Monooxygenase P450BM3 multi-site mutant and application of monooxygenase P450BM3 multi-site mutant in catalyzing steroid compound to generate product C7beta

Abstract

The invention relates to the technical field of enzyme engineering and gene engineering, in particular to a monooxygenase P450BM3 multi-site mutant and application of the monooxygenase P450BM3 multi-site mutant in catalyzing a steroid compound to generate a product C7beta. The mutant is a mutation of one or more of amino acid residues 87, 88, 188 and 330 in an amino acid sequence SEQ ID NO.7 of a wild type P450BM3-LG-23; the mutant is a mutant of one or more of amino acid residues 87, 88, 188 and 330 in the amino acid sequence SEQ ID NO.7; the amino acid residues at each site can be mutated into L, T or A in a same or different manner. According to the invention, LG-23 is taken as an initial template, protein engineering and a directed evolution strategy are combined, and a series of novel combined mutants with relatively high C7beta hydroxylation selectivity and conversion rate on steroid substrates such as testosterone (TES), nandrolone (NAL), 4-androstenedione (4AD), 1, 4-androstenedione (ADD) and the like are successfully obtained through multiple rounds of site-directed saturation mutagenesis. Compared with LG-23, the substrate spectrum of the obtained mutant expands 1, 4-androstenedione, the steroid substrate selectivity is improved to 77%-93%, and the conversion rate is improved to 94.7% or above.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of enzyme engineering and genetic engineering, and particularly relates to a single oxygenase P450BM3 multi-site mutant and application thereof in catalyzing C7beta of a steroid compound to generate a product. BACKGROUND

[0002] Steroid drugs have important medical value and are widely used in various clinical indications such as autoimmune diseases, inflammation, cancer, coronavirus infection and osteoporosis. The biological activity of the drug is closely related to its molecular structure, and the realization of the regioselective and stereoselective hydroxylation on the steroid skeleton is one of the key steps to regulate its pharmacological function. In the steroid synthesis process, the chiral hydroxylation reaction at a specific position often depends on enzyme catalysis. Enzyme catalysis has excellent regioselectivity and stereoselectivity for natural substrates, but also has problems such as insufficient thermal stability and solvent stability, and narrow substrate spectrum, which limits its wide application in industrial conditions. Therefore, developing an enzyme catalyst with high selectivity, high stability and easy to scale-up for the target reaction is an important challenge in the current field of steroid biocatalysis.

[0003] Cytochrome P450BM3 is a naturally fused single-component monooxygenase belonging to the widely existing heme-containing P450 superfamily. Family members exhibit good catalytic activity for multiple types of natural substrates due to their extremely high electron transfer efficiency and rate. P450BM3 and its mutants have been extensively studied in catalyzing steroid hydroxylation. In the past three decades, mutants with good effects include F87A, which exhibits hydroxylation activity at the C2beta and C15beta positions of testosterone and the C2beta and C16beta positions of progesterone; A82W, which has hydroxylation activity at the C16alpha / beta positions of testosterone; and a series of mutants with high stereoselective and diastereoselective hydroxylation activity at the C16 position of various steroids, which are developed by iterative saturation of mutant libraries based on the guidance of mutational landscape and molecular dynamics simulation.

[0004] Among the multiple hydroxylation sites of steroid compounds, C7beta hydroxylation is a key step in the synthesis of important steroid drugs such as the anticancer drug fluvastatin, the cardiovascular disease treatment drug eplerenone, and the diuretic spironolactone. LG-23 obtained by directed evolution of P450BM3 has been reported to be able to realize C7beta hydroxylation of testosterone, nandrolone, androstenone, adrenosterone, epitestosterone and D-ethyl steroid ketone, and has good broad-spectrum substrate activity, but the product concentration is only millimolar, and the catalytic activity is low, which is difficult to meet the needs of industrial production. Therefore, it is urgent to further improve the catalytic efficiency of the enzyme while maintaining its regioselectivity and stereoselectivity. SUMMARY

[0005] The application aims to provide a monooxygenase P450BM3 multi-site mutant and application thereof in catalyzing a steroid compound to generate a product C7β.

[0006] In order to achieve the above-mentioned purpose, the application adopts the following technical scheme: A monooxygenase P450BM3 multi-site mutant, the mutant is a mutation of one or more of the amino acid residues at positions 87, 88, 188 and 330 in the amino acid sequence SEQ ID NO. 7 of wild-type P450BM3-LG-23; the mutation of each site amino acid residue can be the same or different and is L, T or A.

[0007] The mutant is a mutation of one or more of the amino acid residues at positions 87, 88, 188 and 330 in the amino acid sequence SEQ ID NO. 7 of wild-type P450BM3-LG-23, the amino acid residue at position 87 is mutated to A; the amino acid residue at position 88 is mutated to L or T; the amino acid residue at position 188 is mutated to L; the amino acid residue at position 330 is mutated to L.

