Cytochrome p450 enzyme mutants and uses thereof

Abstract

The present application provides a cytochrome P450 enzyme mutant and use thereof. The mutant has any one of the following amino acid mutations in the amino acid sequence shown in SEQ ID NO. 2: G294A, G294A+E89G, G294A+N236H, G294A+F321W, G294A+N236H+F321W, G294A+N236H+F321W+V297A, and G294A+N236H+F321W+I397V. The present application takes the amino acid sequence shown in SEQ ID NO. 2 as a template, mutates and modifies key active sites of the template, constructs a mutant library, and screens a series of mutants with significantly improved catalytic activity and / or selectivity. The mutant can selectively catalyze 7β-hydroxylation of a steroid compound to generate a corresponding 7β-hydroxylated steroid compound, and has excellent selectivity and / or high yield, indicating that the mutant has a good application prospect in catalyzing the steroid compound to generate a 7β-hydroxylated steroid compound.

Description

TECHNICAL FIELD

[0001] The embodiment of the present application relates to the technical field of biological enzymes, and particularly relates to a cytochrome P450 enzyme mutant and application thereof. BACKGROUND

[0002] Ursodeoxycholic acid (UDCA), also known as 3α,7β-dihydroxy-5β-cholestane-24-oic acid, is a steroidal compound with multiple biological activities. Ursodeoxycholic acid has important clinical value in the treatment of hepatobiliary diseases, including cholesterolic cholelithiasis, primary biliary cholangitis, cholestatic liver disease, etc.

[0003] Cytochrome P450 (CYP) is a large family of self-oxidizable heme proteins. P450 enzymes, as widely used biological catalysts, can participate in more than 20 different types of chemical oxidation reactions, including hydroxylation, epoxidation, decarboxylation, N- and O-dealkylation, C-C bond coupling or cleavage, etc.; for example, under mild conditions, P450 enzymes can catalyze the regio- and stereoselective oxidation of non-activated C-H bonds in complex organic molecules to generate hydroxyl groups.

[0004] In the prior art, Grobe et al. (Grobe, S; Badenhorst, C; Bayer, T.; Hamnevik, E; Wu, S; Grathwol, CW; Link, A; Koban, S; Brundiek, H; Grossjohann, B; Engineering regioselectivity of a P450 monooxygenase enables the synthesis of ursodeoxycholic acid via 7beta-hydroxylation of lithocholic acid. Angew. Chem. Int. Ed. 2021, 60, 753-757) used UDCA colorimetry to screen specific residue site combinations and found a triple mutant that could produce 7β-hydroxylation of lithocholic acid (LCA) to generate UDCA in whole cells, but the molar yield of UDCA catalyzed by whole cells was low. Due to the permeability barrier of the cell envelope, the reaction catalyzed by whole cells is reported to be slower than that catalyzed by free enzymes, which is limited by the ability of substrates and products to penetrate the cell membrane barrier and substrate toxicity, and the high-efficiency performance of enzymes cannot be fully demonstrated.

[0005] Therefore, it is of great significance to promote the industrial production of UDCA by mining more P450 enzymes, modifying the structure of the enzymes, and realizing the efficient synthesis of UDCA through free enzyme catalysts. SUMMARY

[0006] The present application provides a cytochrome P450 enzyme mutant and use thereof.

[0007] To achieve the above object, the present application provides the following technical solutions.

[0008] In a first aspect, the present application provides a cytochrome P450 enzyme mutant, wherein the mutant has any one of the following amino acid mutations in the amino acid sequence shown in SEQ ID NO. 2:

[0009] G294A, G294A+E89G, G294A+N236H, G294A+F321W, G294A+N236H+F321W, G294A+N236H+F321W+V297A, G294A+N236H+F321W+I397V.

[0010] In a second aspect, the present application provides a DNA molecule, wherein the DNA molecule is a nucleotide sequence encoding the cytochrome P450 enzyme mutant of the first aspect or a complementary sequence thereof.

[0011] In a third aspect, the present application provides a recombinant plasmid, wherein the recombinant plasmid contains the DNA molecule of the second aspect.

[0012] In a fourth aspect, the present application provides a recombinant strain, wherein the recombinant strain contains the recombinant plasmid of the third aspect.

[0013] As an implementation form, the host cell of the recombinant strain is a prokaryotic cell or a eukaryotic cell, and the eukaryotic cell is a yeast cell.

[0014] As an implementation form, the host cell of the recombinant strain is a competent cell.

[0015] As an implementation form, the competent cell is a Pichia pastoris cell, an Escherichia coli BL21 (DE3) cell, or a Transetta (DE3) cell.

