker and reacted at 28°C, 180 rpm for 1 h. The reaction solution was extracted twice with an equal volume of ethyl acetate, and the upper organic phase was concentrated by vacuum instrument. The precipitated solid was dissolved in 400 μL of methanol, and then the substrate and product were detected by high performance liquid chromatography-differential refractometer detector. The enzyme activity and the relative reduction amount of byproduct MDCA were calculated, and the results are shown in Table 2.

[0062] Enzyme activity unit definition: the amount of enzyme required to convert 1 μmol of substrate in 1 min under the above conditions is 1 enzyme activity unit (U).

[0063] Specific activity: enzyme activity per mg of enzyme protein, unit U / mg.

[0064] Table 2, enzyme activity of dominant single-point mutants

[0065]

[0066] High performance liquid chromatography (HPLC) was UltiMate 3000 (Thermo) with column Omega PSC18. The 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.

[0067] The results showed that the hydrolysis activity of the engineered bacteria E. coli BL21(DE3) / pET28b-OleP-3-S181T / pACYCDuet-Pdr-Pdx (denoted as OleP-3-S181T) was the highest (8.80 U / g), which was 2.2 times that of the wild type.

[0068] Example 6, Iterative mutation of OleP-3

[0069] 1, Double mutation

[0070] Based on the mutant S181T with the highest activity obtained in Example 5, the optimal single-point mutations of S249, V192, and T288, i.e., S249T, V192A, and T288I, were further superimposed in the form of permutation and combination. The pET28b-OleP-3-S181T plasmid was used as a mutation template, and the mutation primer of S249T (Table 3) was designed for whole plasmid PCR amplification.

[0071] Table 3, Mutation primer design table of S249T, V192A, and T288I sites

[0072]

[0073] PCR conditions: (1) 95 °C pre-denaturation for 5 min; (2) 95 °C denaturation for 15 s, 60 °C for 5 s, 72 °C extension for 3.5 min, 35 cycles of step (2); (3) finally 72 °C extension for 5 min, and 4 °C storage.

[0074] The obtained PCR product was digested with endonuclease Dpn I at 37°C for 3 h and inactivated at 65°C for 10 min, and then transformed into the expression host E. coli BL21(DE3) together with pACYCDuet-Pdr-Pdx plasmid according to the method in Example 2 to obtain a co-expression recombinant strain. The strain was coated on an LB plate containing kanamycin (50 μg / mL) and chloramphenicol (25 μg / mL) resistance and cultured at 37°C overnight. The highest activity of the obtained strain E. coli BL21(DE3) / pET28b-OleP-3-S181T / S249T / pACYCDuet-Pdr-Pdx (denoted as OleP-3-S181T / S249T) was screened according to the method in Example 5, and the enzyme activity reached 12.46 U / g, as shown in Table 4.

[0075] 2, three mutations

[0076] The pET28b-OleP-3-S181T / S249T plasmid was used as a mutation template, and a mutation primer (Table 3) of V192A was designed for whole plasmid PCR amplification.

[0077] The PCR conditions were as follows: (1) pre-denaturation at 95°C for 5 min; (2) denaturation at 95°C for 15 s, annealing at 60°C for 5 s, and extension at 72°C for 3.5 min, for a total of 35 cycles; (3) final extension at 72°C for 5 min, and storage at 4°C.

[0078] The obtained PCR product was digested with endonuclease Dpn I at 37°C for 3 h and inactivated at 65°C for 10 min, and then transformed into the expression host E. coli BL21(DE3) together with pACYCDuet-Pdr-Pdx plasmid according to the method in Example 2 to obtain a co-expression recombinant strain. The strain was coated on an LB plate containing kanamycin (50 μg / mL) and chloramphenicol (25 μg / mL) resistance and cultured at 37°C overnight. The highest activity of the obtained strain E. coli BL21(DE3) / pET28b-OleP-3-S181T / S249T / V192A / pACYCDuet-Pdr-Pdx (denoted as OleP-3-S181T / S249T / V192A) was screened according to the method in Example 5, and the enzyme activity reached 19.31 U / g, and the content of byproduct MDCA was reduced by 61.44%, as shown in Table 4.

[0079] 3, four mutations

[0080] The pET28b-OleP-3-S181T / S249T / V192A plasmid was used as a mutation template, and a mutation primer (Table 3) of T288I was designed for whole plasmid PCR amplification.

[0081] PCR conditions: (1) 95℃ pre-denaturation for 5 min; (2) 95℃ denaturation for 15 s, 60℃ for 5 s, 72℃ extension for 3.5 min, step (2) for 35 cycles; (3) last 72℃ extension for 5 min, 4℃ storage.

[0082] The amplified PCR product was digested with endonuclease Dpn I at 37℃ for 3 h and inactivated at 65℃ for 10 min, and then transformed into the expression host E. coli BL21 (DE3) together with pACYCDuet-Pdr-Pdx plasmid according to the method in Example 2 to obtain the co-expression recombinant strain. The strain was coated on LB plates containing kanamycin (50 μg / mL) and chloramphenicol (25 μg / mL) resistance and cultured at 37℃ overnight. The highest activity of the engineering bacteria E. coli BL21 (DE3) / pET28b-OleP-3-S181T / S249T / V192A / T288I / pACYCDuet-Pdr-Pdx (denoted as OleP-3-S181T / S249T / V192A / T288I) was obtained by screening according to the method in Example 5, and 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 byproduct MDCA decreased by 62.71% compared with the wild type, as shown in Table 4.

