Use of P450 monooxygenase CYP68N3 in catalyzing C11 alpha-hydroxylation of steroid substrates

Abstract

The application discloses application of P450 monooxygenase CYP68N3 in catalyzing C11 alpha-hydroxylation of a steroid substrate, wherein the steroid substrate includes 18-methyl nandrolone, 18-methyl diketone, 17-hydroxyprogesterone, canrenone, progesterone and nandrolone, and the like; the expression reaction system of the P450 monooxygenase is optimized, and the whole-cell catalytic efficiency and selectivity are further improved, and the application has important significance for production of C11 alpha-hydroxylation products of various steroid compounds.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of bioengineering technology, and particularly relates to an application of cytochrome P450 monooxygenase CYP68N3 in C11α-hydroxylation of a steroid compound as a substrate. BACKGROUND

[0002] Steroid drugs are widely used in the treatment of rheumatism, cardiovascular disease, lymphatic leukemia, anti-tumor, bacterial encephalitis, skin disease, endocrine disorders and other diseases. C11α-hydroxylated steroid compounds have the effects of sterilization, immune regulation, birth control, increasing drug efficacy, and can also be used as a key intermediate for the synthesis of various important drugs (Greyling, et al., S. Afr. J. Anim. Sci, 1999, 29(3): 179). Among them, the C11α-hydroxylated products of 18-methylnandrolone, nandrolone, 18-methyldihydrotestosterone, 17-hydroxyprogesterone, progesterone and other steroids are precursors of common contraceptives on the market. Methyl dienolone can be used to synthesize the steroid drug trenbolone acetate (veterinary drug) through chemical dehydration and esterification reaction and applied to the prevention of mad cow disease. 11α-hydroxykanamycin is a key intermediate for the synthesis of eplerenone, which is a selective aldosterone receptor antagonist for the treatment of cardiovascular disease, and can effectively block the action of the renin-angiotensin-aldosterone system (RAAS), and has a broad market prospect. The C11α-hydroxylated steroid product of androstenedione (AD) and androstadienedione (ADD) is a key intermediate for the synthesis of halogenated corticosteroids (Petric S., et al., J Biotechnol, 2010, 150(3): 428-437).

[0003] 18-methylnorgestrel is an important intermediate for the synthesis of levonorgestrel, etogestrel and oral contraceptive desogestrel, norelisterat. Desogestrel can also be used to treat female menopausal syndrome and endometriosis, and together with testosterone as a male hormone contraceptive. The progestin activity of desogestrel is 1 times stronger than 18-methylnorgestrel and 18 times stronger than norgestrel. Moreover, this drug has only slight androgenic and protein anabolic activity and no adverse effects on lipid metabolism. In 2020, desogestrel was the 120th most commonly prescribed drug in the United States, with more than 5.44 million prescriptions and sales of about $183 million (https: / / clincalc.com / DrugStats / ). There are currently several reports on the synthesis route of desogestrel. However, for the synthesis of the key intermediate 11a-OH-18-methylnorgestrel, there are problems such as long chemical route, harsh reaction conditions, and expensive catalysts. More seriously, the production process uses a large amount of hydrochloric acid and other solvents, causing serious environmental pollution (US20130123523A1).

[0004] Compared with chemical methods, biocatalysis has obvious advantages, such as mild reaction conditions, less environmental pollution, and overall green and clean process. Biocatalysis is in line with the development direction of “green chemistry” and is currently the simplest and most effective means of synthesizing hydroxylated steroids (Liu et al., Bioresource Technol 2011; 102:9368-9373). In 2017, Liu Xiaoguang et al. from Tianjin University successfully mined a P450 enzyme CYP68J5 with C11a-hydroxylation activity on steroid substrates from Aspergillus ochraceus (Yang X, et al., Adv Appl Microbiol, 2018:203.), but the selectivity of the wild-type enzyme to C11a-hydroxylation of steroid substrates is not high, with a selectivity of 69.7% to progesterone and only 58% to diketone. In addition, a P450 enzyme CYP106A2 existing in Bacillus megaterium ATCC 13368 was found to have C11a-hydroxylation activity, but the wild-type had only 18% selectivity to C11a-hydroxylation of steroid substrates (CN113667650A). In 2012, Nguyen et al. reported that the C11a-hydroxylation selectivity of CYP106A2 (T89N / A395I) mutant to progesterone was improved to 81% (Nguyen K T, et al. ChemBioChem, 2012, 13(8): 1161-1166.), but there has been no report on the C11a-hydroxylation of 18-methylnorgestrel by these enzymes.