[0008] The mutant is S88L, S88T, S88T / Q188L, G87A / S88L, S88L / W330L or G87A / S88L / W330L, wherein the amino acid sequences of each mutant are shown in SEQ ID NO. 1-6.

[0009] That is, the amino acid sequence shown in SEQ ID NO: 1 is the S88L mutant of the initial enzyme LG-23, which is abbreviated as LG-23 / S88L; the amino acid sequence shown in SEQ ID NO: 2 is the S88T mutant of the initial enzyme LG-23, which is abbreviated as LG-23 / S88T; the amino acid sequence shown in SEQ ID NO: 3 is the S88T / Q188L mutant of the initial enzyme LG-23, which is abbreviated as LG-23 / S88T / Q188L; the amino acid sequence shown in SEQ ID NO: 4 is the G87A / S88L mutant of the initial enzyme LG-23, which is abbreviated as LG-23 / G87A / S88L; the amino acid sequence shown in SEQ ID NO: 5 is the S88L / W330L mutant of the initial enzyme LG-23, which is abbreviated as LG-23 / S88L / W330L; the amino acid sequence shown in SEQ ID NO: 6 is the G87A / S88L / W330L mutant of the initial enzyme LG-23, which is abbreviated as LG-23 / G87A / S88L / W330L.

[0010] A recombinant expression plasmid containing the mutant.

[0011] An engineered bacterium containing the recombinant expression plasmid.

[0012] The mutant, the recombinant expression plasmid or the engineered bacterium is used for catalyzing the generation of product C7beta of a steroid compound.

[0013] The steroid compound is one or more of testosterone (TES), nandrolone (NAL), 4-androstenedione (4AD) and 1,4-androstenedione (ADD).

[0014] A method for catalyzing the generation of C7beta product of a steroid compound, wherein the mutant, the recombinant expression plasmid or the engineered bacterium is used for catalyzing reaction with the steroid compound as a substrate to generate the corresponding C7beta product.

[0015] The C7beta hydroxylation modification of the substrate is performed under the conditions of pH 7.0, temperature 25 DEG C and reaction time 24 hours by using the recombinant strain containing the mutant as a catalyst.

[0016] The substrate is a clathrate formed by the substrate and hydroxypropyl-beta-cyclodextrin (HP-beta-CD), and the molar ratio of HP-beta-CD to the substrate is 1:1 to 5:1.

[0017] The present application has the following advantages: The present application uses LG-23 as an initial template, combines protein engineering and directed evolution strategy, and successfully obtains a series of novel combined mutants with high C7beta hydroxylation selectivity and conversion rate for steroid substrates such as testosterone (TES), nandrolone (NAL), 4-androstenedione (4AD) and 1,4-androstenedione (ADD) through multiple rounds of site-directed saturation mutation. Compared with LG-23, the substrate spectrum of the mutant obtained by the present application is expanded to 1,4-androstenedione, the selectivity for steroid substrates is increased to 77% to 93%, and the conversion rate is increased to more than 94.7%. The present application not only has important significance for promoting the industrialization process of cytochrome P450BM3 as a directed catalytic enzyme, but also provides a systematic and efficient strategy for the rational design of similar catalytic enzymes. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the following embodiment or prior art description will be briefly introduced. Obviously, the drawings in the following description are only embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0019] Figure 1 Total energy and constraint penalty score for the mutant in Example 1.

[0020] Figure 2 HPLC chromatogram for the reaction of the mutant G87A / S88L / W330L catalyzing the conversion of 1,4-androstadiene-3,17-one to C7β-1,4-androstadiene-3,17-one in Example 3.

[0021] Figure 3 C7β-1,4-androstadiene-3,17-one (ADD) 1 H NMR (CDCL3, 400MHz) spectrum.

[0022] Figure 4 C7β-1,4-androstadiene-3,17-one (ADD) 13 C NMR (CDCL3, 400MHz) spectrum.

[0023] Figure 5 Testosterone (A), Nandrolone (B), 4-androstene-3,17-dione (C), 1,4-androstadiene-3,17-one (D) and the structure of C7β product formed in the hydroxylation reaction.

[0024] Figure 6 Conversion rate comparison chart of the mutant G87A / S88L / W330L catalyzing the conversion of 1,4-androstadiene-3,17-one in different systems in Example 4. DETAILED DESCRIPTION

[0025] The specific embodiments of the present application are further described in the following examples, it should be noted that the specific embodiments described herein are merely intended to illustrate and explain the present application, and are not limited to the present application.

