P450 enzyme mutant, encoding gene and application thereof

Abstract

The application discloses a P450 enzyme mutant, a coding gene and application thereof in preparation of cortisone, and belongs to the technical field of bioengineering. The P450 enzyme mutant is obtained by amino acid mutation of CYP11B1 from Homo sapiens with an amino acid sequence shown as SEQ ID NO. 1, and the amino acid mutation site is at least one of S146V, H331D and L440F. The P450 enzyme mutant provided by the application has a higher soluble expression level in Escherichia coli than wild-type CYP11B1, the catalytic level of the constructed recombinant expression strain is higher, 11-deoxycortisol can be used as a substrate to prepare cortisone, the yield of the product is high, no by-product is generated, and the P450 enzyme mutant has a good industrial application prospect.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a P450 enzyme mutant modified from Homo sapiens CYP11B1, its encoding gene, and its application in the preparation of cortisol. Background Technology

[0002] Cortisol is an adrenocortical hormone extracted from the adrenal cortex that has the strongest effect on carbohydrate metabolism. It belongs to the class of glucocorticoids and plays a crucial role in stress adaptation, energy mobilization, and the regulation of immune responses. Besides being used as an anti-inflammatory drug and immunosuppressant, cortisol is also used as an intermediate in the synthesis of other glucocorticoids, such as prednisone and prednisolone.

[0003] Cortisol is produced from 11-deoxycortisol by the action of 11β-hydroxylase in the mitochondria of the adrenal cortex. The total chemical synthesis of cortisol involves approximately 40 steps and has a low yield (Woodward R, Sondheimer F, Taub D, et al. The total synthesis of steroids[J]. Journal of the American Chemical Society, 1952, 74(17):4223-4251.). In current industrial production, a semi-synthetic method starting from naturally occurring sterols is mainly adopted, including a multi-step chemical process in the early stage and a final step using the microbial transformation of 11-deoxycortisol or its 21-acetate from a culture of Curvularia or Absidia fungi to produce cortisol (Yang Shunkai. Steroid microbial transformation: Robustness of biotransformation of glucocorticoid hydrocortisone and its research application[J]. Journal of Applied and Environmental Biology, 2022, 28(01):230-238.). However, the selectivity of fungal catalysis is poor, and it is often accompanied by the generation of 11α-OH or 14α-OH byproducts. This requires more cost in the downstream separation steps, and the cultivation and transformation time of fungi is relatively long.

[0004] In mammals, cortisol is primarily synthesized in the adrenal glands, with the final step, 11β-hydroxylation, performed by the mitochondrial P450 enzyme CYP11B1. Based on the specific hydroxylation function of CYP11B1, a recombinant Saccharomyces cerevisiae strain has been constructed using human CYP11B1 to synthesize cortisol de novo, but the achieved productivity and potency are low (1.64 mg·L⁻¹, respectively). -1 ·d -1 and 11.5 mg·L -1(Szczebara FM, Chandelier C, Villeret C, et al. Total biosynthesis of hydrocortisone from a simple carbon source in yeast[J]. Nature Biotechnology, 2003, 21(2):143-149.). Co-expression of human CYP11B1 with the complete electron transfer chain of human cortical ferrugin (Adx) and human cortical ferrugin reductase (AdR) in *Schizosaccharomyces cerevisiae* can produce 1 mM cortisol (approximately 120.8 mg·L⁻¹) within 72 hours. -1 ·d -1 (Hakki T, Zearo S, CA, et al. Coexpression of redox partners increases the hydrocortisone (cortisol) production efficiency in CYP11B1 expressing fission yeast Schizosaccharomycespombe[J]. Journal of Biotechnology, 2008, 133(3):351-359.). These cortisol yields are still far from meeting industrial requirements; therefore, developing high-activity CYP11B1-based biocatalysts for cortisol synthesis is a problem that needs to be solved by those skilled in the art. Summary of the Invention

[0005] The purpose of this invention is to provide a cytochrome P450 enzyme with high catalytic activity and stereoselectivity for the preparation of cortisol, meeting the requirements of industrial production.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] This invention utilizes enzyme molecular engineering to modify the cytochrome P450 enzyme CYP11B1 derived from Homo sapiens, resulting in a P450 enzyme mutant with significantly enhanced catalytic activity. Specifically, the P450 enzyme mutant is a mutant obtained by amino acid mutation of Homo sapiens-derived CYP11B1, as shown in SEQ ID NO. 1. The mutation sites are at least one of positions 146, 331, and 440, with serine at position 146 mutated to valine, histidine at position 331 mutated to aspartic acid, and leucine at position 440 mutated to phenylalanine.

