Cytochrome P450 enzyme mutant and application thereof

Abstract

The embodiment of the invention provides a cytochrome P450 enzyme mutant and application thereof. The mutant is subjected to any one of the following amino acid mutations on an amino acid sequence as shown in SEQ ID NO.2: G294A, G294A + E89G, G294A + N236H, G294A + F321W, G294A + N236H + F321W + V297A, and G294A + N236H + F321W + I397V. An amino acid sequence as shown in SEQ ID NO.2 is taken as a template, a key active site of the template is subjected to mutation modification, a mutant library is constructed, a series of mutants with remarkably improved catalytic activity and / or selectivity are obtained through screening, the mutants can selectively catalyze a steroid compound to generate a 7 beta hydroxylation reaction, and the 7 beta hydroxylation reaction can be carried out through a 7 beta hydroxylation reaction. And the mutant has excellent selectivity and / or relatively high yield, so that the mutant has a good application prospect in the preparation of the 7beta hydroxysteroid compound by catalyzing the steroid compound.

Description

Technical Field

[0001] The embodiments of the present application relate to the field of biological enzyme technology, and specifically to a cytochrome P450 enzyme mutant and its use. Background Art

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

[0003] Cytochrome P450 (CYP) enzymes are a large family of self-oxidizable heme proteins. As widely used biocatalysts, P450 enzymes 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 stereo-selective oxidation of non-activated C-H bonds in complex organic molecules to generate hydroxyl groups.

[0004] In the prior art, researchers such as Grobe (Grobe, S; Badenhorst, C; Bayer, T.; Hamnevik, E; Wu, S; Grathwol, CW; Link, A; Koban, S; Brundiek, H; Grossjohann, B; Engineeringregioselectivity ofa P450 monooxygenase enables the synthesisofursodeoxycholic acid via7beta-hydroxylation oflithocholicacid.Angew.Chem.Int.Ed.2021,60,753-757) used the UDCA colorimetric method to screen the specificity-determining residue site combination and found a triple mutant that can produce 7β-hydroxylation reaction of lithocholic acid (LCA) to produce UDCA in whole cells, but its whole-cell catalytic molar yield of UDCA is low. Due to the permeability barrier of the cell envelope, reactions catalyzed by whole cells are reported to be slower than those catalyzed by free enzymes. Limited by the ability of substrates and products to pass through the cell membrane barrier and substrate toxicity, the efficient refinement performance of enzymes cannot be fully demonstrated.

[0005] In view of this, it is of great significance to explore more P450 enzymes and modify their structures to achieve efficient synthesis of UDCA through free enzyme catalysts, which will promote the industrial production of UDCA. Summary of the invention

[0006] The purpose of the embodiments of the present application is to provide a cytochrome P450 enzyme mutant and its use.

[0007] To achieve the above objectives, the present application proposes the following technical solutions:

[0008] In the 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, an embodiment of the present application provides a DNA molecule, wherein the DNA molecule is a nucleotide sequence encoding the cytochrome P450 enzyme mutant as described in the first aspect or its complementary sequence.

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

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

[0013] As an embodiment, 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 embodiment, the host cell of the recombinant strain is a competent cell.

[0015] As an embodiment, the competent cells are Pichia pastoris cells, Escherichia coli BL21 (DE3) or Transetta (DE3).

[0016] In a fifth aspect, the present application proposes the use of the cytochrome P450 enzyme mutant according to the first aspect, the DNA molecule according to the second aspect, the recombinant plasmid according to the third aspect, or the recombinant strain according to the fourth aspect for catalyzing the 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 embodiment, the preparation method of the 7β-hydroxylated steroid compound comprises:

[0019] preparing a recombinant plasmid containing a cytochrome P450 enzyme mutant;

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

[0021] Using steroid compounds as substrates, in the presence of O2 and NAD(P)H, the recombinant strain is used to catalyze the 7β-hydroxylation reaction of the steroid compounds to obtain 7β-hydroxylated steroid compounds.