[0026] Example 1: Design of mutation sites 1. Molecular docking To obtain the potential binding mode of LG-23 with steroid substrates, the crystal structure of LG-23 (PDB ID: 6LY4) was molecularly docked with steroid compounds using Rosetta's flexible small molecule docking protocol (Rosetta Ligand docking with flexible XML protocols). The structures of 4 steroid compounds (testosterone (A), nandrolone (B), 4-androstene-3,17-dione (C), 1,4-androstadiene-3,17-dione (D)) were obtained through the chemical information database PubChem (https: / / pubchem.ncbi.nlm.nih.gov / ), and all docking results were sorted from low to high according to the binding energy and conformational clustering, and finally the representative cluster with the lowest binding energy and consistent with the catalytic conformation was selected as the representative conformation of the potential binding mode.

[0027] 2. Molecular dynamics simulation To determine the key elements on the enzyme that play a dominant role in the binding of LG-23 and steroid substrates under experimental / production conditions, a 100 ns molecular dynamics (MD) simulation was performed on the representative conformation obtained by molecular docking using the AMBER20 software package. Among them, the LG-23 was parameterized using the AMBER-ff19SB force field; based on the optimization of the geometric structure of the steroid substrate through quantum calculation (QM), the general AMBER force field (GAFF) was used for parameterization to generate the corresponding bond angle and atomic charge parameters. In MD, a SPC cubic water box was used to construct the solvent environment, and periodic boundary conditions were added. At the same time, Na + and Cl - counterions were added to the solvent to neutralize the total charge of the system.

[0028] 3. Computer-aided rational design of catalytic enzymes The Enzyme Design module of Rosetta was used to design mutations for the representative conformation obtained above, and the conformational constraint condition was set according to the near attack conformation (NAC) of the steroid skeleton at C7β. Subsequently, according to the selected key residue sites, the enzyme mutation design was performed on the enzyme-substrate complex system structure, and 100 mutant structures were generated for each mutation site each time to extensively search for the mutant conformation of the input structure. At the same time, the enzyme design without mutation was performed once on the starting input structure under the same conditions as the control.

[0029] 4. Sorting and screening of mutants Each design generated by Rosetta's enzyme design method includes a corresponding total_score term and all_cst term, representing the total energy and total constraint penalty of each mutant, respectively, which can be used for ranking the mutants. In addition, if the designed mutants contain the same amino acid sequence, the one with the lowest total energy is retained. Therefore, the best mutant for experimental characterization is selected based on the following criteria: (1) the mutation probability of the designed mutant should be greater than 50%, and if the mutation site only contains one mutation, it should be at least greater than 20%; (2) the total energy or constraint penalty of the designed mutant should be less than or equal to the unmutated structure; (3) the introduced mutations are not allowed to produce obvious conformational conflicts or form large cavities through visual inspection. The total energy and constraint penalty of the mutants are shown in Table 1. Figure 1

[0030] Based on the above process, 4 mutation sites for the four substrates were selected, and the mutation types and mutation probabilities of each type are shown in Table 1.

[0031] Table 1 Mutation types and mutation probabilities of mutation sites for four substrates

[0032] Note: PIKAA represents the specific set of amino acids allowed to appear at the specified design position.

[0033] Example 2: Construction and site-directed mutagenesis of LG-23 monooxygenase In this example, the LG-23 mutant reported in the literature to have 7β hydroxylation activity on testosterone ((Li A, Acevedo-Rocha C G, D'Amore L, et al., Regio- and Stereoselective Steroid Hydroxylation at the C7-Position by Cytochrome P450 Monooxygenase Mutants [J]. Angewandte Chemie International Edition, 2020) was used as a template; pRSFDuet-1 was used as a vector for synthesis and recombinant expression; the amino acid sequence of the parent template LG-23 is shown in SEQ ID NO. 7.

[0034] ​

[0035] The LG-23 gene was cloned into the pRSFDuet-1 vector Not I and Hind III restriction enzyme sites, with a C-terminal hexahistidine tag, and then transformed into E. coli DH5a or BL21 (DE3) competent cells for gene cloning and heterologous expression, respectively.

[0036] Site-directed mutagenesis was performed according to the method described in the Fast Mutagenesis System Site-Directed Mutagenesis Kit (TransGen Biotech, Beijing, China). The 2x TransStart FastPfu Fly PCR SuperMix polymerase premix recommended in the method (TransGen Biotech, Beijing, China) was used to amplify the full-length plasmid by PCR to construct the variant, and the sequence encoding the mutant amino acid and the homologous sequence for DNA assembly were introduced by PCR primers (BGI, Beijing, China).

[0037] The PCR reaction system was as follows: the total reaction system was 50 μL, 2x TransStart FastPfu Fly PCR SuperMix 25 μL, ddH2O 20 μL, upstream and downstream primers 1 μL each, and template 2 μL were added, respectively. The reaction conditions were 94°C pre-denaturation for 5 min; 94°C denaturation for 20 s, 55°C annealing for 20 s, 57°C extension for 75 s, a total of 25 cycles from denaturation to extension; 72°C for 10 min. Finally, 4°C was stored.