[0008] Specifically, the mutant CYP11B1-S146V, in which serine at position 146 is mutated to valine, has the amino acid sequence shown in SEQ ID NO.2;

[0009] The mutant CYP11B1-H331D, in which histidine at position 331 is mutated to aspartic acid, has the amino acid sequence shown in SEQ ID NO.3.

[0010] The mutant CYP11B1-L440F, in which leucine at position 440 is mutated to phenylalanine, has the amino acid sequence shown in SEQ ID NO.4.

[0011] The mutant CYP11B1-S146V / H331D, in which serine at position 146 is mutated to valine and histidine at position 331 is mutated to aspartic acid, has the amino acid sequence shown in SEQ ID NO.5.

[0012] The mutant CYP11B1-S146V / L440F, in which serine at position 146 is mutated to valine and leucine at position 440 is mutated to phenylalanine, has the amino acid sequence shown in SEQ ID NO.6.

[0013] The mutant CYP11B1-H331D / L440F, in which histidine at position 331 is mutated to aspartic acid and leucine at position 440 is mutated to phenylalanine, has the amino acid sequence shown in SEQ ID NO.7.

[0014] The mutant CYP11B1-S146V / H331D / L440F, in which serine at position 146 is mutated to valine, histidine at position 331 is mutated to aspartic acid, and leucine at position 440 is mutated to phenylalanine, has the amino acid sequence shown in SEQ ID NO.8.

[0015] Studies have shown that, compared to wild-type CYP11B1, the above-mentioned P450 enzyme mutants have significantly increased soluble expression levels in Escherichia coli, and the whole-cell catalytic activity of the genetically engineered bacteria has been significantly enhanced.

[0016] Conserved substitutions, additions or deletions of one or more amino acids, and amino-terminal truncation of other amino acid sites in the P450 enzyme mutant are also included within the scope of this invention.

[0017] To obtain the P450 enzyme mutant of the present invention, the coding gene of CYP11B1 from Homo sapiens (nucleotide sequence as shown in SEQ ID NO.9) can be mutated at a specific site, and the P450 enzyme mutant can be obtained after gene expression.

[0018] The present invention also provides a coding gene for encoding the P450 enzyme mutant, which is obtained by mutating the codons encoding the corresponding amino acids based on the nucleotide sequence shown in SEQ ID NO.9. Specifically, S146V is obtained by mutating the codon TCT encoding serine at position 146 to the codon GTG encoding valine; H331D is obtained by mutating the codon CAT encoding histidine at position 331 to the codon GAT encoding aspartic acid; and L440F is obtained by mutating the codon TTA encoding leucine at position 440 to the codon TTT encoding phenylalanine.

[0019] The present invention also provides a recombinant expression vector comprising a coding gene encoding the amino acid sequence of the aforementioned P450 enzyme mutant. Preferably, the recombinant expression vector uses pET-17b as the vector plasmid.

[0020] Furthermore, the recombinant expression vector also includes the coding genes for adrenodoxin reductase (AdR) and adrenodoxin (Adx). AdR and Adx are responsible for transferring the redox equivalents required for P450 oxidation from NAD(P)H to the P450 enzyme. This invention clones the AdR and Adx coding genes in a recombinant expression vector and co-expresses them with the P450 enzyme mutant coding gene, acting as redox chaperones to transfer the electrons required for the hydroxylation reaction involving CYP11B1.

[0021] Preferably, the redox chaperone is bovine AdR and bovine Adx, the amino acid sequence and encoding gene of AdR are shown in SEQ ID NO.10 and SEQ ID NO.11, respectively, and the amino acid sequence and encoding gene of Adx are shown in SEQ ID NO.12 and SEQ ID NO.13, respectively.

[0022] Preferably, the recombinant expression vector contains an RBS nucleotide sequence as shown in SEQ ID NO.14 inserted upstream of the corticosteroid reductase encoding gene, and an RBS nucleotide sequence as shown in SEQ ID NO.15 inserted upstream of the corticosteroid reductase encoding gene.

[0023] The present invention also provides a genetically engineered bacterium comprising the recombinant expression vector, the genetically engineered bacterium being used to produce the P450 enzyme mutant. The recombinant vector is transformed into host cells to obtain the recombinant genetically engineered bacterium. The host cells can be various conventional host cells in the art; preferably, the host bacterium for the genetically engineered bacterium is *Escherichia coli* C43(DE3).

[0024] Preferably, the genetically engineered bacteria further comprises a pGro7 plasmid expressing the molecular chaperone GroEL / ES. Chaperone proteins help eukaryotic membrane proteins in prokaryotic hosts to fold correctly after translation, ensuring the active expression of CYP11B1 and AdR.