[0016] In a fifth aspect, the present application provides a use of the cytochrome P450 enzyme mutant of the first aspect, the DNA molecule of the second aspect, the recombinant plasmid of the third aspect, or the recombinant strain of the fourth aspect in catalyzing a 7β-hydroxylation reaction of a steroid compound to generate a 7β-hydroxylated steroid compound, wherein the steroid compound is selected from one of the following compounds:

[0017]

[0018] As an implementation form, the method for preparing the 7beta-hydroxylated steroid compound comprises the following steps:

[0019] Preparation of a recombinant plasmid containing a coding sequence of a cytochrome P450 enzyme mutant;

[0020] Transformation of the recombinant plasmid into a host cell to obtain a recombinant strain;

[0021] Catalysis of 7beta-hydroxylation of a steroid compound by the recombinant strain in the presence of O2 and NAD(P)H to obtain a 7beta-hydroxylated steroid compound.

[0022] As an implementation form, the method for preparing the 7beta-hydroxylated steroid compound comprises the following steps:

[0023] Using an expression vector containing the amino acid sequence shown in SEQ ID NO. 2 as a template, mutation is performed at the corresponding site to obtain a recombinant plasmid containing a coding sequence of a cytochrome P450 enzyme mutant.

[0024] Compared with the prior art, the embodiments of the present application have at least the following beneficial effects:

[0025] The embodiments of the present application use the amino acid sequence shown in SEQ ID NO. 2 as a template (basis), mutate the key active sites of the template (basis), construct a mutant library, and screen a series of mutants with significantly improved catalytic activity and / or selectivity. The cytochrome P450 enzyme mutant provided by the embodiments of the present application can selectively catalyze the 7beta-hydroxylation of a steroid compound to generate a corresponding 7beta-hydroxylated steroid compound (such as UDCA), and has excellent selectivity and / or high yield, which indicates that the mutant has good application prospects in the preparation of 7beta-hydroxylated steroid compounds from steroid compounds.

[0026] Additional aspects and advantages of the present application will be given in part in the following description, which will become apparent from the following description, or will be understood by those skilled in the art through practice of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 A reaction schematic diagram of catalysis of a compound LCA by a cytochrome P450 enzyme mutant to obtain a compound UDCA is shown;

[0028] Figure 2 An HPLC chart of catalysis of a compound LCA by a template OleP(F84Q / S240A / V291G) is shown;

[0029] Figure 3 HPLC chart of mutant G294A+N236H+F321W+V297A catalyzing compound LCA is shown. DETAILED DESCRIPTION

[0030] The technical solutions in the embodiments will be described clearly and completely below with reference to the embodiments of the present application and drawings. Obviously, the embodiments described below are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application.

[0031] First, some terms and materials involved in the embodiments will be explained below so as to be understood by those skilled in the art.

[0032] LCA: Lithocholic acid, English name.

[0033] UDCA: Ursodeoxycholic Acid, English name, also referred to as 7βOH-LCA in the embodiments.

[0034] Unless otherwise specified, the culture medium used in the following embodiments has the following specific composition:

[0035] LB liquid medium: 2.5 g of protein peptone, 1.25 g of yeast powder and 2.5 g of sodium chloride are added, stirred and dissolved with an appropriate amount of distilled water, and then diluted to 250 mL. The solution is sterilized by high pressure. LB solid medium: 3.75 g of agar powder is added to the LB liquid medium, and then sterilized by high pressure.

[0036] TB liquid medium: 3.0 g of protein peptone, 6.0 g of yeast powder and 1 mL of glycerol are added, stirred and dissolved with an appropriate amount of distilled water, and then diluted to 225 mL. The solution is divided into 22.5 mL per bottle and sterilized by high pressure. In addition, 16.43 g (0.72 M) of dipotassium hydrogen phosphate trihydrate and 2.31 g (0.17 M) of anhydrous potassium dihydrogen phosphate are stirred and dissolved with an appropriate amount of distilled water, and then diluted to 100 mL to prepare a potassium phosphate buffer. Before use, 2.5 mL of the potassium phosphate buffer is added to each bottle of TB medium.

[0037] The cytochrome P450 enzyme mutants and their uses in the embodiments will be described in detail below.

[0038] First, the cytochrome P450 enzyme mutants of the first aspect of the embodiments will be described.

[0039] Cytochrome P450 enzyme mutants

[0040] In the prior art, steroidal compounds represented by UDCA are mainly prepared by chemical or biosynthetic pathways. Among them, the traditional chemical synthesis pathway needs to synthesize UDCA through multiple steps, and the synthesis route takes cholic acid (CA) or chenodeoxycholic acid (CDCA) as the starting substrate, and CA and CDCA are separated from bovine bile and chicken, duck, goose bile respectively, which are cheap and easy to obtain. However, the above route needs to use toxic and dangerous chemical reagents, and will produce a large amount of waste, and has the problems of harsh conditions, poor selectivity and low recovery rate.