[0038] The PCR product was treated with DMTase to digest the original DNA template. 1 μL of DMTase was added to the amplification product and mixed, and incubated at 37°C for 1 h.

[0039] The incubated product was transferred into E. coli DH5a competent cells (TransGen Biotech, Beijing, China). The E. coli chemical transformation method was used for transformation, and the steps were as follows: (1) The DH5a competent cells were thawed on ice, and 2-5 μL of the DMTase digestion product was added, and ice-bathed for 30 min.

[0040] (2) Heat shock at 42°C for 60 s, and immediately ice-bath for 2 min.

[0041] (3) Add 500 μL of non-resistant LB medium, and then recover at 37°C, 220 rpm in a shaker for 1 h.

[0042] (4) At the end of recovery, 100 μL of the bacterial solution was spread on LB plates containing kana resistance.

[0043] (5) The plates were placed in a 37℃ incubator for overnight culture for about 12-16 h.

[0044] After the initial screening using kanamycin sulfate, well-growing transformant single colonies were picked and inoculated in 5 mL of LB liquid medium containing Kana resistance, and cultured at 37℃, 220 r / min for 12 h. About 3 mL of activated bacterial solution was aspirated, and a plasmid extraction kit (DP105) was used to extract plasmids for sequencing to detect whether the site-directed mutation was successful. Sequencing services were provided by Suzhou Jinyu Zhi Biological Technology Co., Ltd.

[0045] Based on the screening of the mutant site in Example 1, the above-mentioned site-directed mutation kit was used to mutate the P450BM3-LG-23 monooxygenase mutants of S88L, S88T, S88T / Q188L, G87A / S88L, S88L / W330L and G87A / S88L / W330L. The site-directed mutation primers for constructing all the above-mentioned sites are shown in Table 2.

