Steroid hydroxylase and its coding gene in beauveria bassiana and application

Abstract

The present application relates to the field of steroid drugs and biotechnology, and relates to a novel steroid hydroxylase CYP68BE1 from Beauveria bassiana, a coding gene thereof and application. Specifically, the present application provides a novel steroid hydroxylase CYP68BE1 from Beauveria bassiana, a nucleic acid molecule coding the enzyme, a vector containing the nucleic acid molecule, and a recombinant bacterium containing the vector or having the nucleic acid molecule integrated therein. Through recombination of yeast bacteria or microsomal transformation of steroids, it is found that the CYP68BE1 enzyme can catalyze the hydroxylation reaction of C11α and / or C6β and / or C1β and / or C1α of steroid compounds. Among them, the transformation products include various steroid drug intermediates and / or steroid active compounds, and the present application has great application potential and economic value.

Description

Technical Field

[0001] This invention relates to the fields of steroidal drugs and biotechnology, and more specifically, to a steroidal hydroxylase obtained from *Beauveria bassiana* and its encoding gene. This invention also relates to the application of this steroidal hydroxylase in the preparation of steroidal drug intermediates and their steroidal active compounds. Background Technology

[0002] Steroid compounds possess important and broad pharmacological activities, such as anti-infection, anti-tumor, anti-inflammatory, and anti-AIDS effects. Currently, over 400 steroid drugs are widely used to treat diseases such as inflammation, cancer, rheumatism, and arthritis. The hydroxyl group is a crucial functional group on the steroid drug skeleton. Hydroxylation of the steroid nucleus via chemical methods is typically complex and inefficient. However, compared to chemical synthesis, microbial transformation offers advantages such as milder reaction conditions, environmental friendliness, and simpler experimental procedures. Therefore, employing microbial transformation for the hydroxylation of specific positions in steroid compounds has become a key technology in the steroid pharmaceutical industry, laying the foundation for the industrial production and innovative research and development of steroid drugs. Currently reported microbial hydroxylation sites are mainly concentrated at C-1, C-6, C-7, C-9, C-11, C-14, C-15, C-16, etc. (Chen X, Bao YJ, Wang Y, et al., Distinct regioselectivity offungal P450 enzymes for steroidal hydroxylation. Applied and Environmental Microbiology, 2019, 85(18): 1-13).

[0003] Fungi are typically used industrially as biomaterials for the hydroxylation of steroids by microorganisms. However, due to the relatively complex culture conditions of fungi, some fungi can produce toxins and harmful substances or cause environmental pollution. Therefore, identifying relevant steroid hydroxylases within fungi, selecting hosts with simple backgrounds, easy cultivation, high efficiency, and no environmental impact (such as Saccharomyces cerevisiae and Pichia pastoris) to construct related engineered strains, and applying them to the production of key intermediates in steroidal pharmaceuticals will undoubtedly usher in a new era for the industrial manufacturing of steroidal drugs.

[0004] It has been reported that *Beauveria bassiana* can perform microbial hydroxylation reactions on steroidal compounds (11α monohydroxylation, 11α,6β dihydroxylation, and 11α,6β,17β trihydroxylation) with high conversion rates and diverse products (Xiong Z, Wei Q, CHEN H, et al. Microbial transformation of androst-4-ene-3,17-dione by *Beauveria bassiana*. *Steroids*, 2006, 71(11-12): 979-83). *Beauveria bassiana* contains 83 cytochrome P450 monooxygenase (CYP450) genes, but the CYP450 gene associated with steroidal hydroxylation has not yet been identified. Therefore, identifying the CYP450 gene with highly efficient catalytic hydroxylation capabilities within *Beauveria bassiana* and applying it to the production of steroidal drugs or their key intermediates is of great significance. Summary of the Invention

[0005] The technical problem solved by this invention is to provide a highly efficient steroidal hydroxylase from Beauveria bassiana and its amino acid sequence, a nucleic acid molecule encoding the enzyme, a carrier and recombinant cells containing the nucleic acid molecule, and a method for producing steroidal substances by single-site and / or multi-site hydroxylation reactions (positions 11 and / or 6 and / or 1).