[0041] In order to improve the synthesis efficiency of UDCA, biosynthesis method has been developed gradually. Biological UDCA synthesis is mainly free enzyme catalytic synthesis or whole cell synthesis (Song P, Zhang X, Feng W, Xu W, Wu C, Xie S, Yu S, Fu R. Biological synthesis of ursodeoxycholic acid. Front Microbiol. 2023, 14: 1140662.). Among them, free enzyme catalytic synthesis takes CDCA or CA as the substrate, and UDCA is synthesized by multi-enzyme cascade reaction; whole cell synthesis involves adding CDCA or lithocholic acid (LCA) substrate during microbial culture, which is converted to UDCA by microbial cells.

[0042] As known to those skilled in the art, cytochrome P450 enzymes are widely used in the synthesis of various chemicals due to their higher catalytic activity than other P450 enzymes. However, in the synthesis process of using cytochrome P450 enzymes to catalyze lithocholic acid (LCA) to generate ursodeoxycholic acid (UDCA), other non-target products are also generated, and currently cytochrome P450 enzymes generally have the problems of low biological activity and / or poor selectivity. For example, in the literature (Grobe, S; Badenhorst, C; Bayer, T.; Hamnevik, E; Wu, S; Grathwol, CW; Link, A; Koban, S; Brundiek, H; Grossjohann, B;

[0043] Engineering regioselectivity of a P450 monooxygenase enables the synthesis of ursodeoxycholic acid via 7beta-hydroxylation of lithocholic acid. Angew. Chem. Int. Ed. 2021, 60, 753-757) adopts a P450 CYP107D1 (OleP) mutant (F84Q / S240A / V291G) to verify the catalytic effect of 7β hydroxylation reaction on LCA, but the three mutant catalyzes the free enzyme catalytic reaction of LCA to generate 7βOH-LCA, which has the problems of low yield and / or poor selectivity.

[0044] Therefore, the present embodiment carries out mutation and modification on the above-mentioned P450 CYP107D1 (OleP) mutant as an initial template, aiming to find a P450 enzyme mutant with high yield and / or high selectivity for catalyzing 7β hydroxylation reaction of steroidal compounds.

[0045] Specifically, the present embodiment provides a cytochrome P450 enzyme mutant, which has any one of the following amino acid mutations on the amino acid sequence as shown in SEQ ID NO. 2:

[0046] G294A, G294A+E89G, G294A+N236H, G294A+F321W, G294A+N236H+F321W, G294A+N236H+F321W+V297A, G294A+N236H+F321W+I397V.

[0047] Wherein, the amino acid sequence (SEQ ID NO. 2) of cytochrome P450 enzyme is:

[0048] MTDTHTGPTPADAVPAYPFSLPHALDLDPHYAELRRDEPVSRVRLPYGEGTAWLVTRMSDARIVLGDSRFSTAAATDPATPRMFPTPPEPDGVLAQDPPDHTRLRRLVGKAFTARRVEEMRPRVRSLVDSLLDDMVAHGSPADLVEFLAVPFPVAVICELLGVPLEDRDLFRTFSDAMLSSTRLTAAEIQRVQQDFMVYMDGLVAQRRDAPTEDLLGALALATDNDDHLTKGEIVNMGVSLLIAGHETSVNQITNLVHLLLTERKRYESLVADPALVPAAVEEMLRYTPLVSAGSFVRVATEDVELSTVTVRAGEPCVVHFASANRDEEVFDHADELDFHRERNPHIAFGHGAHHCIGAQLGRLELQEALSALVRRFPTLDLAEPVAGLKWKQGMLIRGLERQIVSW.

[0049] It can be understood that the present embodiment is based on the amino acid sequence shown in SEQ ID NO. 2 as a template (basis), and the key active site of the cytochrome P450 enzyme is mutated and reformed based on the amino acid sequence to construct a mutant library. The cytochrome P450 enzyme mutant provided in the present embodiment can selectively catalyze the 7β hydroxylation reaction of a steroid compound (for example, LCA) to generate the corresponding 7β hydroxylated steroid compound (for example, UDCA), and has excellent selectivity and / or higher yield.

[0050] The nucleotide sequence of the cytochrome P450 enzyme in the present embodiment is shown in SEQ ID NO. 1, and the amino acid sequence is shown in SEQ ID NO. 2 as described above.