[0046] Table 2 LG-23 site-directed mutation primer sequences

[0047] Note: The suffix "F" of the primer name represents forward; "R" represents reverse SEQ ID NO. 1: AIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEAPGWVTRYLSSQRLIKEACDESRFDKNLWQALKYLRDILGDGLG LSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLVQKWERLNADEHIEVPEDMTRLTLDTIGLCGFNYRFNSFYRDQPHPFITSTVRALDEAQNKQQRANPDDPAYDENKRQFQEDTKVMNDLVDKIIADRKASGEQSDDLLTHMLNGKDPETGEPLDDENIRYQIITFLIAGHETTSGLLSFALYFLVKNPHVLQKAAEEAARVLVDPVPSYKQVKQLKYVGMVLNEALRLWPTGPWFSLYAKEDTVLGGEYPLEKGDELMVLIPQLHRDKTIWGDDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLKHFDFEDHTNYELDIKETLTLKPEGFVVKAKSKKIPLGGIPSPSTEQSAKKVRKKAENAHNTPLLVLYGSNMGTAEGTARDLADIAMSKGFAPQVATLDSHAGNLPREGAVLIVTASYNGHPPDNAKQFVDWLDQASADEVKGVRYSVFGCGDKNWATTYQKVPAFIDETLAAKGAENIADRGEADASDDFEGTYEEWREHMWSDVAAYFNLDIENSEDNKSTLSLQFVDSAADMPLAKMHGAFSTNVVASKELQQPGSARSTRHLEIELPKEASYQEGDHLGVIPRNYEGIVNRVTARFGLDASQQIRLEAEEEKLAHLPLAKTVSVEELLQYVELQDPVTRTQLRAMAAKTVCPPHKVELEALLEKQAYKEQVLAKRLTMLELLEKYPACEMKFSEFIALLPSIRPRYYSISSSPRVDEKQASITVSVVSGEAWSGYGEYKGIASNYLAELQEGDTITCFISTPQSEFTLPKDPETPLIMVGPGTGVAPFRGFVQARKQLKEQGQSLGEAHLYFGCRSPHEDYLYQEELENAQSEGIITLHTAFSRMPNQPKTYVQHVMEQDGKKLIELLDQGAHFYICGDGSQMAPAVEATLMKSYADVHQVSEADARLWLQQLEEKGRYAKDVWAG SEQ ID NO. 2 AIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEAPGWVTRYLSSQRLIKEACDESRFDKNLWQALKYLRDILGDGLG TSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLVQKWERLNADEHIEVPEDMTRLTLDTIGLCGFNYRFNSFYRDQPHPFITSTVRALDEAQNKQQRANPDDPAYDENKRQFQEDTKVMNDLVDKIIADRKASGEQSDDLLTHMLNGKDPETGEPLDDENIRYQIITFLIAGHETTSGLLSFALYFLVKNPHVLQKAAEEAARVLVDPVPSYKQVKQLKYVGMVLNEALRLWPTGPWFSLYAKEDTVLGGEYPLEKGDELMVLIPQLHRDKTIWGDDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLKHFDFEDHTNYELDIKETLTLKPEGFVVKAKSKKIPLGGIPSPSTEQSAKKVRKKAENAHNTPLLVLYGSNMGTAEGTARDLADIAMSKGFAPQVATLDSHAGNLPREGAVLIVTASYNGHPPDNAKQFVDWLDQASADEVKGVRYSVFGCGDKNWATTYQKVPAFIDETLAAKGAENIADRGEADASDDFEGTYEEWREHMWSDVAAYFNLDIENSEDNKSTLSLQFVDSAADMPLAKMHGAFSTNVVASKELQQPGSARSTRHLEIELPKEASYQEGDHLGVIPRNYEGIVNRVTARFGLDASQQIRLEAEEEKLAHLPLAKTVSVEELLQYVELQDPVTRTQLRAMAAKTVCPPHKVELEALLEKQAYKEQVLAKRLTMLELLEKYPACEMKFSEFIALLPSIRPRYYSISSSPRVDEKQASITVSVVSGEAWSGYGEYKGIASNYLAELQEGDTITCFISTPQSEFTLPKDPETPLIMVGPGTGVAPFRGFVQARKQLKEQGQSLGEAHLYFGCRSPHEDYLYQEELENAQSEGIITLHTAFSRMPNQPKTYVQHVMEQDGKKLIELLDQGAHFYICGDGSQMAPAVEATLMKSYADVHQVSEADARLWLQQLEEKGRYAKDVWAG SEQ ID NO. 3 AIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEAPGWVTRYLSSQRLIKEACDESRFDKNLWQALKYLRDILGDGLG T SWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLVQKWERLNADEHIEVPEDMTRLTLDTIGLCGFNYRFNSFYRDQPHPFITSTVRALDEAQNK LQRANPDDPAYDENKRQFQEDTKVMNDLVDKIIADRKASGEQSDDLLTHMLNGKDPETGEPLDDENIRYQIITFLIAGHETTSGLLSFALYFLVKNPHVLQKAAEEAARVLVDPVPSYKQVKQLKYVGMVLNEALRLWPTGPWFSLYAKEDTVLGGEYPLEKGDELMVLIPQLHRDKTIWGDDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLKHFDFEDHTNYELDIKETLTLKPEGFVVKAKSKKIPLGGIPSPSTEQSAKKVRKKAENAHNTPLLVLYGSNMGTAEGTARDLADIAMSKGFAPQVATLDSHAGNLPREGAVLIVTASYNGHPPDNAKQFVDWLDQASADEVKGVRYSVFGCGDKNWATTYQKVPAFIDETLAAKGAENIADRGEADASDDFEGTYEEWREHMWSDVAAYFNLDIENSEDNKSTLSLQFVDSAADMPLAKMHGAFSTNVVASKELQQPGSARSTRHLEIELPKEASYQEGDHLGVIPRNYEGIVNRVTARFGLDASQQIRLEAEEEKLAHLPLAKTVSVEELLQYVELQDPVTRTQLRAMAAKTVCPPHKVELEALLEKQAYKEQVLAKRLTMLELLEKYPACEMKFSEFIALLPSIRPRYYSISSSPRVDEKQASITVSVVSGEAWSGYGEYKGIASNYLAELQEGDTITCFISTPQSEFTLPKDPETPLIMVGPGTGVAPFRGFVQARKQLKEQGQSLGEAHLYFGCRSPHEDYLYQEELENAQSEGIITLHTAFSRMPNQPKTYVQHVMEQDGKKLIELLDQGAHFYICGDGSQMAPAVEATLMKSYADVHQVSEADARLWLQQLEEKGRYAKDVWAG SEQ ID NO. 4 AIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEAPGWVTRYLSSQRLIKEACDESRFDKNLWQALKYLRDILGDGL ALSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLVQKWERLNADEHIEVPEDMTRLTLDTIGLCGFNYRFNSFYRDQPHPFITSTVRALDEAQNKQQRANPDDPAYDENKRQFQEDTKVMNDLVDKIIADRKASGEQSDDLLTHMLNGKDPETGEPLDDENIRYQIITFLIAGHETTSGLLSFALYFLVKNPHVLQKAAEEAARVLVDPVPSYKQVKQLKYVGMVLNEALRLWPTGPWFSLYAKEDTVLGGEYPLEKGDELMVLIPQLHRDKTIWGDDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLKHFDFEDHTNYELDIKETLTLKPEGFVVKAKSKKIPLGGIPSPSTEQSAKKVRKKAENAHNTPLLVLYGSNMGTAEGTARDLADIAMSKGFAPQVATLDSHAGNLPREGAVLIVTASYNGHPPDNAKQFVDWLDQASADEVKGVRYSVFGCGDKNWATTYQKVPAFIDETLAAKGAENIADRGEADASDDFEGTYEEWREHMWSDVAAYFNLDIENSEDNKSTLSLQFVDSAADMPLAKMHGAFSTNVVASKELQQPGSARSTRHLEIELPKEASYQEGDHLGVIPRNYEGIVNRVTARFGLDASQQIRLEAEEEKLAHLPLAKTVSVEELLQYVELQDPVTRTQLRAMAAKTVCPPHKVELEALLEKQAYKEQVLAKRLTMLELLEKYPACEMKFSEFIALLPSIRPRYYSISSSPRVDEKQASITVSVVSGEAWSGYGEYKGIASNYLAELQEGDTITCFISTPQSEFTLPKDPETPLIMVGPGTGVAPFRGFVQARKQLKEQGQSLGEAHLYFGCRSPHEDYLYQEELENAQSEGIITLHTAFSRMPNQPKTYVQHVMEQDGKKLIELLDQGAHFYICGDGSQMAPAVEATLMKSYADVHQVSEADARLWLQQLEEKGRYAKDVWAG SEQ ID NO. 5 AIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEAPGWVTRYLSSQRLIKEACDESRFDKNLWQALKYLRDILGDGLG L SWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLVQKWERLNADEHIEVPEDMTRLTLDTIGLCGFNYRFNSFYRDQPHPFITSTVRALDEAQNKQQRANPDDPAYDENKRQFQEDTKVMNDLVDKIIADRKASGEQSDDLLTHMLNGKDPETGEPLDDENIRYQIITFLIAGHETTSGLLSFALYFLVKNPHVLQKAAEEAARVLVDPVPSYKQVKQLKYVGMVLNEALRLWPTGP LFSLYAKEDTVLGGEYPLEKGDELMVLIPQLHRDKTIWGDDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLKHFDFEDHTNYELDIKETLTLKPEGFVVKAKSKKIPLGGIPSPSTEQSAKKVRKKAENAHNTPLLVLYGSNMGTAEGTARDLADIAMSKGFAPQVATLDSHAGNLPREGAVLIVTASYNGHPPDNAKQFVDWLDQASADEVKGVRYSVFGCGDKNWATTYQKVPAFIDETLAAKGAENIADRGEADASDDFEGTYEEWREHMWSDVAAYFNLDIENSEDNKSTLSLQFVDSAADMPLAKMHGAFSTNVVASKELQQPGSARSTRHLEIELPKEASYQEGDHLGVIPRNYEGIVNRVTARFGLDASQQIRLEAEEEKLAHLPLAKTVSVEELLQYVELQDPVTRTQLRAMAAKTVCPPHKVELEALLEKQAYKEQVLAKRLTMLELLEKYPACEMKFSEFIALLPSIRPRYYSISSSPRVDEKQASITVSVVSGEAWSGYGEYKGIASNYLAELQEGDTITCFISTPQSEFTLPKDPETPLIMVGPGTGVAPFRGFVQARKQLKEQGQSLGEAHLYFGCRSPHEDYLYQEELENAQSEGIITLHTAFSRMPNQPKTYVQHVMEQDGKKLIELLDQGAHFYICGDGSQMAPAVEATLMKSYADVHQVSEADARLWLQQLEEKGRYAKDVWAG SEQ ID NO. 6 AIKEMPQPKTFGELKNLPLLNTDKPVQALMKIADELGEIFKFEAPGWVTRYLSSQRLIKEACDESRFDKNLWQALKYLRDILGDGL ALSWTHEKNWKKAHNILLPSFSQQAMKGYHAMMVDIAVQLVQKWERLNADEHIEVPEDMTRLTLDTIGLCGFNYRFNSFYRDQPHPFITSTVRALDEAQNKQQRANPDDPAYDENKRQFQEDTKVMNDLVDKIIADRKASGEQSDDLLTHMLNGKDPETGEPLDDENIRYQIITFLIAGHETTSGLLSFALYFLVKNPHVLQKAAEEAARVLVDPVPSYKQVKQLKYVGMVLNEALRLWPTGP L FSLYAKEDTVLGGEYPLEKGDELMVLIPQLHRDKTIWGDDVEEFRPERFENPSAIPQHAFKPFGNGQRACIGQQFALHEATLVLGMMLKHFDFEDHTNYELDIKETLTLKPEGFVVKAKSKKIPLGGIPSPSTEQSAKKVRKKAENAHNTPLLVLYGSNMGTAEGTARDLADIAMSKGFAPQVATLDSHAGNLPREGAVLIVTASYNGHPPDNAKQFVDWLDQASADEVKGVRYSVFGCGDKNWATTYQKVPAFIDETLAAKGAENIADRGEADASDDFEGTYEEWREHMWSDVAAYFNLDIENSEDNKSTLSLQFVDSAADMPLAKMHGAFSTNVVASKELQQPGSARSTRHLEIELPKEASYQEGDHLGVIPRNYEGIVNRVTARFGLDASQQIRLEAEEEKLAHLPLAKTVSVEELLQYVELQDPVTRTQLRAMAAKTVCPPHKVELEALLEKQAYKEQVLAKRLTMLELLEKYPACEMKFSEFIALLPSIRPRYYSISSSPRVDEKQASITVSVVSGEAWSGYGEYKGIASNYLAELQEGDTITCFISTPQSEFTLPKDPETPLIMVGPGTGVAPFRGFVQARKQLKEQGQSLGEAHLYFGCRSPHEDYLYQEELENAQSEGIITLHTAFSRMPNQPKTYVQHVMEQDGKKLIELLDQGAHFYICGDGSQMAPAVEATLMKSYADVHQVSEADARLWLQQLEEKGRYAKDVWAG Example 3: Characterization of the optimal mutant catalyzing 1, 4-androstadien- 17-one The mutant strain (G87A / S88L / W330L) induced to express in Example 2 was collected by centrifugation at 4000 r / min for 10 min, washed twice with 0.1 M potassium phosphate buffer (KPi, pH 7.0), and resuspended in a buffer containing 10% glycerol. Then, 1 mM of 1, 4-androstadien-17-one was added, and the catalytic reaction was carried out at 25°C and 220 r / min for 24 h. The reaction solution was extracted with an equal volume of ethyl acetate three times, and the combined organic phase was dried by rotary evaporation. HPLC analysis was performed using a WondaSil C18 column (5 μm x 250 mm x 4.6 mm) with acetonitrile-water (60:40, V / V) as the mobile phase (flow rate 0.8 mL / min, detection wavelength 254 nm, column temperature 40°C) (see Figure 2 、 3 and 4).