[0006] To address the aforementioned technical problems, this invention has extracted a steroid hydroxylase gene, CYP68BE1, from Beauveria bassiana. Recombinant bacterial cells obtained through heterologous expression catalyze hydroxylation reactions at positions 11 and / or 6 and / or 1 of steroid compounds, producing various steroid drugs and / or steroid drug intermediates.

[0007] Therefore, in a first aspect, the present invention provides a steroidal hydroxylase CYP68BE1, the amino acid sequence of which is shown in SEQ ID NO:1, or the amino acid sequence shown in SEQ ID NO:1 obtained by substitution, deletion or addition of one or more amino acids to obtain a sequence with the same function.

[0008] The steroid hydroxylase CYP68BE1 is derived from Beauveria bassiana and can catalyze the hydroxylation of sterols at positions 11 and / or 6 and / or 1.

[0009] The protein provided by this invention is any one of the following proteins (P1)-(P4):

[0010] (P1) defines the amino acid sequence of the protein as shown in SEQ ID NO:1 of the sequence listing;

[0011] (P2) A protein that has the same function as the amino acid sequence defined by (P1) by substitution and / or deletion and / or addition of one or more amino acid residues.

[0012] (P3) is a protein that has 99%, 95%, 90%, 85%, or 80% homology with the amino acid sequence defined by (P1) or (P2) and has the same function.

[0013] (P4) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of any of the proteins defined in (P1)-(P3).

[0014] In a second aspect, the present invention provides a nucleic acid molecule that encodes the CYP68BE1 described in the present invention.

[0015] The nucleic acid molecule may be DNA obtained from Beauveria bassiana.

[0016] The nucleic acid molecule of the present invention can be in DNA or RNA form. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand. The coding region sequence encoding the protein of the present invention can be identical to the nucleotide sequence shown in SEQ ID NO: 2 or a degenerate variant. As used herein, "degenerate variant" refers to a nucleotide sequence encoding the protein having SEQ ID NO: 1 but differing from the nucleotide sequence shown in SEQ ID NO: 2. That is, the nucleic acid molecule of the present invention can be any nucleic acid molecule sequence capable of encoding the amino acid sequence having SEQ ID NO: 1. The preferred nucleotide sequence is SEQ ID NO: 2.

[0017] In this invention, the nucleic acid molecule encoding the steroid hydroxylase CYP68BE1 (e.g., optionally a DNA sequence encoding a spacer sequence) can be obtained by chemical synthesis.

[0018] Furthermore, the nucleic acid molecule may specifically be any one of the following (S1)-(S3) DNA molecules:

[0019] (S1) A nucleic acid sequence as shown in SEQ ID NO:2 or a nucleic acid sequence optimized by microbial codons that can encode any of the amino acid sequences shown in (P1)-(P4);

[0020] (S2) A DNA molecule that hybridizes under stringent conditions with the DNA molecule defined by (S1) and encodes any of the proteins shown in (P1)-(P4) as described in the first aspect above;

[0021] (S3) is a DNA molecule that has 99%, 95%, 90%, 85% or more or 80% homology with any of the DNA sequences defined in (S1) and (S2) and encodes any of the proteins shown in (P1)-(P4) as described in the first aspect above.

[0022] Furthermore, the nucleic acid molecule may specifically be a DNA molecule as shown in any of (S1) and (S3) above, or a DNA molecule with a nucleotide sequence as shown in SEQ ID No: 2.

[0023] In a third aspect, the present invention provides a carrier containing the nucleic acid molecule described in the second aspect of the present invention.

[0024] According to embodiments of the present invention, the carrier may include an expression carrier, a shuttle carrier, or an integration carrier. For example, the carrier may be a commercially available carrier, or any carrier having the same function. In a preferred embodiment, the carrier is an expression carrier.

[0025] Expression vectors, shuttle vectors, or integration vectors containing the CYP68BE1 coding DNA sequence of this invention and suitable transcription / translation control signals can be constructed using methods well known to those skilled in the art. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology.

[0026] For example, various yeast cell expression vectors well known to those skilled in the art can be used, including but not limited to pESC-HIS, pESC-LEU, pESC-URA, pESC-TRP, pYES2, or pAUR123. Preferably, the yeast expression vector is pESC-HIS, pESC-LEU, pESC-URA, or pESC-TRP. For example, Escherichia coli cell expression vectors well known to those skilled in the art can be used, including but not limited to pET-32(a) and pET-28(a). Preferably, the Escherichia coli expression vector is pET-32(a).