[0025] The present invention also provides a method for constructing the P450 enzyme mutant, the method comprising the following steps:

[0026] (1) Design site-directed mutagenesis primers, and use a plasmid containing a gene fragment with a nucleotide sequence as shown in SEQ ID NO.9 as a template to perform reverse PCR to obtain a single point mutation product in CYP11B1 where S at position 146 is mutated to V, H at position 331 is mutated to D, or L at position 440 is mutated to F.

[0027] (2) Using the single-site mutation product as a template, reverse PCR is performed using the site-directed mutation primers to obtain the double-site mutation product; using the double-site mutation product as a template, reverse PCR is performed using the site-directed mutation primers to obtain the triple-site mutation product.

[0028] (3) The single point mutation product, double point mutation product or triple point mutation product is introduced into the host bacteria, and the P450 enzyme mutant expression strain is screened and induced to express to obtain the P450 enzyme mutant.

[0029] The primer required for the S-to-V mutation at position 146 is:

[0030] S146V-F: 5'-TTGCTCCGGATTTTGTGCAGGCACTGAAAAAAAAAGTGC-3';

[0031] S146V-R: 5'-TTTTTTTTCAGTGCCTGCACAAAATCGCGAGCAACGGC-3';

[0032] Primer required for the H-to-D mutation at position 331:

[0033] H331D-F: 5'-GTATTAGCGAAGATCCACAGAAAGCTACGAC-3';

[0034] H331D-R: 5'-GTCGTAGCTTTCTGTGGATCTTCGCTAATAC-3';

[0035] Primer required for the L-to-F mutation at position 440:

[0036] L440F-F: 5'-AGAAATGCTGTTATTTTTACATCATGTGCTG-3';

[0037] L440F-R: 5'-CAGCACATGATGTAAAAATAACAGCATTTCT-3'.

[0038] Preferably, the plasmid containing the gene fragment with nucleotide sequences as shown in SEQ ID NO. 9 also contains cloned gene fragments with nucleotide sequences as shown in SEQ ID NO. 11 and SEQ ID NO. 13.

[0039] Preferably, the original vector for the recombinant plasmid is pET-17b; the host bacterium is Escherichia coli C43(DE3).

[0040] Another object of the present invention is to provide the application of the P450 enzyme mutant in the preparation of cortisol, the application comprising: the P450 enzyme mutant catalyzing the 11β-hydroxylation of 11-deoxycortisol to cortisol under the combined action of corticosteroid reductase and corticosteroid.

[0041] The P450 enzyme mutant provided by this invention can catalyze the 11β-hydroxylation of 11-deoxycortisol under the combined action of redox chaperones AdR / Adx, generating cortisol with high optical purity (ee > 99%), and has good prospects for industrial application.

[0042] Furthermore, the application includes: using wet bacterial cells obtained by centrifugation after fermentation of genetically engineered bacteria containing recombinant expression plasmids encoding P450 enzyme mutant genes, AdR and Adx genes, and wet bacterial cells immobilized as catalysts, 11-deoxycortisol as substrate, and a buffer solution with a pH ≤ 8 containing a co-solvent as the reaction medium, reacting at 25-37℃ and 150-300 rpm. After the reaction is completed, the reaction solution is separated and purified to obtain cortisol.

[0043] Preferably, the host bacterium of the genetically engineered bacteria is Escherichia coli, and the host bacterium contains a pGro7 plasmid expressing the molecular chaperone GroEL / ES and a recombinant expression plasmid containing the P450 enzyme mutant encoding gene, the AdR encoding gene, and the Adx encoding gene.

[0044] Preferably, in the reaction system, the amount of catalyst used is 25-100 g / L based on the weight of wet bacterial cells, wherein the water content of the wet bacterial cells is 70-90% by mass. More preferably, it is 25 g / L.

[0045] Preferably, the concentration of the substrate in the reaction system is 3-10 mM, more preferably 10 mM.

[0046] Preferably, the pH buffer solution is a potassium phosphate buffer, i.e., a KH₂PO₄-K₂HPO₄ buffer, with a concentration of 50 mM and a pH of 7.4, containing 0.1-1 mM isopropyl-β-D-thiogalactoside (IPTG), 1-5 mg / mL arabinose (L-ara), 0.1-1.5 mM δ-aminolevulinic acid (δ-ALA), 0-200 μg / mL ampicillin, and 1-5% glycerol. More preferably, it contains 1 mM IPTG, 4 mg / mL L-ara, 1 mM δ-ALA, 50 μg / mL ampicillin, and 2% glycerol.