[0005] Metarhizium anisopliae has the advantages of strong pathogenicity to pests, wide host range, and no toxin residues, and has a broad application prospect as a broad-spectrum biological insecticide. However, current research on Metarhizium anisopliae mainly focuses on agricultural application and genetic breeding. In 1997, Shi Jiping et al. of Shanghai Medical University successfully C11α-hydroxylated 19-nor-13-ethyl-androst-4-ene-3, 17-dione (FA) with Metarhizium anisopliae at a substrate concentration of 0.2% (w / v), but the conversion rate was only 36% (Shi Jiping et al., Journal of Shanghai Medical University; 1997, 04). In addition, in CN107974481A, Zhao Xiaojuan, Yang Fang, et al. used Metarhizium anisopliae to convert 18-methylnorgestrel to 11α-OH-18-methylnorgestrel, but the yield was not ideal, only 70%-80%. It can be seen that the current application of microorganism transformation to synthesize 11α-OH-18-methylnorgestrel still has the problem of low activity. Moreover, the genetic background of the fungal production strain is not clear, and the molecular operation is difficult. These problems limit the directional molecular genetic modification of the strain, and seriously affect its application in the synthesis of steroid drugs. Therefore, if the enzyme for 11α-hydroxylation of 18-methylnorgestrel can be found, and the function of the enzyme is strengthened through enzyme molecular modification technology, and the reaction system is optimized, then the important intermediate 11α-OH-18-methylnorgestrel can be synthesized with high efficiency and high selectivity, which is expected to better understand the structural basis of steroid stereoselectivity and substrate specificity, significantly improve the synthesis efficiency of various global universal contraceptives including desogestrel, and promote the innovation of the production technology of various contraceptives including desogestrel Figure 1 ). In addition, the C11α-hydroxylated products of 18-methylnorgestrel, 18-methyldihydrotestosterone, and canrenone and other steroid compounds have very important medicinal value. If the C11α-hydroxylase found has a relatively wide substrate spectrum, the research and development speed of these drugs will be accelerated, and the production cost and price will be reduced. SUMMARY

[0006] Therefore, the present application aims to provide an enzyme with high selectivity for catalyzing C11α-hydroxylation of 18-methylnorgestrel, construct a high-efficiency catalytic reaction system of the enzyme, and further explore and expand the steroid substrate spectrum of the enzyme on this basis.

[0007] The technical scheme of the present application is as follows:

[0008] The first aspect of the present application provides a P450 monooxygenase CYP68N3 with high selectivity for catalyzing C11α-hydroxylation, and the amino acid sequence of the enzyme is a1) or a2):

[0009] a1) as shown in SEQ ID NO. 1;

[0010] a2) a sequence having at least 80% homology with the sequence shown in SEQ ID NO. 1 and having the same function as the sequence shown in SEQ ID NO. 1.

[0011] Based on the observation that Metarhizium anisopliae EEG016 can hydroxylate C11 of 18-methylnandrolone, the inventors explored a P450 gene with up-regulated expression through a large number of experiments such as steroid substrate induction, transcriptome sequencing and candidate gene cloning, and mined the amino acid sequence (as shown in SEQ ID NO. 1) encoded by the gene was submitted to the P450 naming website (https: / / drnelson.uthsc.edu) and was named CYP68N3. Currently, the function of the P450 enzyme encoded by the gene and its high selectivity for C11α-hydroxylation of steroids have not been reported.

[0012] The second aspect of the present application provides a nucleic acid molecule encoding the P450 monooxygenase CYP68N3, and the nucleic acid molecule is b1) or b2):

[0013] b1) is a P450 gene of Metarhizium anisopliae EEG016;

[0014] b2) is a DNA molecule having at least 50% homology with the sequence defined in b1) and encoding the P450 monooxygenase CYP68N3.