[0027] In a fourth aspect, the present invention provides a recombinant cell containing the vector described in the third aspect of the present invention, or having the nucleic acid molecule described in the second aspect of the present invention integrated into its genome.

[0028] According to some embodiments of the present invention, the recombinant cells are eukaryotic or prokaryotic cells. Preferably, the recombinant cells are *Saccharomyces cerevisiae* cells, *Pichia pastoris* cells, or *Escherichia coli* cells. The yeast may be *Saccharomyces cerevisiae* (e.g., strain BY4742), *Yersinia lipolytica* (e.g., strain PO1G), *Schizosaccharomyces cerevisiae* (e.g., strain CICC1762), or *Pichia pastoris* (e.g., strain GS115), etc.

[0029] In a preferred embodiment, the recombinant cells are yeast cells, preferably Saccharomyces cerevisiae cells.

[0030] The appropriate method for introducing the vector or nucleic acid molecule into the cell can be selected depending on the type of recombinant cell. These methods are well known to those skilled in the art. For example, methods known in the art can be used to transform the vector into Saccharomyces cerevisiae cells, or to integrate nucleic acid molecule fragments into the genome of Saccharomyces cerevisiae through homologous recombination. The transformation of Saccharomyces cerevisiae can be achieved using various transformation methods well known to those skilled in the art, such as electroconversion, lithium acetate chemical transformation, etc.

[0031] In a fifth aspect, the present invention provides a method for hydroxylating steroidal drugs and / or steroidal drug intermediates, the method comprising: performing a hydroxylation reaction of the steroidal compound in the presence of the hydroxylase CYP68BE1.

[0032] According to some embodiments of the present invention, the method causes hydroxylation at positions 11 and / or 6 and / or 1 of the steroid compound. Furthermore, the hydroxylation method of the present invention can catalyze hydroxylation at one and / or two sites at positions 11 and / or 6 and / or 1 of the steroid compound.

[0033] According to some embodiments of the present invention, the method may be in the form of whole-cell catalysis or enzymatic catalysis, with whole-cell catalysis being preferred.

[0034] In a specific embodiment of the present invention, a whole-cell catalysis method is employed. Further, engineered bacteria containing the cyp68be1 gene are fermented and cultured, and a steroidal compound is added as a substrate to carry out a catalytic reaction. Specifically, the catalytic reaction conditions are 28°C, 200–250 rpm (e.g., 220 rpm), and shaking culture for 1–4 days (e.g., 2 days); the substrate in the reaction system has a concentration of 10 mg–200 mg (e.g., 150 mg / L).

[0035] In a sixth aspect, the present invention provides the use of CYP68BE1 in the production of hydrocortisone and / or eplerenone and / or strophanthidin G and / or trenbolone.

[0036] Beneficial technical effects:

[0037] The steroid hydroxylase CYP68BE1 from *Beauveria bassiana* provided in this invention can catalyze the hydroxylation of steroid precursor compounds at the C11 and / or C6 and / or C1 positions, thereby generating steroid drugs and / or steroid drug intermediates. Furthermore, CYP68BE1 can also serve as a basic element in the metabolic pathways for constructing steroid drugs and / or steroid drug intermediates. Developing new resources for generating steroid drugs and / or steroid drug intermediates through synthetic biology techniques has significant economic value and good application potential.

[0038] The following will further explain the concept, specific structure, and technical effects of the present invention in conjunction with the accompanying drawings, so as to fully understand the purpose, features, and effects of the present invention. Attached Figure Description

[0039] Figure 1 SDS-PAGE image of cyp68be1 gene amplification.

[0040] Figure 2 A schematic diagram of the pESC-LEU-cyp68be1 recombinant plasmid is shown.

[0041] Figure 3 The LC-MS spectrum of the reaction products of 4AD catalyzed by recombinant yeast SC-1 is shown. Figure 3 A shows the HPLC chromatograms of 4AD catalyzed by the control group SC-5 (S. cerevisiae INVSc1) and the experimental group recombinant yeast SC-1. Figure 3 B shows the product structure and transformation route of 4AD catalyzed by the yeast recombinant strain SC-1.

[0042] Figure 4 The application of CYP68BE1 protein in the synthesis of hydrocortisone, eplerenone, and strophanthidin G is shown (solid arrows indicate catalysis by recombinant SC-1 bacteria, and dashed arrows indicate chemical catalysis).