[0047] Preferably, the co-solvent is methyl-β-cyclodextrin or (2-hydroxypropyl)-γ-cyclodextrin, and the molar ratio of cyclodextrin to substrate in the pH buffer solution is 3 to 5:1, more preferably methyl-β-cyclodextrin with a molar ratio of 3:1 to substrate.

[0048] The concentrations of the above-mentioned raw materials, such as wet bacterial cells, substrates, and cyclodextrin, are all calculated based on a final volume of 1L of pH buffer.

[0049] Preferably, the reaction temperature is 27.5℃.

[0050] The reaction time is 12-32 hours, preferably 24 hours.

[0051] Preferably, the oscillation rate is 170 rpm.

[0052] Preferably, the wet bacterial cell is E. coli C43(DE3) / pET-17b-CYP11B1-S146V / H331D / L440F. This mutant can achieve a cortisol yield of 2756.3 mg·L⁻¹. -1 ·d -1 It is 1.87 times that of wild-type CYP11B1 under the same catalytic conditions, and the optical purity ee is 99%.

[0053] The fermentation culture method was as follows: the recombinant engineered bacteria were inoculated into TB medium containing ampicillin (final concentration 100 μg / mL) and chloramphenicol (final concentration 20 μg / mL) (with potassium phosphate concentration of 0.017 M KH2PO4 + 0.072 M K2HPO4), and cultured at 37℃ and 220 rpm until the bacterial cell concentration reached OD500. 600 When the concentration reaches approximately 1.0, add 1 mM IPTG, 4 mg / mL L-ara, 1 mM δ-ALA, and 50 μg / mL ampicillin. Induce culture at 30°C and 200 rpm for 21 h, and collect bacterial cells by centrifugation at 4°C and 3500 rpm for 10 min.

[0054] The beneficial effects of this invention are as follows:

[0055] (1) The P450 enzyme mutant provided by this invention has a higher soluble expression level in Escherichia coli than wild-type CYP11B1. The constructed recombinant expression strain has a higher catalytic level and maintains specific stereoselectivity. It can prepare cortisol with 11-deoxycortisol as a substrate, with high product yield and no by-products.

[0056] (2) This invention utilizes whole cells expressing P450 enzyme mutants as biocatalysts for the hydroxylation of steroidal drug intermediates, making it more economical and convenient to obtain high optical purity chiral products. The production method has the advantages of simple operation and low cost, which greatly reduces the production cost and has a good prospect for industrial application. Attached Figure Description

[0057] Figure 1 This is a plasmid map of the recombinant mutant.

[0058] Figure 2 To compare the relative viability of recombinant strains constructed using RBS sequences of different intensities.

[0059] Figure 3 This study compares the relative viability of wild-type CYP11B1 engineered bacteria under different induction expression temperature conditions.

[0060] Figure 4 Wild-type CYP11B1 engineered bacteria cultured to different OD values 600 Comparison of relative vitality under different conditions.

[0061] Figure 5 This is a color comparison image of the bacterial cells harvested after the culture of some recombinant mutant strains.

[0062] Figure 6 The effect of different co-solvents on the catalytic activity of wild-type CYP11B1 engineered bacteria in the reaction system was investigated.

[0063] Figure 7 The effect of substrate concentration in the reaction system on the catalytic activity of wild-type CYP11B1 engineered bacteria was investigated.

[0064] Figure 8 The liquid chromatograms are of a mixture of standard cortisol and substrate 11-deoxycortisol (Standard), as well as blank control of E. coli C43(DE3) (Blank), CYP11B1 and its mutant (S146V) and the product obtained by reacting with substrate 11-deoxycortisol. Detailed Implementation

[0065] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0066] Unless otherwise specified, all raw materials used in this invention are commercially available or commonly used in the art. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art. Nucleic acids are written from left to right in a 5' to 3' direction, and amino acid sequences are written from left to right in a direction from the amino terminus to the carboxyl terminus.

[0067] Example 1: Construction of engineered bacteria capable of expressing various mutants

[0068] 1. Based on the amino acid sequence of the P15538 protein (Homo sapiens-derived CYP11B1, www.uniprot.org / uniprotkb / P15538 / entry) in the UniProt database, the corresponding coding gene was synthesized by Shanghai Jierui Biotechnology Co., Ltd. The first 27 amino acids at the N-terminus of the P15538 amino acid sequence were replaced with MATK, i.e., the N-terminal sequence was changed from MALRAKAEVCMAVPWLSLQRAQALGTR to MATK. The amino acid sequence of the wild-type Homo sapiens-derived CYP11B1 is shown in SEQ ID NO.1, and the nucleotide sequence of the coding gene is shown in SEQ ID NO.9.