[0022] As an embodiment, the preparation of a recombinant plasmid containing a cytochrome P450 enzyme mutant comprises:

[0023] Using the 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 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 example of the present application uses the amino acid sequence shown in SEQ ID NO.2 as a template (basis), performs mutation modification on the key active sites of the template (basis), constructs a mutant library, and screens a series of mutants with significantly improved catalytic activity and / or selectivity; the cytochrome P450 enzyme mutant provided in the example of the present application can selectively catalyze the 7β-hydroxylation reaction of steroid compounds to generate corresponding 7β-hydroxylated steroid compounds (such as UDCA), and has excellent selectivity and / or high yield, indicating that the mutant has good application prospects in catalyzing steroid compounds to prepare 7β-hydroxysteroid compounds.

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

[0027] Figure 1 A schematic diagram of the reaction of the cytochrome P450 enzyme mutant catalyzing the compound LCA to obtain the compound UDCA;

[0028] Figure 2 HPLC graph of LCA of the catalytic compound of template OleP (F84Q / S240A / V291G) is shown;

[0029] Figure 3 The HPLC profile of the LCA of the compound catalyzed by mutants G294A+N236H+F321W+V297A is shown. DETAILED DESCRIPTION

[0030] The technical solutions in the embodiments will be described clearly and completely below in combination with the embodiments and drawings of the present application. Obviously, the embodiments described below are only part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0031] Some of the terms and materials involved in this embodiment are explained below to facilitate understanding by those skilled in the art.

[0032] LCA: Lithocholic acid, English name is Lithocholic acid.

[0033] UDCA: Ursodeoxycholic acid, also known as Ursodeoxycholic Acid in English, or 7βOH-LCA in this embodiment.

[0034] Unless otherwise specified, the specific composition of the culture medium used in the following examples is as follows:

[0035] LB liquid medium: add 2.5g peptone, 1.25g yeast powder and 2.5g sodium chloride, stir and dissolve with appropriate amount of distilled water, make up to 250mL, and sterilize by autoclave. LB solid medium: add 3.75g agar powder to LB liquid medium and sterilize by autoclave.

[0036] TB liquid medium: Add 3.0g peptone, 6.0g yeast powder and 1mL glycerol, stir and dissolve with appropriate amount of distilled water, dilute to 225mL, dispense into 22.5mL bottles, and sterilize by high pressure. In addition, weigh 16.43g (0.72M) potassium dihydrogen phosphate trihydrate and 2.31g (0.17M) anhydrous potassium dihydrogen phosphate, stir and dissolve with appropriate amount of distilled water, dilute to 100mL to make potassium phosphate buffer. Before use, add 2.5mL potassium phosphate buffer to each bottle of TB medium.

[0037] The cytochrome P450 enzyme mutant of this example and its use will be described in detail below.

[0038] First, the cytochrome P450 enzyme mutant according to the first aspect of this embodiment is 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 requires multiple steps to synthesize UDCA. The synthesis route uses bile acid (CA) or chenodeoxycholic acid (CDCA) as the starting substrate, and separates CA and CDCA from cheap and easily available ox bile and chicken, duck, and goose bile, respectively. However, the above route requires the use of toxic and dangerous chemical reagents, and will produce a large amount of waste, and there are problems such as harsh conditions, poor selectivity, and low recovery rate.

[0041] In order to improve the synthesis efficiency of UDCA, biosynthesis has been gradually developed. Bio-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 uses CDCA or CA as substrates, and UDCA is synthesized by multi-enzyme cascade reaction; whole cell synthesis involves adding CDCA or lithocholic acid (LCA) substrates during microbial culture, which are converted into UDCA by microbial cells.

[0042] It is known to those skilled in the art that cytochrome P450 enzymes are widely used in the synthesis of various chemicals because of their higher catalytic activity than other P450 enzymes. However, in the process of synthesizing ursodeoxycholic acid (UDCA) from lithocholic acid (LCA) catalyzed by cytochrome P450 enzymes, other non-target products will be generated at the same time, and currently cytochrome P450 enzymes generally have the problems of low biological activity and / or poor selectivity. For example, 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) A P450 CYP107D1 (OleP) mutant (F84Q / S240A / V291G) was used to verify its catalytic effect on the 7β-hydroxylation of LCA, but the free enzyme-catalyzed reaction of LCA to produce 7βOH-LCA by the triple mutant had problems of low yield and / or poor selectivity.