[0043] Figure 5 The LC-MS spectrum of the reaction products of Testosterone catalyzed by recombinant yeast SC-1 is shown. Figure 5 A shows the HPLC chromatograms of Testosterone catalyzed by the control group SC-5 (S. cerevisiae INVSc1) and the experimental group recombinant yeast SC-1. Figure 5 B shows the product structure and transformation route of Testosterone catalyzed by yeast recombinant strain SC-1.

[0044] Figure 6The LC-MS spectrum of the reaction products of 1,4-Androstenedione catalyzed by recombinant yeast SC-1 is shown. Figure 6 A shows the HPLC chromatograms of 1,4-Androstenedione catalyzed by the control group SC-5 (S. cerevisiae INVSc1) and the experimental group recombinant yeast SC-1. Figure 6 B shows the product structure and transformation route of 1,4-Androstenedione catalyzed by the yeast recombinant strain SC-1.

[0045] Figure 7 The LC-MS spectrum of the reaction products of Progesterone catalyzed by recombinant yeast SC-1 is shown. Figure 7 A shows the HPLC chromatograms of Progesterone catalyzed by the control group SC-5 (S. cerevisiae INVSc1) and the experimental group recombinant yeast SC-1. Figure 7 B shows the product structure and transformation route of Progesterone catalyzed by yeast recombinant strain SC-1.

[0046] Figure 8 The LC-MS spectrum of the reaction products of recombinant yeast SC-1 catalyzing Estrone is shown. Figure 8 A shows the HPLC chromatograms of Estrone catalyzed by the control group SC-5 (S. cerevisiae INVSc1) and the experimental group recombinant yeast SC-1. Figure 8 B shows the product structure and transformation route of Estrone catalyzed by the yeast recombinant strain SC-1.

[0047] Figure 9 The LC-MS spectrum of the reaction products of recombinant yeast SC-1 catalyzing Estra-4,9-diene-3,17-dione is shown. Figure 9 A shows the HPLC chromatograms of Estra-4,9-diene-3,17-dione catalyzed by the control group SC-5 (S. cerevisiae INVSc1) and the experimental group recombinant yeast SC-1. Figure 9 B shows the product structure and transformation route of Estra-4,9-diene-3,17-dione catalyzed by the yeast recombinant strain SC-1.

[0048] Figure 10The application of the CYP68BE1 protein in the synthesis of hydrocortisone is illustrated. Estra-4,9-diene-3,17-dione was catalyzed using the SC-1 recombinant strain to generate the 11α-hydroxylated intermediate 6c, which can serve as an important intermediate in the industrial synthesis of trenbolone (solid arrows indicate SC-1 recombinant bacterial catalysis, dashed arrows indicate chemical catalysis).

[0049] Figure 11 The LC-MS spectra of the reaction products of sterol substrates 1-6 catalyzed by microsomes of Saccharomyces cerevisiae containing CYP68BE1 are shown. Figure 11 The substrate in A is 4AD; Figure 11 The substrate in B is Testosterone; Figure 11 The substrate in C is 1,4-Androstenedione; Figure 11 The substrate in D is Progesterone; Figure 11 The substrate in E is Estrone; Figure 11 The substrate in F is Estra-4,9-diene-3,17-dione. Detailed Implementation

[0050] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Those skilled in the art should understand that these embodiments are for illustrative purposes only and do not limit the scope of the invention. The scope of protection of the present invention is limited only by the claims. Without departing from the scope of the claims, those skilled in the art can make various modifications and improvements to various aspects of the present invention, and these modifications and improvements also fall within the scope of protection of the present invention.

[0051] Additionally, it should be noted that, unless otherwise specified, all materials and reagents used in the following embodiments are commonly used in the art and can be obtained through conventional commercial means; all methods used are conventional methods known to those skilled in the art.

[0052] Example 1: Cloning of the Beauveria bassiana steroid hydroxylase gene cyp68be1

[0053] Gene cloning involves the following two steps:

[0054] 1) Extraction of total RNA from Beauveria bassiana (total RNA was extracted using the Fungal RNA Kit, Omega Bio-tek).