[0015] The third aspect of the present application provides a reaction system in which the P450 monooxygenase CYP68N3 catalyzes C11α-hydroxylation of a steroid substrate efficiently, and specifically comprises: inducing culture of a transformant carrying the cyp68n3 gene, and then adding a steroid substrate to perform whole-cell biocatalytic reaction.

[0016] In view of the limitations of the enzyme in the application of steroid drug synthesis due to unclear genetic background of fungal production strains, difficulties in molecular manipulation and other problems, the present application optimizes and improves the catalytic activity of the enzyme on steroid substrates by replacing the cyp68n3 gene expression cell chassis.

[0017] The transformant is obtained by transforming the recombinant plasmid with the cyp68n3 gene into the host cell in the above reaction system. It should be noted that the recombinant plasmid can be constructed by connecting the gene fragment encoding CYP68N3 in Metarhizium anisopliae EEG016 to various vectors by the conventional method in the art. The vector can be various plasmid vectors in the art, as long as the recombinant plasmid can be normally replicated in the corresponding expression host and the corresponding P450 monooxygenase can be expressed. The host cell is the conventional host cell in the art, as long as the recombinant plasmid can be stably replicated by itself and the gene cyp68n3 encoding CYP68N3 can be effectively expressed. Moreover, the preferred plasmid vector is different for different expression hosts.

[0018] When the host cell is Saccharomyces cerevisiae, the plasmid vector is preferably a pYES2 plasmid. In an embodiment of the present application, the transformant is prepared by the following method: the nucleic acid product obtained by PCR amplification and the expression vector pYES2 are respectively amplified by primers, and then the complementary cohesive ends are formed by T5 exonuclease cleavage, and then the recombinant plasmid containing the gene cyp68n3 of Metarhizium anisopliae EEG016 is obtained by incubation at 42°C after ice storage, and then the recombinant plasmid is transformed into Saccharomyces cerevisiae INVSc1.

[0019] When the host cell is Pichia pastoris, the plasmid vector is preferably a pPICZA plasmid. In an embodiment of the present application, the transformant is constructed by using the pPICZA plasmid and Pichia pastoris X33.

[0020] Experiments show that the recombinant Saccharomyces cerevisiae transformant constructed in the present application has a conversion rate of 84.5% and a C11α-selectivity of 85.3% for 18-methylnandrolone after reacting with 1 mM substrate for 72 h in a whole-cell catalytic system. More preferably, the recombinant Pichia pastoris transformant constructed in the present application has a conversion rate of 99.0% for 18-methylnandrolone and a selectivity of 98.5% for the target product after reacting with 1 mM substrate for 72 h in a whole-cell biocatalysis. It can be seen that the catalytic activity and selectivity of the enzyme for the steroid substrate can be significantly improved after changing the cyp68n3 gene expression cell chassis in the present application.

[0021] The fourth aspect of the present application expands the steroid substrate spectrum of P450 monooxygenase CYP68N3. Experiments show that the enzyme has good catalytic activity and C11α-hydroxylation selectivity for nandrolone, 17-hydroxyprogesterone, progesterone, 18-methyl diketone and canrenone and other steroid drugs, and the substrate spectrum is wide.

[0022] Compared with the prior art, the present application has the beneficial effects that the P450 monooxygenase CYP68N3 is cloned and characterized from fungi for the first time, and the enzyme has high selectivity C11a-hydroxylation function of steroids, and the efficiency and selectivity of whole cell catalysis are further improved by heterologous expression of the enzyme in a yeast system, such as that the conversion rate of 18-methyl nandrolone can be as high as 99.0% and the C11a-hydroxylation product selectivity can be as high as 98.5% under the condition of 1mM substrate concentration and reaction for 72h; moreover, CYP68N3 has good catalytic activity and C11a-hydroxylation selectivity for a variety of steroid drugs such as nandrolone, 17-hydroxyprogesterone and canrenone, and the substrate spectrum is extensive, so the P450 enzyme CYP68N3 has good industrial application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 A technical roadmap for biocatalysis of 18-methyl nandrolone to synthesize desoxymethyltestosterone.

[0024] Figure 2 HPLC analysis results of recombinant Saccharomyces cerevisiae catalysis of 18-methyl nandrolone reaction in Example 1, wherein blank is transformant without Metarhizium anisopliae cyp68n3 gene, 68N3 is Saccharomyces cerevisiae transformant with Metarhizium anisopliae cyp68n3 gene, 1 is the peak of 18-methyl nandrolone, and 2 is the peak of 11OH-18-methyl nandrolone.