[0044] In view of this, this example uses the above-mentioned P450 CYP107D1 (OleP) mutant as an initial template for mutation modification, aiming to find a P450 enzyme mutant with high yield and / or high selectivity for catalyzing the 7β-hydroxylation reaction of steroid compounds.

[0045] Specifically, this embodiment provides a cytochrome P450 enzyme mutant, which has any one of the following amino acid mutations in the amino acid sequence 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 of cytochrome P450 enzyme (SEQ ID NO.2) is:

[0048] MTDTHTGPTPADAVPAYPFSLPHALDLDPHYAELRRDEPVSRVRLPYGEGTAWLVTRMSDARIVLGDSRFSTAAATDPATPRMFPTPPEPDGVLAQDPPDHTRLRRLVGKAFTARRVEEMRPRVRSLVDSLLDDMVAHGSPADLVEFLAVPFPVAVICELLGVPLEDRDLFRTFSDAMLSSTRLTAAEIQRVQQDFMVYMDGLV AQRRDAPTEDLLGALALATDNDDHLTKGEIVNMGVSLLIAGHETSVNQITNLVHLLLTERKRYESLVADPALVPAAVEEMLRYTPLVSAGSFVRVATEDVELSTVTVRAGEPCVVHFASANRDEEVFDHADELDFHRERNPHIAFGHGAHHCIGAQLGRLELQEALSALVRRFPTLDLAEPVAGLKWKQGMLIRGLERQIVSW.

[0049] It can be understood that this embodiment uses the amino acid sequence shown in SEQ ID NO.2 as a template (basis), and on the basis of this amino acid sequence, the key active site of the cytochrome P450 enzyme is mutated and modified to construct a mutant library; the cytochrome P450 enzyme mutant provided in this embodiment can selectively catalyze the 7β-hydroxylation reaction of steroid compounds (such as LCA) to generate corresponding 7β-hydroxylated steroid compounds (such as UDCA), and has excellent selectivity and / or high yield.

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

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

[0052]

[0053] As a preferred embodiment of this embodiment, the mutant of this embodiment undergoes any one of the following amino acid mutations in the amino acid sequence shown in 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 in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4.

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

[0056]

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

[0058] MTDTHTGPTPADAVPAYPFSLPHALDLDPHYAELRRDEPVSRVRLPYGEGTAWLVTRMSDARIVLGDSRFSTAAATDPATPRMQPTPPEPDGVLAQDPPDHTRLRRLVGKAFTARRVEEMRPRVRSLVDSLLDDMVAHGSPADLVEFLAVPFPVAVICELLGVPLEDRDLFRTFSDAMLSSTRLTAAEIQRVQQDFMVYMDGLV AQRRDAPTEDLLGALALATDNDDHLTKGEIVHMGVALLIAGHETSVNQITNLVHLLLTERKRYESLVADPALVPAAVEEMLRYTPLGSAASFARVATEDVELSTVTVRAGEPCVVHWASANRDEEVFDHADELDFHRERNPHIAFGHGAHHCIGAQLGRLELQEALSALVRRFPTLDLAEPVAGLKWKQGMLIRGLERQIVSW.

[0059] It is understandable that the cytochrome P450 enzyme mutant of this embodiment can be mutated at the corresponding site by existing site-directed mutagenesis technology to obtain the target mutant. 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, a mutation is performed at another amino acid mutation site to obtain a mutant with multiple mutation points.

[0060] In summary, the cytochrome P450 enzyme mutant provided in this example can catalyze the 7β-hydroxylation reaction of steroid compounds with excellent selectivity and / or high yield, indicating that the mutant has good application prospects in the preparation of 7β-hydroxysteroid compounds from steroid compounds catalyzed by biological enzymes.

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

[0062] The selectivity is defined as: 7β-hydroxylation product / total product×100%; the yield is defined as: actual yield of target product (moles) / theoretical yield of target product (moles)×100%.

[0063] It should be noted that the mutant provided in this example is not limited to catalyzing LCA to produce UDCA, and the mutant can also catalyze the 7β-hydroxylation reaction of other steroid compounds to produce the corresponding target product.