[0051] Specifically, the nucleotide sequence of the cytochrome P450 enzyme (SEQ ID NO. 1) is as follows:

[0052]

[0053] As a preferred embodiment of the present embodiment, the mutant of the present embodiment has any one of the following amino acid mutations in the amino acid sequence shown as SEQ ID NO. 2: G294A+N236H, G294A+N236H+F321W, G294A+N236H+F321W+V297A, G294A+N236H+F321W+I397V.

[0054] Specifically, the nucleotide sequence of the mutant G294A+N236H+F321W+V297A is shown as SEQ ID NO. 3, and the amino acid sequence is shown as SEQ ID NO. 4.

[0055] wherein the nucleotide sequence (SEQ ID NO. 3) of the mutant G294A+N236H+F321W+V297A is:

[0056]

[0057] The amino acid sequence (SEQ ID NO. 4) of the mutant G294A+N236H+F321W+V297A is:

[0058] MTDTHTGPTPADAVPAYPFSLPHALDLDPHYAELRRDEPVSRVRLPYGEGTAWLVTRMSDARIVLGDSRFSTAAATDPATPRMQPTPPEPDGVLAQDPPDHTRLRRLVGKAFTARRVEEMRPRVRSLVDSLLDDMVAHGSPADLVEFLAVPFPVAVICELLGVPLEDRDLFRTFSDAMLSSTRLTAAEIQRVQQDFMVYMDGLVAQRRDAPTEDLLGALALATDNDDHLTKGEIVHMGVALLIAGHETSVNQITNLVHLLLTERKRYESLVADPALVPAAVEEMLRYTPLGSAASFARVATEDVELSTVTVRAGEPCVVHWASANRDEEVFDHADELDFHRERNPHIAFGHGAHHCIGAQLGRLELQEALSALVRRFPTLDLAEPVAGLKWKQGMLIRGLERQIVSW.

[0059] It can be understood that the cytochrome P450 enzyme mutants of the present embodiment can be mutated at the corresponding sites by existing site-directed mutation techniques to obtain target mutants. Among them, the mutant with multiple mutation points can be superimposed on the basis of a single mutant, that is, on the basis of a single mutant, mutation is carried out at another amino acid mutation site, so as to obtain a mutant with multiple mutation points.

[0060] In summary, the cytochrome P450 enzyme mutants provided in the present embodiment can catalyze the 7β-hydroxylation reaction of steroid compounds, and have excellent selectivity and / or high yield, suggesting that the mutants have good application prospects in the preparation of 7β-hydroxysteroid compounds by biological enzyme catalysis of steroid compounds.

[0061] It should be understood that the cytochrome P450 enzyme mutants provided in the present embodiment can individually improve the selectivity of steroid compounds to generate 7β-hydroxysteroid compounds, can individually improve the yield of steroid compounds to generate 7β-hydroxysteroid compounds, or can simultaneously improve the selectivity and yield of steroid compounds to generate 7β-hydroxysteroid compounds.

[0062] The selectivity is defined as: 7beta-hydroxylated product / total product x 100%; and the yield is defined as: actual yield of target product (mole) / theoretical yield of target product (mole) x 100%.

[0063] It should be noted that the mutant provided in the present embodiment is not limited to catalyzing the generation of UDCA from LCA, and the mutant can also catalyze the 7beta-hydroxylation of other steroid compounds to generate corresponding target products.

[0064] Next, the DNA molecule of the second aspect of the present embodiment will be described.

[0065] DNA molecule

[0066] It can be understood that the DNA molecule provided in the present embodiment is a DNA molecule encoding the nucleotide sequence of any of the above-mentioned cytochrome P450 enzyme mutants or the complementary sequence thereof.

[0067] Next, the recombinant plasmid of the third aspect of the present embodiment will be described.

[0068] Recombinant plasmid

[0069] The recombinant plasmid provided in the present embodiment is a DNA molecule encoding the nucleotide sequence of any of the above-mentioned cytochrome P450 enzyme mutants or the complementary sequence thereof.

[0070] Exemplarily, the recombinant plasmid of the present embodiment can be selected from any of the following:

[0071] pET-21b(+), pET-22b(+), pET-3a(+), pET-3d(+), pET-11a(+), pET-12a(+), pET-14b, pET-15b(+), pET-16b(+), pET-17b(+), pET-19b(+), pET-20b(+), pET-21a(+), pET-23a(+), pET-23b(+), pET-24a(+), pET-25b(+), pET-26b(+), pET-27b(+), pET-28a(+), pET-29a(+), pET-30a(+), pET-31b(+), pET-32a(+), pET-35b(+), pET-38b(+), pET-39b(+), pET-40b(+), pET-41a(+), pET-41b(+), pET-42a(+), pET-43a(+), pET-43b(+), pET-44a(+), pET-49b(+), pQE2, pQE9, pQE30, pQE31, pQE32, pQE40, pQE70, pQE80, pRSET-A, pRSET-B, pRSET-C, pGEX-5X-1, pGEX-6p-1, pGEX-6p-2, pBV220, pBV221, pBV222, pTrc99A, pTwin1, pEZZ18, pKK232-8, pUC-18, pUC-19, pPICZA, pPICZαB, pPICZαC, pPIC3K, pPIC3.5K.