[0025] Figure 3 HPLC analysis results of recombinant Pichia pastoris catalysis of 18-methyl nandrolone reaction in Example 2, wherein blank is transformant without Metarhizium anisopliae cyp68n3 gene, 68N3 is Pichia pastoris transformant with Metarhizium anisopliae cyp68n3 gene, 1 is the peak of 18-methyl nandrolone, and 2 is the peak of 11a-OH-18-methyl nandrolone.

[0026] Figure 4 HPLC analysis results of recombinant Pichia pastoris catalysis of nandrolone reaction in Example 3, wherein blank is transformant without Metarhizium anisopliae cyp68n3 gene, 68N3 is Pichia pastoris transformant with Metarhizium anisopliae cyp68n3 gene, 1 is the peak of nandrolone, and 2 is the peak of 11a-OH-nandrolone.

[0027] Figure 5 HPLC analysis results of recombinant Pichia pastoris catalysis of 17-hydroxyprogesterone reaction in Example 3, wherein blank is transformant without Metarhizium anisopliae cyp68n3 gene, 68N3 is Pichia pastoris transformant with Metarhizium anisopliae cyp68n3 gene, 1 is the peak of 17-hydroxyprogesterone, and 2 is the peak of 11a, 17-OH-progesterone.

[0028] Figure 6 HPLC analysis results of recombinant Pichia catalyzing 18-methylgonadion reaction in Example 3, wherein blank is transformant without Metarhizium cyp68n3 gene, 68N3 is Pichia transformant with Metarhizium cyp68n3 gene, 1 is 18-methylgonadion peak, and 2 is 11a-OH-18-methylgonadion peak.

[0029] Figure 7 HPLC analysis results of recombinant Pichia catalyzing kanamycin reaction in Example 3, wherein blank is transformant without Metarhizium cyp68n3 gene, 68N3 is Pichia transformant with Metarhizium cyp68n3 gene, 1 is kanamycin peak, and 2 is 11a-OH-kanamycin peak.

[0030] Figure 8 HPLC analysis results of recombinant Pichia catalyzing progesterone reaction in Example 3, wherein blank is transformant without Metarhizium cyp68n3 gene, 68N3 is Pichia transformant with Metarhizium cyp68n3 gene, 1 is progesterone peak, and 2 is 11a-OH-progesterone peak.

[0031] Figure 9 HPLC analysis results of different recombinant Pichia catalyzing 18-methylgonadion reaction in Comparative Example 1, wherein blank is transformant without Metarhizium cyp68n3 gene, 68N3 is Pichia transformant with Metarhizium cyp68n3 gene, 68J5 is Pichia transformant with Aspergillus cyp68j5 gene, 1 is 18-methylgonadion peak, and 2 is 11a-OH-18-methylgonadion peak. DETAILED DESCRIPTION

[0032] For a better understanding of the present application, reference will be made to the following embodiments and drawings thereof. It is to be understood that the specific embodiments described herein are merely illustrative and explanatory of the present application and are not intended to limit the scope of the present application.

[0033] The experimental methods used in the following examples are conventional methods in the art unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0034] Example 1 Construction of Saccharomyces cerevisiae heterologous expression vector plasmid and whole cell biotransformation of steroid substrate

[0035] In this example, the cyp68n3 gene mined from Metarhizium EEG016 was constructed in the vector pYES2 and introduced into Saccharomyces cerevisiae to catalyze the reaction of the substrate 18-methylgonadion. The specific process is as follows:

[0036] The cDNA of the Metarrhizium anisopliae RNA was reverse transcribed to obtain the gene fragment of interest, and the PCR primers were as shown in SEQ ID NO. 2-3 in Table 1.

[0037] Table 1

[0038]

[0039]

[0040] The target gene fragment was recovered, and the recovered target fragment and the pYES2 fragment of the plasmid vector were ligated using T5 exonuclease, and then transformed into the E. coli DH5a competent cells, and then coated on the solid LB plate with the final concentration of ampicillin of 50 μg / mL. After the colonies grew on the plate, a single colony was picked and cultured at 37°C, and the plasmid was extracted to obtain the pYES2-CYP68N3 expression plasmid with the up-regulated gene. Colony PCR was performed using primers with 15 bp homologous ends (the sequences are shown in SEQ ID NO. 6-7), thereby proving that the target gene of the fungus was successfully constructed into the plasmid pYES2.