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

[0065] DNA molecules

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

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

[0068] Recombinant plasmid

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

[0070] Illustratively, the recombinant plasmid of this embodiment can be selected from any one of the following:

[0071] pET-21b(+), pET-22b(+), pET-3a(+), pET-3d(+), pET-11a(+), pET-12a(+), pET-1 4b, pET-15b(+), pET-16b(+), pET-17b(+), pET-19b(+), pET-20b(+), pET-21a(+), p ET-23a(+),p ET-23b(+),pET-24a(+),p ET-25b(+), pET-26b(+), pET-27b(+), pET-28a(+), pET-29a(+), pET-30a(+), pET-31b(+), pET-32a(+), pET-35b(+), pET-3 8b(+), 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, p BV220, pBV221, pBV222, pTrc99A, pTwin1, pEZZ18, pKK232-8, pUC-18, pUC-19, pPICZA, pPICZαB, pPICZαC, pPIC3K, pPIC3.5K.

[0072] It is understandable that the recombinant plasmid of this embodiment can be prepared by a known preparation method, for example:

[0073] Using the 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 cytochrome P450 enzyme mutant.

[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 into the pET-28a vector by a gene synthesis company (such as Tianyi Huiyuan Gene Technology Co., Ltd.) to obtain the corresponding template (such as pET28a-OleP (F84Q / S240A / V291G) in Example 1).

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

[0076] Recombinant strains

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

[0078] In this 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 may be a competent cell, and the competent cell is preferably a Pichia pastoris cell, Escherichia coli BL21 (DE3) or Transetta (DE3).

[0080] The recombinant strain of this embodiment can be prepared by using existing preparation methods, for example:

[0081] The prepared recombinant plasmid was transferred into Escherichia coli BL21 (DE3) for cultivation to obtain a recombinant strain.

[0082] The prepared recombinant strain usually needs to be stored in a refrigerator (such as -80°C).

[0083] Next, the use of the cytochrome P450 enzyme mutant according to the fifth aspect of this embodiment will be described.

[0084] Uses of cytochrome P450 enzyme mutants

[0085] As mentioned above, the cytochrome P450 enzyme mutant of this example can be used to catalyze the 7β-hydroxylation reaction of steroid compounds (such as LCA) to generate 7β-hydroxylated steroid compounds.

[0086] It is understandable that, based on the use of cytochrome P450 enzyme mutants, the DNA molecules, recombinant plasmids, and recombinant strains of this embodiment can also be used to catalyze the 7β-hydroxylation reaction of steroid compounds to generate 7β-hydroxylated steroid compounds.

[0087] The preparation method of 7β-hydroxylated steroidal compounds will be described in detail below.

[0088] Illustratively, the method for preparing the 7β-hydroxylated steroid compound in this embodiment comprises:

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

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

[0091] (3) Using steroid compounds as substrates, in the presence of O2 and NAD(P)H, the recombinant strain catalyzes the steroid compounds to undergo 7β-hydroxylation to obtain 7β-hydroxylated steroid compounds.

[0092] Wherein, the steroid compound is selected from any one of the following compounds 1 to 6:

[0093]

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

[0095] In step (1), preparing a recombinant plasmid encoding a cytochrome P450 enzyme mutant comprises:

[0096] Using the expression vector containing the amino acid sequence shown in SEQ ID NO.2 as a template, mutation of the corresponding site (site-directed mutagenesis) is performed to obtain a recombinant plasmid containing the encoded cytochrome P450 enzyme mutant.

[0097] Among them, site-directed mutagenesis can adopt existing mutagenesis 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, comprising:

[0099] The recombinant plasmid obtained in step (1) was transferred into Escherichia coli BL21 (DE3) for cultivation 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 the steroid compound to undergo 7β-hydroxylation to obtain the 7β-hydroxylated steroid compound, comprising:

[0101] The obtained recombinant bacteria were inoculated into TB liquid culture medium, and the bacteria were collected by centrifugation after shaking culture;

[0102] The bacteria were resuspended in a buffer (100 mM potassium phosphate buffer, pH = 7.5), and a steroid compound was added in the presence of O2 and NAD(P)H to react and obtain a 7β-hydroxylated steroid compound.