[0069] The coding genes for bovine AdR (P00257, https: / / www.uniprot.org / uniprotkb / P00257 / entry) and Adx (P08165, https: / / www.uniprot.org / uniprotkb / P08165 / entry) were synthesized by Shanghai Jierui Biotechnology Co., Ltd. The amino acid sequence of AdR and the nucleotide sequence of its coding gene are shown in SEQ ID NO. 10 and SEQ ID NO. 11, respectively, and the amino acid sequence of Adx and the nucleotide sequence of its coding gene are shown in SEQ ID NO. 12 and SEQ ID NO. 13, respectively.

[0070] Initial RBS sequences for each gene were predicted using RBS design software (https: / / www.denovodna.com / ). The RBS sequence for CYP11B1 was fixed as the sequence inherent on the expression plasmid. The initial RBS intensities for CYP11B1, AdR, and Adx were 8400au, 5000au, and 20000au, respectively. RBS sequences of different intensities were designed for AdR and Adx. The RBS intensities for AdR were designed to be 10000au, 50000au, and 100000au, respectively, and the RBS intensities for Adx were designed to be 20000au, 80000au, 150000au, and 430000au, respectively. Primers were designed based on the specific RBS sequences, as shown in Table 1.

[0071] Table 1. RBS sequences of different intensities encoding AdR and Adx genes

[0072]

[0073]

[0074] Note: The RBS base sequence portion is indicated by underscores.

[0075] Using the synthesized AdR gene fragment and Adx gene fragment as templates, respectively, PCR amplification was performed using corresponding primer pairs to obtain gene fragments with different RBS sequences.

[0076] Then, using plasmid pET-17b as a vector, a recombinant plasmid pET-17b containing the CYP11B1 encoding gene, AdR encoding gene, and Adx encoding gene was constructed through PCR, double enzyme digestion, and in vitro ligation. Figure 1 The recombinant plasmid was transformed into E. coli C43(DE3) to obtain the wild-type CYP11B1 recombinant strain. The recombinant strain was activated on LB agar plates containing 100 μg / mL ampicillin resistance and cultured at 37°C for 12 h. Single colonies were picked and cultured in 5 mL LB tubes containing 100 μg / mL ampicillin resistance and cultured at 37°C and 220 rpm for about 12 h. The plasmid was extracted according to the instructions of the plasmid miniprep kit.

[0077] The correctly sequenced recombinant plasmid pET-17b and the pGro7 plasmid expressing the molecular chaperone GroEL / ES were co-transformed into *E. coli* C43(DE3) to obtain an engineered recombinant strain. This recombinant strain was activated on LB agar plates containing 100 μg / mL ampicillin-resistant and 20 μg / mL chloramphenicol-resistant plasmids and cultured at 37°C for 12 h. Single colonies were picked and placed in 5 mL LB tubes containing the same 100 μg / mL ampicillin-resistant and 20 μg / mL chloramphenicol-resistant plasmids and cultured at 37°C and 220 rpm for approximately 12 h. Then, a 1% inoculum (v / v) was inoculated into 50 mL TB medium containing 100 μg / mL ampicillin and 20 μg / mL chloramphenicol (with a potassium phosphate concentration of 0.034 M KH₂PO₄ + 0.144 M K₂HPO₄) and cultured at 37°C and 220 rpm until the bacterial concentration reached OD₀. 600 When the concentration reaches approximately 0.5, add IPTG to a final concentration of 1 mM, L-ara at 4 mg / mL, δ-ALA at 1 mM, and ampicillin at 50 μg / mL. Induce culture at 27.5℃ and 200 rpm for 21 h. Collect bacterial cells by centrifugation at 4℃ and 3500 rpm for 10 min to obtain wet bacterial cells of engineered bacteria expressing wild-type enzymes.

[0078] The results are as follows Figure 2 As shown, when the RBS intensities of AdR and Adx were 50,000 au and 80,000 au, respectively (i.e., the RBS sequences are as shown in SEQ ID NO.14 and SEQ ID NO.15), the whole-cell catalytic activity reached its highest level, which was 18.6% higher than that of the original strain (Ori). The space yield for cortisol production was increased to 407.0 mg·L⁻¹. -1 ·d -1 The plasmids constructed under these conditions were used in subsequent experiments.

[0079] 2. Construction of CYP11B1 mutants at points 146, 331, and 440

[0080] Using the plasmid constructed in step 1 as a template, the mutant plasmid was constructed using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent, United States). Single point mutations were performed on serine (S) at position 146, histidine (H) at position 331, and leucine (L) at position 440 in the wild-type amino acid sequence. Corresponding primers were designed, as shown in Table 2.