[0041] The successfully constructed recombinant plasmid was transformed into the S. cerevisiae INVSc1 competent cells. A single colony was picked and cultured in a liquid medium, and then 1 mL of the bacterial solution was transferred into 50 mL of uracil-free SC medium, and cultured at 28°C and 220 rpm until the OD 600 was 5-6. The uracil-free SC medium was replaced with uracil-free SC-galactose induction medium, centrifuged at 4°C and 4000 rpm to remove the supernatant, and then resuspended with an equal volume of uracil-free SC-galactose induction medium. After induction at 28°C for 12 h, 1 mM 18-methylnorgestrel was added for whole-cell reaction.

[0042] The 72 h reaction sample was subjected to HPLC analysis, and the analysis results are shown in Table 2: Figure 2 The recombinant S. cerevisiae transformant reacted with 18-methylnorgestrel in the whole-cell biotransformation, and generated a product peak consistent with the peak time of the 11α-OH-18-methylnorgestrel standard, indicating that CYP68N3 is an enzyme that regulates the C11α-hydroxylation of the steroid substrate in the Metarrhizium anisopliae.

[0043] Construction of Pichia pastoris heterologous expression vector plasmid and whole-cell biotransformation of steroid substrate

[0044] Referring to Example 1, the P450 enzyme cyp68n3 gene is ligated with the pPICZA plasmid to prepare the recombinant plasmid pPICZA-68N3; wherein the upstream and downstream primer sequences of the pPICZA plasmid are GGTACCTCGAGCCGCGGC (SEQ ID NO. 8) and GATCCGAGACGGCCGGCT (SEQ ID NO. 9), respectively.

[0045] The constructed pPICZA-68N3 plasmid is transformed into E. coli DH5a competent cells, which are coated on a solid LB plate containing 35 μg / mL of bleomycin. Single colonies are picked for culture and plasmid extraction, and the obtained plasmid is the recombinant plasmid pPICZA-68N3 carrying CYP68N3. Subsequently, colony PCR (the primer sequences used are shown in SEQ ID NO. 6-7) is used to verify whether the recombinant plasmid is successfully constructed.

[0046] The successfully constructed recombinant plasmid is then transformed into Pichia pastoris X33 competent cells, and single colonies are picked into 3 mL of liquid YPD containing 300 μg / mL of bleomycin for culture for 48 h. The supernatant is removed by centrifugation, and then the Pichia pastoris cells are resuspended in 25 mL of BMMY induction medium, and induced at 28°C for 72 h. During the induction, methanol (1% (v / v)) is supplemented every 24 h. After the induction is completed, 18-methylnorgestrel is added for Pichia pastoris whole-cell reaction.

[0047] The 72 h sample is taken for HPLC analysis, and the analysis results are shown in Table 2 and Figure 3 As shown, the Pichia pastoris transformant containing the cyp68n3 gene produces a product peak consistent with the elution time of the 11a-OH-18-methylnorgestrel standard, and the conversion rate reaches 99.0%, and the C11a-hydroxylation selectivity reaches 98.5%.

[0048] Compared with S. cerevisiae, the conversion rate and C11a-hydroxylation selectivity of the Pichia pastoris catalytic system in this example are greatly improved.

[0049] Example 3 Exploration of the Steroid Substrate Spectrum of C11a-Hydroxylase CYP68N3

[0050] In this example, the recombinant Pichia pastoris X33 prepared in Example 2 is used to detect the catalytic effect of CYP68N3 on norgestrel, 17-hydroxyprogesterone, 18-methylnorgestrel, canrenone and progesterone, respectively.

[0051] The experimental process is the same as that of Example 2. After the recombinant Pichia pastoris carrying the cyp68n3 gene catalyzes norgestrel, 17-hydroxyprogesterone, 18-methylnorgestrel, canrenone and progesterone for 72 h, respectively, the products are analyzed by HPLC, and the results are shown in Table 2 and Figures 4-8 .