[0048] As can be seen from the above, the main product of the mutant catalyzing 1, 4-androstadien-17-one is C7β-hydroxy-1, 4-androstadien-17-one. Figure 2 Figure 3 and Figure 4 show the 1H NMR (DMSO-d, 400 MHz) and 13C NMR (DMSO-d, 400 MHz) spectra of C7β-hydroxy-1, 4-androstadien-17-one, respectively.

[0049] As can be seen from the above, the main product of the mutant catalyzing 1, 4-androstadien-17-one is C7β-hydroxy-1, 4-androstadien-17-one.

[0050] Example 4: Determination of selectivity and conversion rate of the mutant The six mutants obtained in Example 2 were induced to express according to the procedure described in Example 2. After the bacterial solution was collected and stored for 24 h, it was washed twice with 0.1 M potassium phosphate buffer (KPi, pH 7.0), resuspended in a buffer containing 10% glycerol, and 1 mM of 1, 4-androstadien-17-one (ADD) was added. The catalytic reaction was carried out at 25°C and 220 r / min for 24 h. The reaction solution was extracted with an equal volume of ethyl acetate three times, and the combined organic phase was dried by rotary evaporation. HPLC analysis was performed using a WondaSil C18 column (5 μm x 250 mm x 4.6 mm) with acetonitrile-water (60:40, V / V) as the mobile phase (flow rate 0.8 mL / min, detection wavelength 254 nm, column temperature 40°C). Meanwhile, the wild-type LG-23 was used as a control.

[0051] wherein the selectivity is defined as 7β-hydroxylation product / total product x 100%; and the conversion rate is defined as (initial substrate concentration - substrate concentration at the end of the reaction) / initial substrate concentration x 100%.​

[0052] Table 3. Conversion and selectivity results of mutants catalyzing the conversion of 1,4-androstadiene-3,17-dione

[0053] Note: Conv.: conversion, 1a, 1b, etc. represent the selectivity of the corresponding product as shown. Figure 1

[0054] From Table 3, it can be seen that among the 6 mutants, 5 can increase the conversion of LG-23 by 24.77%~31.75%. At the same time, all mutants significantly increase the conversion of C7β-hydroxylation of 1,4-androstadiene-3,17-dione, with an increase of 68.86%~103.95%.

[0055] At the same time, according to the above description, the substrate is replaced with testosterone (TES), nandrolone (NAL) or 4-androstenedione (4AD), and the conversion and selectivity results of each mutant catalyzing different substrates (see Tables 4-6).