[0081] Table 2. Mutant Primers

[0082]

[0083] The constructed mutant plasmid was transformed into E. coli C43(DE3) competent cells, mixed well, and placed on ice for 15 min. After that, the E. coli C43(DE3) competent cells were heat-shocked at 42℃ for 90 s and then placed on ice for 5 min. Then, 1 mL of LB medium was added and cultured at 37℃ for 50 min. After that, the cells were centrifuged at 12000 rpm for 1 min, and 100 μL of the supernatant was taken to resuspend the cells. The cells were then spread on LB plates containing 100 μg / mL ampicillin and cultured at 37℃ for 12 h.

[0084] Pick a single colony from the plate and place it in a test tube containing 200 μL of LB medium. After incubating for 8 hours, take 100 μL of the culture for sequencing. If the sequencing results are correct, add an equal volume of 40% glycerol solution to the remaining bacterial culture and store it in a -80℃ freezer for later use.

[0085] The CYP11B1 mutant engineered bacteria E. coli C43(DE3) / pET-17b-CYP11B1-S146V, E. coli C43(DE3) / pET-17b-CYP11B1-H331D, and E. coli C43(DE3) / pET-17b-CYP11B1-L440F were obtained, respectively. Sequencing results showed that the codon TCT encoding serine (S) at position 146 was mutated to the codon GTG encoding valine (V); the codon CAT encoding histidine (H) at position 331 was mutated to the codon GAT encoding aspartic acid (D); and the codon TTA encoding leucine (L) at position 440 was mutated to the codon TTT encoding phenylalanine (F). The amino acid sequences of mutants S146V, H331D, and L440F are SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4, respectively.

[0086] 3. Construction of CYP11B1 combinatorial mutants

[0087] Using pET-17b-CYP11B1-S146V and pET-17b-CYP11B1-H331D constructed in step 2 as templates, mutant plasmids were constructed using a point mutation kit, following the same method as above. Sequencing results correctly yielded the CYP11B1 mutant engineered bacteria E. coli C43(DE3) / pET-17b-CYP11B1-S146V / H331D, E. coli C43(DE3) / pET-17b-CYP11B1-S146V / L440F, and E. coli C43(DE3) / pET-17b-CYP11B1-H331D / L440F, with corresponding amino acid sequences of SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO.7.

[0088] Using the plasmid described above as a template, the mutant plasmid was constructed using a point mutation kit, following the same method as above. Sequencing results correctly yielded the CYP11B1 mutant engineered E. coli C43(DE3) / pET-17b-CYP11B1-S146V / H331D / L440F, with the corresponding amino acid sequence SEQ ID NO. 8.

[0089] Example 2: Induced expression of various mutants

[0090] 1. Optimization of cultivation conditions

[0091] (1) Following the cell culture conditions of Example 1, the effects of different induction temperatures (30℃, 27.5℃, 24℃, 20℃, and 17℃) on the wild-type CYP11B1 engineered bacteria were compared. The results are as follows: Figure 3 As shown, the strain cultured at 30℃ exhibited the best catalytic effect, with a relative activity of 118.4%.

[0092] (2) Culture medium composition: When the concentration of potassium phosphate in the TB culture medium in Example 1 was adjusted to 0.017M KH2PO4 + 0.072M K2HPO4, the catalytic effect of the cultured strain was better, with a relative activity of 167.1%.

[0093] (3) Comparison of different induced OD (OD 600 =0.5, 0.6, 0.7, 0.8, 1.0, 1.2), the results are as follows Figure 4 As shown, where in OD 600 The strain cultured after induction at a concentration of 1.0 showed the best catalytic effect, with a relative activity of 132.7%.

[0094] The optimized single-factor levels were combined as follows: (30℃ + TB medium (0.017M KH2PO4 + 0.072M K2HPO4) + OD) 600=1.0), the whole-cell catalytic activity of the cultured strain increased from 407.0 mg·L⁻¹. -1 ·d -1 Increased to 584.2 mg·L -1 ·d -1 .

[0095] 2. The engineered bacteria carrying the gene expression plasmids of each mutant constructed in Example 1 were inoculated into 5 mL LB liquid medium tubes containing 100 μg / mL ampicillin and cultured at 37°C for 12 h. The plasmids were then extracted according to the instructions of the plasmid miniprep kit.