[0072] It can be understood that the recombinant plasmid of the embodiment can be prepared by using the known preparation method, for example:

[0073] The expression vector containing the amino acid sequence shown in SEQ ID NO. 2 is used as a template to mutate the corresponding site, and a recombinant plasmid containing a cytochrome P450 enzyme mutant is obtained.

[0074] For example, the above-mentioned expression vector can be selected from pET-28a; the above-mentioned expression vector containing the amino acid sequence shown in SEQ ID NO. 2 can be synthesized by a gene synthesis company (such as Tianyi Huiyuan Gene Technology Co., Ltd.) to synthesize the corresponding gene sequence on the pET-28a vector to obtain the corresponding template (such as pET28a-OleP(F84Q / S240A / V291G) in Example 1).

[0075] Next, the recombinant strain of the fourth aspect of the embodiment will be described.

[0076] Recombinant strains

[0077] It can be understood that the recombinant strain provided in the embodiment contains the recombinant plasmid of the third aspect.

[0078] In the embodiment, the host cell of the recombinant strain is a prokaryotic cell or a eukaryotic cell, and preferably the eukaryotic cell is a yeast cell.

[0079] The host cell can be a competent cell, and preferably the competent cell is a Pichia pastoris cell, Escherichia coli BL21 (DE3) or Transetta (DE3).

[0080] The recombinant strain of the embodiment can be prepared by using an existing preparation method, for example:

[0081] The prepared recombinant plasmid is transformed into Escherichia coli BL21 (DE3) for culture to obtain the recombinant strain.

[0082] The prepared recombinant strain is generally stored in a refrigerator (for example, -80°C).

[0083] Secondly, the use of the cytochrome P450 enzyme mutant of the fifth aspect of the embodiment will be described.

[0084] Use of the cytochrome P450 enzyme mutant

[0085] As described above, the cytochrome P450 enzyme mutant of the embodiment can be used to catalyze the 7β-hydroxylation reaction of a steroid compound (for example, LCA) to generate a 7β-hydroxylated steroid compound.

[0086] It can be understood that, based on the use of the cytochrome P450 enzyme mutant, the DNA molecule, the recombinant plasmid and the recombinant strain of the embodiment can also be used to catalyze the 7β-hydroxylation reaction of a steroid compound to generate a 7β-hydroxylated steroid compound.

[0087] The preparation method of the 7β-hydroxylated steroid compound will be described in detail below.

[0088] Exemplarily, the preparation method of the 7β-hydroxylated steroid compound in the embodiment includes:

[0089] (1) preparing a recombinant plasmid containing a coding cytochrome P450 enzyme mutant;

[0090] (2) transforming the recombinant plasmid into a host cell to obtain a recombinant strain;

[0091] (3) using the recombinant strain to catalyze the 7β-hydroxylation of a steroid compound in the presence of O2 and NAD(P)H, to obtain a 7β-hydroxylated steroid compound.

[0092] The steroid compound is selected from any one of the following compounds 1-6:

[0093]

[0094] The steps of the above preparation method will be further described below.

[0095] In step (1), the preparation of a recombinant plasmid containing a coding cytochrome P450 enzyme mutant includes:

[0096] The expression vector containing the amino acid sequence shown in SEQ ID NO. 2 is used as a template to perform mutation (site-directed mutation) at the corresponding site to obtain a recombinant plasmid containing a coding cytochrome P450 enzyme mutant.

[0097] The site-directed mutation can use existing mutation techniques, such as polymerase chain reaction (PCR) method.

[0098] In step (2), the recombinant plasmid is transformed into a host cell to obtain a recombinant strain, including:

[0099] The recombinant plasmid obtained in step (1) is transformed into Escherichia coli BL21 (DE3) for culture to obtain a recombinant strain.

[0100] In step (3), the steroid compound is used as a substrate, and in the presence of O2 and NAD(P)H, the recombinant strain is used to catalyze 7β-hydroxylation of the steroid compound to obtain a 7β-hydroxylated steroid compound, including:

[0101] The obtained recombinant strain is inoculated into TB liquid medium and shaken for culture, and then the bacterial cells are collected by centrifugation;

[0102] The bacterial cells are resuspended by buffer (100 mM potassium phosphate buffer, pH = 7.5), and in the presence of O2 and NAD(P)H, the steroid compound is added for reaction to obtain a 7β-hydroxylated steroid compound.