[0055] (1) After culturing Beauveria bassiana on a plate for several days, a certain number of spores were collected and inoculated into 50mLYM (yeast extract-maltose) medium;

[0056] (2) Collect the mycelium cultured for 3 days into a 7 ml tube, centrifuge to collect the cells, wash and set aside;

[0057] (3) Take several 1.5 mL RNase-free Ep tubes into an Ep tube rack, add 500 μL LRB buffer to each tube, and then add 10 μL β-mercaptoethanol (4°C refrigerator);

[0058] (4) Pour a small amount of liquid nitrogen into a mortar (wrapped in aluminum foil and placed in an oven at 180℃ for more than 3 hours to remove RNase) to cool it down. Use the handle of a spoon to pick up the bacteria into the mortar and crush them. After adding liquid nitrogen, grind quickly, repeating 4-5 times until a fine powder is formed. Quickly transfer about 100 mg of powder to RB buffer (prevent the sample from melting during this process), and immediately shake for 30 seconds to 1 minute (to ensure that the sample is completely resuspended and there are no clumps).

[0059] (5) Place the gDNA filter column (blue) into a 2mL collection tube, transfer the lysate into the column, and centrifuge at 14,000 rpm for 5 min;

[0060] (6) Transfer the clarified lysis solution in the collection tube to a new Ep tube and measure the volume;

[0061] (7) Add 0.5 times the volume of anhydrous ethanol and vortex at maximum speed for 20 seconds (precipitation may occur at this time, but it will not affect subsequent operations);

[0062] (8) Insert HiBind RNAcolumn (yellow) into a 2mL collection tube, transfer 700μL of sample (including the precipitate formed) into the column, centrifuge at 12,000rpm for 1min, discard the waste liquid and recover the collection tube, repeat until all samples are transferred into the column;

[0063] (9) Add 400 μL of RWF buffer, centrifuge at 10,000 rpm for 30 s, and discard the waste liquid and collection tube;

[0064] (10) Transfer the HiBind RNA column to a new collection tube, add 500 μL RNA washbuffer II, centrifuge at 10,000 rpm for 30 s, discard the waste liquid, and recover the collection tube;

[0065] (11) Add 500 μL RNAwashbuffer II, centrifuge at 10,000 rpm for 30 s, discard the waste liquid, recover the collection tube, centrifuge at the maximum speed for 2 min, and evaporate the ethanol.

[0066] (12) Transfer the HiBind RNA column to a new Eppendorf tube, air dry for 3 min, add 50 μL of DEPC water to wash for 5 min, centrifuge at maximum speed for 1 min to obtain RNA, and take 5 μL to determine the RNA concentration and purity using NanoDrop. Usually, the A260 / A280 ratio of RNA samples with good purity is between 1.9 and 2.0, and the A260 / A230 is usually greater than 2.

[0067] 2) Reverse transcription of cDNA and amplification of fragments using cDNA as a template (the reverse transcription experiment was performed using the TransScriptOne-Step gDNA Removal and cDNA Synthesis SuperMix kit).

[0068] (1) Reverse transcription of cDNA: RNase-free PCR tubes were used to amplify cDNA using a reverse transcription kit. Reaction system:

[0069]

[0070] To achieve higher synthesis efficiency, the RNA template, primers, and RNase-free water were first mixed and incubated in a PCR instrument at 65°C for 5 min, followed by an ice bath for 2 min. Then, other reaction components were added. The mixture was incubated at 42°C for 30 min, and then heated at 85°C for 5 s to inactivate TransScript RT / RI and gDNA Remover. The concentration of cDNA was detected using a UV spectrophotometer.

[0071] (2) PCR amplification: Using the cDNA obtained from reverse transcription as a template, PCR amplification was performed using primers CYP68BE1-F1 (SEQ ID NO: 3) and CYP68BE1-R1 (SEQ ID NO: 4) to obtain the hydroxylase gene CYP68BE1 (SEQ ID NO: 2). The sequences of the primers CYP68BE1-F1 (SEQ ID NO: 3) and CYP68BE1-R1 (SEQ ID NO: 4) used are listed in Table 1. The PCR products were detected by 1.0% agarose gel electrophoresis. Figure 1 ).