[0096] The pET-17b plasmid carrying the coding genes of each mutant was co-transformed with the pGro7 plasmid expressing the molecular chaperone GroEL / ES into *E. coli* C43(DE3) to obtain an engineered recombinant strain. This recombinant strain was activated on LB agar plates containing 100 μg / mL ampicillin resistance and 20 μg / mL chloramphenicol resistance, and cultured at 37°C for 12 h. Single colonies were picked and placed in 5 mL LB tubes containing the same 100 μg / mL ampicillin resistance and 20 μg / mL chloramphenicol resistance, and cultured at 37°C and 220 rpm for approximately 12 h. Then, a 1% inoculum (v / v) was inoculated into 50 mL TB medium (0.017 M KH₂PO₄ + 0.072 M K₂HPO₄) containing 100 μg / mL ampicillin and 20 μg / mL chloramphenicol, and cultured at 37°C and 220 rpm until the bacterial concentration reached OD₀. 600 When the concentration reaches approximately 1.0, add 1 mM IPTG, 4 mg / mL L-ara, 1 mM δ-ALA, and 50 μg / mL ampicillin. Induce culture at 30°C and 200 rpm for 21 h. Collect bacterial cells by centrifugation at 4°C and 3500 rpm for 10 min to obtain wet bacterial cells of the engineered bacteria expressing the mutant.

[0097] Experimental results analysis: Compared with wild type (WT), the engineered strain expressing the mutant was redder (e.g., Figure 5 This is a significant characteristic of increased expression of active P450, indicating that the Homo sapiens-derived CYP11B1 mutant provided by this invention has a higher soluble expression level.

[0098] Example 3: Preparation of cortisol from various mutants

[0099] 1. Optimization of whole-cell reaction conditions

[0100] (1) Cosolvent

[0101] A 50 mM dimethyl sulfoxide (DMSO) solution containing 11-deoxycortisol was prepared as the substrate stock solution. The wild-type wet cells constructed in Example 1 were resuspended in potassium phosphate buffer, and the substrate stock solution and stock solutions of IPTG, L-ara, δ-ALA, and ampicillin were added to achieve a final wet cell concentration of 25 g / L and a final substrate concentration of 3 mM. This was prepared as the reaction system solution and incubated at 27.5 °C and 170 rpm for 24 h in a constant temperature shaker. After the reaction, the cells were extracted with ethyl acetate and centrifuged, then rotary evaporated under vacuum at 35 °C for 1 h, and subsequently reconstituted with the liquid mobile phase (acetonitrile).

[0102] Methyl-β-cyclodextrin (M-β-CD) and (2-hydroxypropyl)-γ-cyclodextrin [(2-OH)-γ-CD] were prepared as stock solutions at molar ratios of 3:1, 4:1, and 5:1 with the substrate, respectively. These were added to the reaction system at a volume ratio of 10%, with a final substrate concentration of 3 mM for all solutions.

[0103] like Figure 6 As shown, the application of both types of CDs significantly improved catalytic activity. Among them, M-β-CD showed the best effect when added at a low molar ratio (3:1), with a relative activity increase of 51.0% and a space-time yield of 881.9 mg·L⁻¹. -1 ·d -1 When (2-OH)-γ-CD was added at a molar ratio of 4:1, the relative activity increased by 71.8%, and the space-time yield reached 1003.4 mg·L⁻¹. -1 ·d -1 Given that M-β-CD is significantly cheaper than (2-OH)-γ-CD, M-β-CD (3:1 molar ratio) was chosen as the cosolvent for the whole-cell reaction system in subsequent experiments.

[0104] (2) Substrate concentration

[0105] The substrate supply amounts were 3 mM, 5 mM, 7 mM, and 10 mM, respectively. Figure 7 As shown, the cortisol yield increased significantly with increasing substrate concentration, reaching a space-time yield of 1470.0 mg·L⁻¹ at a substrate concentration of 10 mM. -1 ·d -1 This concentration was then fixed for use in subsequent experiments.

[0106] 2. Prepare an aqueous solution containing 100 mM 11-deoxycortisol and 300 mM M-β-CD as the substrate stock solution.

[0107] The wet bacterial cells of each mutant obtained in Example 2 were resuspended in potassium phosphate buffer, and substrate stock solution and stock solutions of IPTG, L-ara, δ-ALA, and ampicillin were added to prepare a final wet bacterial cell concentration of 25 g / L and a final substrate concentration of 10 mM. The reaction system was prepared and reacted at 27.5 °C and 170 rpm for 24 h in a constant temperature shaker. After the reaction was completed, the cells were extracted with ethyl acetate and centrifuged, then rotary evaporated under vacuum at 35 °C for 1 h, and subsequently reconstituted with liquid mobile phase (acetonitrile).

[0108] The yield and ee value were determined by high performance liquid chromatography (HPLC). The HPLC chromatograms of cortisol and 11-deoxycortisol standards are shown below. Figure 8 As shown in the figure. The final measured product ee value and yield are shown in Table 3.