[0103] In the above reaction, oxygen is required to allow the cytochrome P450 enzyme mutant to catalyze the steroid compound to generate the corresponding 7β-hydroxylated steroid compound, so the reaction can be directly performed in an air environment; at the same time, NAD(P)H is used as an electron donor, which is a condition for the cytochrome P450 enzyme mutant to exert activity, so the reaction usually needs to be performed in an NAD(P)H regeneration reduction system.

[0104] Generally, the reaction conditions of step (3) are as follows: the reaction temperature is 20±10℃, the rotation speed is 200±20 rpm, and the reaction time is 20-24 h.

[0105] It is understood that the steroidal compound described in this embodiment is the substrate for the reaction.

[0106] For example, please refer to Figure 1 When the substrate is LCA (compound 1), in this embodiment, the cytochrome P450 enzyme mutant can selectively catalyze the direct 7β-hydroxylation of BA to obtain the corresponding 7β-hydroxylated steroid compound, namely product UDCA.

[0107] It should be noted that the mutant in this embodiment is not limited to catalyzing the conversion of lithocholic acid (LCA) to ursodeoxycholic acid (UDCA). The mutant can also catalyze the 7β-hydroxylation of other steroidal compounds. For example, compounds 2-6 can be catalyzed to generate the corresponding 7β-hydroxylated steroidal compounds.

[0108] In summary, the cytochrome P450 enzyme mutant provided in this embodiment can catalyze the 7β-hydroxylation reaction of a variety of steroidal compounds with excellent selectivity and / or high yield, suggesting that this mutant has good application prospects in the preparation of 7β-hydroxy steroidal compounds (7β-hydroxylation products) by bioenzymatic catalysis of steroidal compounds.

[0109] The present application will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustration / explanation only and are not intended to limit the scope of the present application.

[0110] Unless otherwise specified, all materials, reagents and instruments used in the following embodiments are commercially available.

[0111] Example 1

[0112] Synthesis and recombinant expression of template pET28a-OleP (F84Q / S240A / V291G)

[0113] This embodiment is based on the OleP (F84Q / S240A / V291G) mutant (Grobe, S; Badenhorst, C; Bayer, T.; Hamnevik, E; Wu, S; Grathwol, CW; Link, A; Koban, S; Brundiek, H; Grossjohann, B) mutant with low 7β-hydroxylation activity for lithocholic acid, as reported in the literature.

[0114] 7 beta-hydroxylation of lithocholic acid. Angew. Chem.-Int. Edit. 2021, 60, 753-757) was used as a template; given that the template OleP (F84Q / S240A / V291G) has problems with low yield and / or poor selectivity in the reaction of free enzyme biocatalysis of LCA to 7βOH-LCA.

[0115] In this embodiment, this was used as the parental template for the synthesis and recombinant expression of template pET28a-OleP(F84Q / S240A / V291G); wherein the nucleotide sequence of the parental template OleP(F84Q / S240A / V291G) is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0116] Specifically:

[0117] The gene sequence of OleP(F84Q / S240A / V291G) was synthesized into the pET28a vector and named pET28a-OleP(F84Q / S240A / V291G).

[0118] pET28a-OleP (F84Q / S240A / V291G) and the redox chaperone plasmid pACYCDuet-pdX-pdR were co-transformed into E. coli BL21(DE3) competent cells. After recovery culture at 37°C and 200 rpm for 1 h, an appropriate amount of bacterial culture was plated onto LB agar plates containing 50 μg / mL Kana and 34 μg / mL Cm resistance, and incubated overnight at 37°C. The next day, single colonies from the plates were picked for activation. The activated bacterial culture was transferred to 25 mL TB medium containing 50 μg / mL Kana and 34 μg / mL Cm resistance at a 1:50 dilution ratio and cultured at 37°C and 220 rpm until the logarithmic growth phase (OD50). 600 Add IPTG (0.6-1.0), heme precursor (5-ALA), and FeCl3 solution to final concentrations of 0.2 mM, 0.5 mM, and 0.6 mM, respectively, and induce culture at 25 °C and 180 rpm for 20 h.

[0119] The bacterial cells were collected by centrifugation at 6500 rpm and 4℃, washed twice with potassium phosphate buffer at pH 7.5, and then sonicated to disrupt the cells. The cells were then centrifuged at 12000 rpm for 10 min at 4℃, and the supernatant was collected as a control for subsequent screening of mutant activity. For a 1 mM catalytic substrate, the wet cell volume was 66 mg / mL.