[0072] Table 1 Primer sequences

[0073]

[0074] After the PCR reaction, 4 μL of the PCR product was ligated to 1 μL of the cloning vector pEASY-Blunt simple vector. The ligation conditions were as follows: room temperature (20℃-37℃) for 20 min. The ligation product was directly transformed into Trans1-T1 competent cells, and single colonies were picked for PCR verification. Transformants with correct PCR results were selected for sequencing confirmation. The sequencing results showed that the glycosyltransferase gene CYP68BE1 was 1563 bp in length, consistent with the agarose gel electrophoresis results, and had the nucleotide sequence shown in SEQ ID NO: 2. The obtained amplified product was named CYB68BE1.

[0075] Example 2: Construction of gene expression plasmid pESC-LEU-CYP68BE1

[0076] The plasmid pESC-LEU was double-digested with NheI and XhoI. After complete digestion was detected by electrophoresis, the linearized vector was recovered by ethanol precipitation and quantified for later use.

[0077] Using plasmid pEASY-Blunt-simple-CYP68BE1 as a template, PCR amplification of the fragment containing the pESC-LEU homologous arm was performed using primers pESC-LEU-CYP68BE1-F1 (SEQ ID NO: 5) and pESC-LEU-CYP68BE1-R1 (SEQ ID NO: 6). The sequences of the primers pESC-LEU-CYP68BE1-F1 (SEQ ID NO: 5) and pESC-LEU-CYP68BE1-R1 (SEQ ID NO: 6) used are listed in Table 2.

[0078] Table 2 Primer sequences

[0079]

[0080] The fragment was ligated into the NheI / XhoI double-digested pESC-LEU vector using the homologous recombinase Exnase II (Novitamin). The molar ratio of vector to insert was 1:2. The optimal amount of cloning vector used was (0.02 × number of base pairs of cloning vector) ng, and the optimal amount of insert used was (0.04 × number of base pairs of insert) ng. The reaction mixture was prepared on ice and collected to the bottom of the tube after brief centrifugation.

[0081]

[0082] React at 37°C for 30 min, cool on ice, transfer 5 μL of recombinant product to 50 μL of competent cells, and incubate on ice for 30 min. Heat shock at 42°C for 45 s, cool on ice for 2 min, rejuvenate for 1 h, spread on LB agar plates with the corresponding antibiotics, and incubate overnight at 37°C.

[0083] The following day, colony PCR was performed using universal primers GAL1-F / R. Positive transformants selected by colony PCR were sent for sequencing. Transformants with correct sequencing results were cultured and the plasmid pESC-LEU-cyp68be1 was extracted. Figure 2 ).

[0084] Example 3. Construction of engineered Saccharomyces cerevisiae SC-1

[0085] (1) Select a single clone of Saccharomyces cerevisiae INVSc1 and inoculate it into an appropriate amount of liquid yeast extract peptone glucose (YPD) medium. Incubate on a full-temperature shaking incubator at 30℃ and 220 rpm for 16-18 hours until OD reaches the target value. 600 =0.8-1.0. The YPD medium composition is as follows: 2.1% yeast extract, 2% peptone, 2% glucose. Glucose is prepared separately, filtered and sterilized, and 1.5% agar is added to the solid medium. After dispensing, it is sterilized at high temperature. When using, glucose solution is added to make the final concentration 2% (all percentages represent g / 100mL).

[0086] (2) Dispense the bacterial culture into several 1.5 mL EP tubes (4 tubes are the amount of competent cells for one transformation), centrifuge at 4000 rpm for 2 min, and discard the supernatant.

[0087] (3) Combine the bacterial cells, wash twice with sterile double-distilled water, centrifuge at 4000 rpm for 2 min, and discard the supernatant.

[0088] (4) Resuspend each tube with 0.1mM LiAc, blow evenly and let stand, centrifuge at 4000rpm for 1-2min, and discard the supernatant.

[0089] (5) Take 2 mg / ml of ssDNA from -20℃, denature it at 100℃ for 10 min, and then quickly place it on ice to cool.

[0090] (6) Add them in order:

[0091]

[0092] Mix well with a pipette.

[0093] (7) Incubate at 30℃ for 30 min, then heat shock in a water bath at 42℃ for 60 min, inverting and mixing several times during the process.

[0094] (8) Centrifuge at 5000 rpm for 2 min, discard the supernatant, wash twice with sterile double-distilled water, centrifuge again and discard the supernatant. Add an appropriate amount of sterile double-distilled water and mix well. Spread the mixture onto a plate (SD-LEU-), seal the edges of the plate with sealing film, and incubate at 30℃ for 2-3 days.