[0103] Among them, the above reaction requires the participation of oxygen so that the cytochrome P450 enzyme mutant catalyzes the steroid compound to generate the corresponding 7β-hydroxylated steroid compound, so the reaction can be carried out directly in an air environment; at the same time, NAD(P)H acts as an electron donor and is a condition for the cytochrome P450 enzyme mutant to be active, so the reaction usually needs to be carried out in an NAD(P)H regeneration and reduction system.

[0104] Typically, the reaction conditions of step (3) are: reaction temperature of 20±10°C, rotation speed of 200±20rpm, and reaction time of 20-24h.

[0105] It can be understood that the steroid compound described in this embodiment is the substrate of the reaction.

[0106] For example, see Figure 1 When the substrate is LCA (Compound 1), the cytochrome P450 enzyme mutant can selectively catalyze BA to directly undergo 7β-hydroxylation to obtain the corresponding 7β-hydroxylated steroid compound, namely, the product UDCA.

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

[0108] In summary, the cytochrome P450 enzyme mutant provided in this example can catalyze the 7β-hydroxylation reaction of various steroid compounds with excellent selectivity and / or high yield, indicating that the mutant has good application prospects in the preparation of 7β-hydroxysteroid compounds (7β-hydroxylation products) from steroid compounds by bio-enzyme catalysis.

[0109] The present application will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate / explain the present application and are not used to limit the scope of the present application.

[0110] In the following examples, the materials, reagents and instruments used can be purchased from commercial sources unless otherwise specified.

[0111] Example 1

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

[0113] This example is based on the OleP (F84Q / S240A / V291G) mutant with low 7β-hydroxylation activity for lithocholic acid reported in the literature (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

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

[0115] In this example, this was used as a parent template to synthesize and recombinantly express the template pET28a-OleP (F84Q / S240A / V291G); wherein the nucleotide sequence of the parent 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 1 hour of recovery culture at 37°C and 200rpm, an appropriate amount of bacterial solution was spread on a solid LB plate containing 50μg / mL Kana and 34μg / mL Cm resistance, and inverted and cultured at 37°C overnight. On the second day, a single colony on the plate was picked for activation, and the activated bacterial solution was transferred to 25mL TB medium containing 50μg / mL Kana and 34μg / mL Cm resistance at a dilution ratio of 1:50, and cultured at 37°C and 220rpm until the logarithmic growth phase (OD 600 0.6-1.0), add inducer IPTG, heme precursor (5-ALA) and FeCl3 solution to the final concentration of 0.2mM, 0.5mM and 0.6mM respectively, and induce culture at 25℃, 180rpm for 20h.

[0119] The cells were collected by centrifugation at 6500 rpm and 4°C, washed twice with potassium phosphate buffer at pH 7.5, and ultrasonically disrupted. The cells were centrifuged at 4°C and 12000 rpm for 10 min, and the supernatant was taken as a control for the subsequent activity screening of mutants; for 1 mM catalytic substrate, the amount of wet cells used was 66 mg / mL.

[0120] Example 2

[0121] Construction of cytochrome P450 enzyme mutants

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

[0123] Among them, the PCR amplification system (25 μL): 0.5 μL (5 ng) pET28a-OleP (F84Q / S240A / V291G), 0.5 μL (10 μM) of the corresponding upstream and downstream mutation primers, 12.5 μL 2×PhantaFlash Master Mix, and finally add sterile distilled water to make up the volume to 25 μL.

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

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

[0126] After the PCR reaction, the PCR product was digested with DpnI enzyme and then transferred into E. coli DH5α competent cells. After the culture was resumed, an appropriate amount of bacterial solution was spread on an LB plate containing 50 μg / mL Kana resistance and incubated at 37°C overnight. Single colonies on the culture plate were verified, 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 strains with correct sequencing.

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

[0129] Example 4

[0130] Activity screening of mutants

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

[0132] The wet cells obtained above were resuspended according to 1.0 g (wet cells): 7.5 mL (potassium phosphate buffer, pH = 7.5), 600 μL of the cell solution was used for ultrasonic disruption, the cell disruption solution was centrifuged at 4 ° C, 12000 rpm for 10 min, and the supernatant was collected; the supernatant (560 μL) was added with 1 mM LCA (dissolved in methanol to prepare a mother solution of 100 mM), 1 mM NAD(P)H, 1 mM NAD(P) + , 100 mM glucose and 1 mg / mL GDH. The reaction mixture was transferred to a 1 mL reaction tube and shaken at 220 rpm and 25°C for 24 hours.