[0120] Example 2

[0121] Construction of cytochrome P450 enzyme mutants

[0122] In this embodiment, plasmid pET28a-OleP(F84Q / S240A / V291G) was used as a template to design mutation primers, with the bases to be mutated placed in the middle of the primers.

[0123] The PCR amplification system (25 μL) consisted of: 0.5 μL (5 ng) pET28a-OleP (F84Q / S240A / V291G), 0.5 μL (10 μM) of each of the corresponding upstream and downstream mutant primers, 12.5 μL of 2×PhantaFlash Master Mix, and finally, sterile distilled water was added to bring the volume to 25 μL.

[0124] The sequences of the upstream and downstream mutation primers used in this embodiment are shown in Table 1 below.

[0125] Table 1: Sequences of upstream and downstream mutant primers The PCR reaction procedure is as follows: (1) 94℃ pre-denaturation for 1 min; (2) 98℃ denaturation for 30 s; (3) annealing temperature 67-73℃, annealing time 30 s; (4) 72℃ extension for 1 min, denaturation-annealing-extension for 32 cycles, and finally 72℃ extension for 1 min, and PCR products are stored at 4℃.

[0126] After the PCR reaction, the PCR product was digested with DpnI enzyme and then transformed into E. coli DH5α competent cells. After recovery culture, an appropriate amount of bacterial culture was plated onto LB agar plates containing 50 μg / mL Kana antibiotics and incubated upside down at 37°C overnight. Single colonies on the culture plates were validated, and 1-3 single colonies were selected for gene sequencing.

[0127] The sequencing results were compared with the gene sequence of the template using Snap Gene software, and plasmids were extracted from the correctly sequenced strains.

[0128] Among them, mutants with multiple mutation points are obtained by superimposing mutations on the basis of a single mutant using the same method described above; the induced expression of the mutant is consistent with the induced expression of the template described above.

[0129] Example 4

[0130] Activity screening of mutants

[0131] In this embodiment, LCA was used as an exemplary substrate to determine the catalytic activity of the mutant.

[0132] The obtained wet bacterial cells were resuspended at a ratio of 1.0 g (wet bacterial cells): 7.5 mL (potassium phosphate buffer, pH = 7.5). 600 μL of the bacterial suspension was used for sonication. The cell lysate was centrifuged at 12000 rpm for 10 min at 4°C, and the supernatant was collected. 560 μL of the supernatant was then added to 1 mM LCA (prepared with methanol to a 100 mM stock solution), 1 mM NAD(P)H, and 1 mM NAD(P) + 100 mM glucose and 1 mg / mL GDH. Transfer the reaction mixture to a 1 mL reaction tube and shake at 220 rpm and 25 °C for 24 hours.

[0133] The above reaction system was reacted in air at 25°C and 220 rpm for 24 h. After the reaction was completed, an equal volume of ethyl acetate was added for extraction, and the organic phase was collected. The extraction operation was repeated three times, and the organic phases were combined. The solvent was dried by nitrogen blowing, and then 1 mL of the methanol-dissolved sample was filtered through a 0.22 μm organic filter membrane. The content was detected by HPLC, and the results are shown in Table 2.

[0134] Table 2: Results of activity tests on mutants

[0135] Mutants Yield (%) Selectivity (%) Template OleP (F84Q / S240A / V291G) 18.52±2.76 52.08±1.62 G294A 32.08±2.03 58.34±2.94 G294A + E89G 19.81±0.30 59.27±2.08 G294A + N236H 43.53±2.01 74.70±1.68 G294A + F321W 20.50±4.10 88.96±1.41 G294A + N236H + F321W 46.46±3.75 84.58±2.07 G294A + N236H + F321W + V297A 68.43±6.02 90.05±2.40 G294A + N236H + F321W + I397V 22.69±8.17 88.91±7.86

[0136] Note: The test results in Table 2 were obtained after three parallel reactions under the same reaction conditions. Selectivity was defined as: 7β-hydroxylated product / total product × 100%; yield was defined as: actual yield (moles) of target product / theoretical yield (moles) of target product × 100%.

[0137] Figure 2 The HPLC chromatogram of LCA catalyzed by template OleP (F84Q / S240A / V291G) is shown. Figure 2 According to the test results in Table 2, the template OleP (F84Q / S240A / V291G) has the disadvantages of low yield and poor selectivity in the reaction of free enzyme catalyzing LCA to 7βOH-LCA.

[0138] This embodiment modifies key active sites based on a template to obtain mutants with significantly improved yields and / or selectivity for the 7β-hydroxylation of steroidal compounds. Specifically, the yield and selectivity catalyzed by mutant G294A are significantly improved compared to the template, with the yield improvement being more pronounced.