[0052] The results show that when the steroid substrate is nandrolone, 17-hydroxyprogesterone, 18-methyldiketone, canrenone and progesterone, chromatographic peaks completely consistent with the peak time of the product standard are produced, proving that the recombinant Pichia pastoris containing the cyp68n3 gene has good conversion activity and C11α-hydroxylation selectivity for 18-methyldiketone, 17-hydroxyprogesterone, canrenone and progesterone, and has certain reaction activity and C11α-hydroxylation selectivity for nandrolone.

[0053] Table 2

[0054] Substrate Product Substrate conversion (%) C11α-hydroxylation selectivity (%) Nandrolone 11α-OH-nandrolone 20.1％ 67.8％ 17-Hydroxyprogesterone 11α, 17 OH-progesterone 99.9％ 93.1％ 18-Methyl dione 11α-OH-18-methyl dione 99.9％ 74.8％ Kanreton 11α-OH-kanreton 80.5％ 95.6％ Progesterone 11α-OH-progesterone 99.6％ 96.6％

[0055] Whole-cell biotransformation of C11α-hydroxylase CYP68J5 and steroid substrates

[0056] Different from Example 2, the gene of C11α-hydroxylase CYP68J5 excavated by Liu Xiaoguang et al. of Tianjin University from Aspergillus ochraceus is used to replace the cyp68n3 gene in this example, and the others are consistent with Example 2.

[0057] The sample taken at 72 h of reaction is subjected to HPLC analysis, and the analysis results are shown in Table 3 and FIG. 2: Figure 9 Under the same conditions, the substrate conversion rate of CYP68J5 in the whole-cell reaction of Pichia pastoris is 52.6% and the C11α-hydroxylation selectivity is 6.3% when 1 mM 18-methylnandrolone is converted for 72 h, which are significantly poorer than the results of CYP68n3 in converting 18-methylnandrolone. Figure 9 It can be clearly seen that CYP68J5 converts 18-methylnandrolone to generate a large amount of by-products.

[0058] Table 3

[0059] P450 Substrate conversion (%) C11α-hydroxylation selectivity (%) CYP68N3 99.0％ 98.5％ CYP68J5 52.6％ 6.3％

[0060] In summary, the P450 enzyme CYP68N3 provided by the application has the function of specifically C11α-hydroxylating steroid substrates, and more importantly, has excellent catalytic reaction activity and high selectivity compared with existing C11α-hydroxylases, can greatly reduce the production cost of C11α-hydroxylated products, and has important significance for the preparation of a variety of C11α-hydroxylated steroid products.

[0061] The above is the preferred embodiment of the application, which cannot be used to limit the scope of the application, and it should be pointed out that any modification, equivalent replacement and improvement made within the spirit and principle of the application should be included in the protection scope of the application.

Claims

1. Use of the P450 monooxygenase CYP68N3 in the catalysis of the hydroxylation of a steroidal substrate C11 α characterized in that The amino acid sequence of the P450 monooxygenase CYP68N3 is shown as SEQ ID NO. 1, and the steroid substrate is 18-methylnandrolone, 18-methyldiketone, 17 hydroxyprogesterone, canrenone, progesterone or nandrolone.

2. Use according to claim 1, characterized in that, A gene encoding the P450 monooxygenase CYP68N3 cyp68n3 From Metarhizium anisopliae EEG016.

3. Use according to claim 2, characterized in that, Induction of cultures harboring transformants with genes cyp68n3 followed by whole-cell biocatalysis with the addition of a steroid substrate.

4. Use according to claim 3, characterized in that, The transformant is obtained by transforming a host cell with a recombinant plasmid having a gene cyp68n3 of the present application.

5. Use according to claim 4, characterized in that, The host cell is a yeast cell.

6. Use according to claim 5, characterized in that, The host cell is a Pichia pastoris cell.

7. Use according to claim 6, characterized in that, The recombinant plasmid is obtained by ligating the gene cyp68n3 with the pPICZA plasmid.

8. Use according to claim 5, characterized in that, The host cell is a Saccharomyces cerevisiae cell, and the recombinant plasmid is derived from the pYES2 plasmid cyp68n3 obtained by ligating the pYES2 plasmid.

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