[0109] Table 3. ee values ​​and yields of cortisol prepared by each mutant

[0110] Product ee value Yield Wild type CYP11B1 >99％ <![CDATA[1470.0±21.2mg·L -1 ·d -1 ]]> mutant CYP11B1-S146V >96％ <![CDATA[2560.1±116.8mg·L -1 ·d -1 (**)]]> mutant CYP11B1-H331D ＞99％ <![CDATA[2338.6±81.3mg·L -1 ·d -1 (**)]]> mutant CYP11B1-L440F ＞99％ <![CDATA[2111.0±42.1mg·L -1 ·d -1 (**)]]> Mutant CYP11B1-S146V / H331D ＞99％ <![CDATA[2523.2±170.3mg·L -1 ·d -1 (**)]]> Mutant CYP11B1-S146V / L440F ＞99％ <![CDATA[1561.0±116.8mg·L -1 ·d -1 (ns)]]> Mutant CYP11B1-H331D / L440F ＞99％ <![CDATA[2297.0±54.3mg·L -1 ·d -1 (**)]]> Mutant CYP11B1-S146V / H331D / L440F ＞99％ <![CDATA[2756.3±104.2mg·L -1 ·d -1 (**)]]>

[0111] Note: Compared with the wild type, ns indicates P>0.05, ** indicates P≤0.01. ± represents standard deviation.

[0112] Analysis of experimental results: The recombinant expressed genetically engineered bacteria exhibit superior catalytic activity. The highest space-time yield of cortisol synthesized by the CYP11B1 mutant reached 2756.3 mg·L⁻¹. -1 ·d -1 The catalyst exhibits an ee value of 99% and high optical purity. Furthermore, it is easy to prepare, operates under mild reaction conditions, and is environmentally friendly. It can efficiently catalyze the highly selective 11β-hydroxylation of steroidal compounds, demonstrating promising prospects for industrial application.

Claims

1. A P450 enzyme mutant, characterized in that, The P450 enzyme mutant is a mutant obtained by amino acid mutation of CYP11B1 from Homo sapiens, as shown in SEQ ID NO.1, wherein the amino acid mutation site is at least one of position 146, position 331, and position 440, and the serine at position 146 is mutated to valine, the histidine at position 331 is mutated to aspartic acid, and the leucine at position 440 is mutated to phenylalanine.

2. The P450 enzyme mutant as described in claim 1, characterized in that, The amino acid sequence of the P450 enzyme mutant is shown in any one of SEQ ID NO.2 to SEQ ID NO.

8.

3. A coding gene for encoding the P450 enzyme mutant as described in claim 1 or 2.

4. A recombinant expression vector, characterized in that, The recombinant expression vector includes the coding gene as described in claim 3.

5. The recombinant expression vector as described in claim 4, characterized in that, The recombinant expression vector further includes a corticosteroid reductase encoding gene and a corticosteroid encoding gene, wherein an RBS nucleotide sequence as shown in SEQ ID NO.14 is inserted upstream of the corticosteroid reductase encoding gene, and an RBS nucleotide sequence as shown in SEQ ID NO.15 is inserted upstream of the corticosteroid encoding gene.

6. A genetically engineered bacterium for producing the P450 enzyme mutant as described in claim 1 or 2, characterized in that, The genetically engineered bacteria contain the recombinant expression vector as described in claim 4 or 5.

7. The genetically engineered bacteria as described in claim 6, characterized in that, The genetically engineered bacteria also contain the pGro7 plasmid expressing the molecular chaperone GroEL / ES.

8. The application of the P450 enzyme mutant as described in claim 1 or 2 in the preparation of cortisol, characterized in that, The application includes: the P450 enzyme mutant catalyzes the 11β-hydroxylation of 11-deoxycortisol to cortisol under the combined action of corticosteroid reductase and corticosteroid.

9. The application as described in claim 8, characterized in that, The application includes: using the wet bacterial cells or wet bacterial cell immobilized cells obtained by centrifugation after fermentation culture of genetically engineered bacteria containing recombinant expression plasmids containing P450 enzyme mutant encoding genes, corticosteroid reductase encoding genes and corticosteroid encoding genes as catalysts, 11-deoxycortisol as substrate, and a buffer solution with a pH value ≤8 containing a cosolvent as the reaction medium, the reaction is carried out at 25-37℃ and 150-300rpm. After the reaction is completed, the reaction solution is separated and purified to obtain cortisol.

10. The application as described in claim 9, characterized in that, In the reaction system, the concentration of the substrate is 3-10 mM, the co-solvent is methyl-β-cyclodextrin or (2-hydroxypropyl)-γ-cyclodextrin, the molar ratio of the co-solvent to the substrate is 3-5:1, and the amount of catalyst is 25-100 g / L based on the weight of wet bacterial cells, wherein the wet bacterial cells contain 70-90% water by mass.

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