[0056] Table 4. Conversion and selectivity results of mutants

[0057] Table 5. Conversion and selectivity results of mutants

[0058] Table 6. Conversion and selectivity results of mutants

[0059] Example 5 ​In order to further provide a reference for the industrial development of the enzyme in the later stage, the yield of the reaction catalyzed by the steroid substrate in the whole-cell catalytic system (Whole-Cell), the crude enzyme extraction system of cell disruption (Crude Enzyme), the whole-cell catalytic reaction after the substrate and hydroxypropyl-β-cyclodextrin (HP-β-CD) form an inclusion body (Whole-Cell / HP-β-CD), and the crude enzyme extraction system after the substrate and hydroxypropyl-β-cyclodextrin (HP-β-CD) form an inclusion body (Crude Enzyme / HP-β-CD) is compared. Specifically, the establishment of the whole-cell catalytic system and the substrate catalytic process are as described in Embodiments 2 and 3. The crude enzyme extraction system of cell disruption is that the bacteria are resuspended according to Embodiments 2 and 3, and then the resuspended bacteria liquid is ultrasonically broken (operation power 40%, operation 3 s intermittently 5 s, total time 12 min) to release the catalytic enzyme from the cells, and then the substrate is added for catalysis. The whole-cell catalytic reaction after the substrate and hydroxypropyl-β-cyclodextrin (HP-β-CD) form an inclusion body is that HP-β-CD and the substrate are dissolved in an appropriate amount of water in a proportion (5:1, W / W) to obtain a substrate final concentration of 100 mM, 121 ℃ moist heat sterilization for 20 min, 0.22 μm filter membrane is used to remove insoluble substances, and then the substrate concentration in the cyclodextrin substrate solution is detected by HPLC according to the substrate standard curve to make it the same as the substrate final concentration in other systems, and then the reaction is carried out in the whole-cell catalytic system according to Embodiments 2 and 3. The crude enzyme extraction system after the substrate and hydroxypropyl-β-cyclodextrin (HP-β-CD) form an inclusion body is that the cyclodextrin substrate solution is prepared in the same way, and then the reaction is carried out in the crude enzyme extraction system of cell disruption. All the reaction conditions of the systems are the same.

[0060] By Figure 6 It can be seen that the whole-cell catalytic reaction after the substrate and hydroxypropyl-β-cyclodextrin (HP-β-CD) form an inclusion body can obtain the best effect.

[0061] The mutant of the application can improve the selectivity of the 7β-hydroxylated product of the steroid compound alone, can improve the conversion rate of the 7β-hydroxylated product of the steroid compound alone, or can improve the selectivity and yield of the 7β-hydroxylated product of the steroid compound. Thus, the present embodiment overcomes the defects of low conversion rate and / or poor selectivity in the 7β-hydroxylated reaction of the template (LG-23) catalyzing the steroid compound in the prior art.

Claims

1. A multi-site mutant of monooxygenase P450BM3, characterized in that, The mutant is a mutation of one or more amino acid residues at positions 87, 88, 188 and 330 in the amino acid sequence of wild-type P450BM3-LG-23 SEQ ID NO.7; the amino acid residues at each position may be mutated to L, T or A in the same or different ways.

2. The monooxygenase P450BM3 multisite mutant according to claim 1, characterized in that, The mutant is the amino acid sequence of wild-type P450BM3-LG-23 in SEQ ID NO.

7. The 87th amino acid residue is mutated to A; The 88th amino acid residue is mutated to L or T; The 188th amino acid residue is mutated to L; The 330th amino acid residue is mutated to L.

3. The monooxygenase P450BM3 multisite mutant according to claim 1 or 2, characterized in that, The mutants are S88L, S88T, S88T / Q188L, G87A / S88L, S88L / W330L or G87A / S88L / W330L, wherein the amino acid sequences of each mutant are shown in SEQ ID NO.1-6 respectively.

4. A recombinant expression plasmid, characterized in that: The recombinant expression plasmid contains the mutant described in claim 1.

5. A genetically engineered bacterium, characterized in that: The genetically engineered bacteria contain the recombinant expression plasmid as described in claim 4.

6. The application of the mutant of claim 1, the recombinant expression plasmid of claim 4, or the genetically engineered bacterium of claim 5, characterized in that: The application of the mutants, recombinant expression plasmids, or genetically engineered bacteria in the catalytic production of steroidal compound product C7β.

7. The application according to claim 6, characterized in that: The steroidal compound is one or more of testosterone (TES), nandrolone (NAL), 4-androstenedione (4AD), and 1,4-androstenedione (ADD).

8. A method for catalytically generating C7β products from steroidal compounds, characterized in that, Using steroidal compounds as substrates, the mutants of claim 1, the recombinant expression plasmids of claim 4, or the genetically engineered bacteria of claim 5 are used to catalyze a reaction to generate the corresponding C7β product.

9. The method for catalytically generating C7β products from steroidal compounds according to claim 8, characterized in that, Using a recombinant strain containing the mutant described in claim 1 as a catalyst, the substrate was modified by C7β hydroxylation under the conditions of pH 7.0, temperature 25°C, and reaction time 24 hours.

10. The method for catalytically generating C7β products from steroidal compounds according to claim 9, characterized in that, The substrate is formed by combining the substrate with hydroxypropyl-β-cyclodextrin (HP-β-CD) to form an inclusion complex, wherein the molar ratio of HP-β-CD to the substrate is 1:1 to 5:1.

Images