[0139] Furthermore, in this embodiment, a superposition mutation was performed on the mutant G294A to obtain a mutant with multiple mutation points, and the effects of other mutants on the yield and selectivity of catalyzing LCA to 7βOH-LCA were simultaneously verified. According to the test results in Table 2, the multiple mutants obtained in this embodiment based on mutant G294A significantly improved the yield and / or selectivity of catalyzing the 7β hydroxylation reaction of LCA compared with mutant G294A.

[0140] in, Figure 3 The HPLC chromatogram of the LCA catalyzed by the mutant G294A+N236H+F321W+V297A is shown; further, in conjunction with Table 2 and... Figure 3 The test results show that the mutant G294A+N236H+F321W+V297A not only exhibits better catalytic activity, but also significantly improved selectivity, with efficiency increased by about 50% compared to the original template and product selectivity increased to 90%.

[0141] It is understood that the mutant provided in this embodiment can individually improve the selectivity of LCA to generate 7β-hydroxylated products, or individually improve the yield of LCA to generate 7β-hydroxylated products, or simultaneously improve both the selectivity and yield of LCA to generate 7β-hydroxylated products. Therefore, this embodiment overcomes the shortcomings of low yield and / or poor selectivity in the template-catalyzed 7β-hydroxylation reaction of LCA in the prior art.

[0142] In summary, this embodiment used the amino acid sequence shown in SEQ ID NO.2 as a template to mutate the key active sites of the template, constructing a mutant library and screening a series of mutants with significantly improved catalytic activity and / or selectivity. The cytochrome P450 enzyme mutant provided in this embodiment can selectively catalyze the 7β-hydroxylation reaction of steroidal compounds to generate the corresponding 7β-hydroxylated steroidal compounds (such as UDCA), and has excellent selectivity and / or high yield, suggesting that this mutant has good application prospects in catalyzing the preparation of 7β-hydroxylated steroidal compounds from steroidal compounds.

[0143] The technical solutions provided by the embodiments of this application have been described in detail above. Specific examples have been used in this document to illustrate the principles and implementation methods of the embodiments of this application. The descriptions of the embodiments above are only for helping to understand the principles of the embodiments of this application. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the embodiments of this application. Therefore, the content of this specification should not be construed as a limitation of this application.

Claims

1. A cytochrome P450 enzyme mutant, characterized in that, The mutant undergoes any one of the following amino acid mutations in the amino acid sequence shown in SEQ ID NO. 2: G294A, G294A+E89G, G294A+N236H, G294A+F321W, G294A+N236H+F321W, G294A+N236H+F321W+V297A, G294A+N236H+F321W+I397V.

2. A DNA molecule, characterized in that, The DNA molecule is a nucleotide sequence encoding the cytochrome P450 enzyme mutant as described in claim 1.

3. A recombinant plasmid, characterized in that, The recombinant plasmid contains the DNA molecule as described in claim 2.

4. A recombinant bacterial strain, characterized in that, The recombinant strain contains the recombinant plasmid as described in claim 3.

5. The recombinant strain according to claim 4, characterized in that, The host cell of the recombinant strain is a prokaryotic cell or a eukaryotic cell, and the eukaryotic cell is a yeast cell.

6. The recombinant strain according to claim 4, characterized in that, The host cell of the recombinant strain is a competent cell.

7. The recombinant strain according to claim 6, characterized in that, The competent cells are Pichia pastoris cells, Escherichia coli BL21(DE3) or Transetta(DE3).

8. The use of the cytochrome P450 enzyme mutant according to claim 1, the DNA molecule according to claim 2, the recombinant plasmid according to claim 3, or the recombinant strain according to any one of claims 4-7 in catalyzing the 7β-hydroxylation reaction of steroid compounds to generate 7β-hydroxylated steroid compounds, characterized in that, The steroid compound is selected from one of the following compounds: 。 9. The use according to claim 8, characterized in that, The method for preparing the 7β-hydroxylated steroid compound includes: Prepare recombinant plasmids containing mutants encoding cytochrome P450 enzymes; The recombinant plasmid was transformed into the host cell to obtain the recombinant bacterial strain; Using steroidal compounds as substrates, 7β-hydroxylation of steroidal compounds was catalyzed by recombinant strains in the presence of O2 and NAD(P)H to obtain 7β-hydroxylated steroidal compounds.

10. The use according to claim 9, characterized in that, The preparation of the recombinant plasmid containing a mutant encoding cytochrome P450 enzyme includes: Using an expression vector containing the amino acid sequence shown in SEQ ID NO. 2 as a template, mutations were performed at the corresponding sites to obtain a recombinant plasmid containing a mutant encoding cytochrome P450 enzyme.

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