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

[0134] Table 2: Activity test results of 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. The selectivity is defined as: 7β-hydroxylation product / total product × 100%; the yield is defined as: actual yield of target product (moles) / theoretical yield of target product (moles) × 100%.

[0137] Figure 2 The HPLC graph 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 defects of low yield and poor selectivity in the reaction of free enzyme catalyzing LCA to 7βOH-LCA.

[0138] In this example, the key active sites are mutated and modified based on the template, and a mutant with significantly improved yield and / or selectivity in catalyzing the 7β hydroxylation reaction of steroid compounds can be obtained. Among them, the yield and selectivity after catalysis by the mutant G294A are significantly improved compared with the template, and the improvement in yield is more obvious.

[0139] Furthermore, in this example, superimposed mutations were performed on the mutant G294A to obtain mutants with multiple mutation points, and the effects of other mutants on the yield and selectivity of catalyzing LCA to produce 7βOH-LCA were simultaneously verified; combined with the test results in Table 2, the multiple mutants obtained in this example on the basis of mutant G294A, relative to mutant G294A, each multiple mutant catalyzed LCA 7β hydroxylation reaction The yield and / or selectivity are significantly improved.

[0140] in, Figure 3 The HPLC graph of the mutant G294A+N236H+F321W+V297A catalytic compound LCA is shown; further combined 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 improves selectivity. The efficiency is increased by about 50% compared with the original template, and the product selectivity is increased to 90%.

[0141] It is understandable that the mutant provided in this embodiment can improve the selectivity of LCA to generate 7β-hydroxylated products alone, can also improve the yield of LCA to generate 7β-hydroxylated products alone, or can simultaneously improve the selectivity and yield of LCA to generate 7β-hydroxylated products. Therefore, this embodiment overcomes the defects of low yield and / or poor selectivity in the 7β-hydroxylation reaction of LCA catalyzed by templates in the prior art.

[0142] In summary, this example uses the amino acid sequence shown in SEQ ID NO.2 as a template, performs mutation modification on the 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 cytochrome P450 enzyme mutant provided in this example can selectively catalyze the 7β-hydroxylation reaction of steroid compounds to generate corresponding 7β-hydroxylated steroid compounds (such as UDCA), and has excellent selectivity and / or high yield, indicating that the mutant has good application prospects in catalyzing steroid compounds to prepare 7β-hydroxysteroid compounds.

[0143] The technical solutions provided in the embodiments of the present application are introduced in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the embodiments of the present application. The description of the above embodiments is only applicable to help understand the principles of the embodiments of the present application. At the same time, for those skilled in the art, according to the embodiments of the present application, there will be changes in the specific implementation methods and application scopes. In summary, the contents of this specification should not be understood as limiting the present application.

Claims

1. A cytochrome P450 enzyme mutant, characterized in that: 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, G294A+N236H+F321W+I397V.

2. A DNA molecule, characterized in that The DNA molecule is a nucleotide sequence encoding the cytochrome P450 enzyme mutant according to claim 1 or its complementary sequence.

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

4. A recombinant strain, characterized in that: The recombinant strain contains the recombinant plasmid as claimed 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 cells of the recombinant strain are competent cells.

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. 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 to 7 for 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 preparation method of the 7β-hydroxylated steroid compound comprises: preparing a recombinant plasmid containing a cytochrome P450 enzyme mutant; Transforming the recombinant plasmid into a host cell to obtain a recombinant strain; Using steroid compounds as substrates, in the presence of O2 and NAD(P)H, the recombinant strain is used to catalyze the 7β-hydroxylation reaction of the steroid compounds to obtain 7β-hydroxylated steroid compounds.

10. The use according to claim 9, characterized in that The method for preparing a recombinant plasmid containing a cytochrome P450 enzyme mutant comprises: Using the 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 cytochrome P450 enzyme mutant.

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