[0083] Table 4, activity comparison of each dominant mutant of OleP-3

[0084]

[0085] Example 7, application of co-expression recombinant E. coli containing cytochrome P450 enzyme in preparation of ursodeoxycholic acid

[0086] The wet bacterial cells of E. coli BL21 (DE3)-OleP-3 / pACYCDuet-Pdr-Pdx obtained in Example 2, E. coli BL21 (DE3)-OleP-3-S181T / pACYCDuet-Pdr-Pdx obtained by the method in Example 5, E. coli BL21 (DE3)-OleP-3-S181T / S249T / pACYCDuet-Pdr-Pdx, E. coli BL21 (DE3)-OleP-3-S181T / S249T / V192A / pACYCDuet-Pdr-Pdx, and E. coli BL21 (DE3)-OleP-3-S181T / S249T / V192A / T288I / pACYCDuet-Pdr-Pdx were used as catalysts.

[0087] The transformation system was added with final concentration composition: 20 mM NaCl, 100 g / L glucose, 4 g / L glycerol, 0.8 g of wet bacteria, 4 g / L of substrate lithocholic acid (LCA), 8% of DMSO by volume, 10 mL reaction system was composed of 200 mM KH2PO4 / K2HPO4 buffer solution with pH 7.4, and the reaction was carried out at 28°C and 120 rpm. Sampling was carried out every 2-4 hours, and the reaction progress was detected by high performance liquid chromatography-differential refractive detector as described in Example 5.

[0088] The results are shown in Table 1. Figure 3 After 24 hours of reaction, the OleP-3 product concentration was 0.3 g / L, the product concentration of the optimal single mutant OleP-3-S181T was 0.37 g / L, which was 1.2 times that of the wild type, the product concentration of the optimal double mutant OleP-3-S181T / S249T was 0.51 g / L, which was 1.7 times that of the wild type, the product concentration of the optimal triple mutant OleP-3-S181T / S249T / V192A was 1.28 g / L, which was 4.26 times that of the wild type, and the product concentration of the optimal quadruple mutant OleP-3-S181T / S249T / V192A / T288I was 1.49 g / L, which was 4.97 times that of the wild type.

[0089] The present application is not limited by the above specific textual description, and various changes can be made within the scope outlined in the claims, which are all within the scope of the present application.

Claims

1. A Streptomyces-derived cytochrome P450 enzyme mutant, characterized in that, The amino acid sequence of the cytochrome P450 enzyme mutant is shown in one of SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8 or SEQ ID NO.

10.

2. A recombinant genetically engineered bacterium comprising the coding gene of the cytochrome P450 enzyme mutant of claim 1.

3. Use of the cytochrome P450 enzyme mutant of claim 1 in catalyzing the preparation of ursodeoxycholic acid from chenodeoxycholic acid.

4. The use according to claim 3, wherein the compound is ###0002### The method of the use is as follows: the wet bacterium obtained by fermentation culture and centrifugation of the engineered bacterium co-expressing the cytochrome P450 enzyme mutant coding gene and the redox partner gene is used as a catalyst, chenodeoxycholic acid is used as a substrate, glucose, NaCl, dimethyl sulfoxide and glycerol are added, a buffer solution with a pH of 5-10 is used as a reaction medium to form a reaction system, the reaction is carried out in a water bath under the conditions of 20-55 ℃ and 100-400 rpm, after the reaction, the reaction solution is extracted with ethyl acetate, the upper organic phase is taken, and the ethyl acetate is volatilized by a vacuum concentration instrument to obtain the product ursodeoxycholic acid; the redox partner gene is a combination of the ferredoxin reductase Pdr and the ferredoxin Pdx genes from Pseudomonas putida.

5. The use according to claim 4, wherein the compound is ###0002### The amino acid sequence of the ferredoxin reductase Pdr is shown in SEQ ID NO: 12; the amino acid sequence of the ferredoxin Pdx is shown in SEQ ID NO:

14.

6. The use according to claim 4, wherein the compound is ###0002### The engineered bacterium co-expressing the cytochrome P450 enzyme mutant coding gene and the redox partner gene is constructed by the following steps: the cytochrome P450 enzyme mutant is inserted into the T7 promoter of pET-28a(+), and the recombinant plasmid pACYCDuet-Pdr-Pdx expressing the ferredoxin reductase Pdr and the ferredoxin Pdx is co-transferred into the host bacterium to obtain the co-expression engineered bacterium.

7. The use according to claim 4, wherein the compound is ###0002### In the reaction system, the concentration of the substrate added is 1-5 g / L, the amount of the catalyst is 50-150 g / L in terms of the weight of the wet bacterium, the concentration of NaCl added is 10-30 mM, the concentration of glucose added is 50-150 g / L, the concentration of glycerol added is 1-5 g / L, and the concentration of dimethyl sulfoxide added is 1-10%.

8. The use according to claim 4, wherein The reaction medium is a potassium phosphate buffer with a pH of 7.4 and a concentration of 200 mM.

9. The use according to claim 4, wherein the compound is ###0002### The wet bacterium is prepared by the following method: The engineering bacteria co-expressing the cytochrome P450 enzyme mutant coding gene and the oxidoreductase partner gene are inoculated into TB culture medium containing 50 μg / mL kanamycin and 25 μg / mL chloramphenicol, and cultured at 37 ℃ for 6-8 h to obtain a seed liquid; then the seed liquid is inoculated into fresh TB liquid culture medium containing 50 μg / mL kanamycin and 25 μg / mL chloramphenicol at a volume concentration of 2%, and cultured at 37 ℃, 180-200 rpm until the bacterial concentration OD600 reaches 0.3-0.45; then 0.1-0.5 mM isopropyl-β-D-thiogalactopyranoside is added to the culture medium at a final concentration, and the culture is induced at 28 ℃, 180-200 rpm for 10-12 h; and then the wet bacteria are collected by centrifugation at 4 ℃, 8000-12000 rpm for 10 min.

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