[0095] (9) The correct positive clone was identified by PCR and named strain SC-1.

[0096] Example 4. Saccharitoyl sacchariculture engineered strain SC-1 catalyzes 4-androst-4-en-3,17-dione (abbreviated as 4AD,1).

[0097] Shake-flask fermentation catalysis: On solid selective medium (formulation: solid yeast selection medium SD (Glu+, LEU-), 10% 10×SD (200mg adenine sulfate, 100mg L-arginine, 500mg L-aspartic acid, 500mg L-glutamic acid, 150mg L-lysine, 100mg L-methionine, 250mg L-phenylalanine, 1875mg L-serine, 1000mg L-threonine, 150mg L-tyrosine, 750mg...) L-valine (dissolved in distilled water, brought to a final volume of 500 mL, filtered through a 0.22 μm filter for sterilization), 2% glucose, 0.005% His, 0.005% Ura, 0.005% Trp, 1.5% agar; all percentages represent g / 100 mL) were used to activate yeast strain SC-1. The yeast strain was then placed in the appropriate liquid selective medium (formulation: liquid yeast selection medium SD (Glu... + Seed culture was prepared in 10% 10×SD, 2% glucose, 0.005% His, 0.005% Ura, and 0.005% Trp (all percentages represent g / 100mL) at 28℃, 220rpm, for 12h. 1mL of this solution was then inoculated into three bottles containing 100mL of liquid yeast selection medium (Glu). + In a 500ml Erlenmeyer flask containing LEU-, incubate at 28℃ with shaking at 220rpm until OD. 600 Yeast cells were collected at 2.5 rpm and washed twice with sterile water. The cells were then resuspended in an equal volume of liquid yeast selection medium SD (Gal). + ,LEU -(Formulation: 10% 10×SD, 2% galactose, 0.005% His, 0.005% Ura, 0.005% Trp; all percentages represent g / 100mL) After induction at 28℃ and 220rpm for 6 h with shaking, substrate 4AD was added to a final concentration of 150mg / L for catalytic reaction. The mixture was then incubated at 28℃ and 220rpm for 2 days with shaking. Three times the volume of extraction solvent (e.g., ethyl acetate) was added, the ethyl acetate layer was concentrated under reduced pressure, reconstituted with 1mL methanol, and the supernatant was centrifuged and filtered through a 0.22μm organic filter into a liquid chromatography bottle for HPLC-MS analysis.

[0098] HPLC-MS conditions: HPLC mobile phase: 0 min, 20% methanol-water; 15 min, 50% methanol-water; 35 min, 100% methanol; flow rate: 0.3 mL / min; column: Shim-pack GIST C18 reversed-phase column (2.1 × 100 mm); UV detection wavelength: 244 nm. MS conditions:

[0099] HPLC-MS results (see) Figure 3 A) showed that, compared with the control group (S. cerevisiae INVSc1), the experimental group (S. cerevisiae INVSc1-CYP68BE1) produced six hydroxylated products (1a, 1b, 1e, 1f, 1g, 1h), with 1b and 1f being the main products. These six products were separated by HPLC using a semi-preparative process. 1 H-NMR, 13 C-NMR and 2D-NMR confirmed their structures; the product structures and transformation pathways are shown in [see...]. Figure 3 B. The spectral data of the products are shown below, where products 1e and 1g are new compounds. Therefore, it can be concluded that the CYP68BE1 protein can catalyze monohydroxylation or dihydroxylation of the substrate 4AD at the 11α and / or 6β and / or 1β positions, and is a hydroxylase at the 11α and / or 6β and / or 1β positions of the steroidal substance 4AD.

[0100] Compound 1a-1h 1 H NMR and 13 The signal attribution for C NMR is as follows:

[0101] 11α-hydroxyandrosta-4,6-diene-3,17-dione (1α): ESI-MS (+): m / z 301.30 [M+H] + ; 1H-NMR(500MHz,CDCl3)δ:6.23(1H,dd,J9.5,2.5Hz,H-7),6.17(1H,dd,J9.5,2.5Hz,H-6),5.7 4(1H,s,H-4), 4.13(1H,ddd,J10.5,10.5,5.0Hz,H-11β),1.29(3H,s,H-19),1.02(3H,s,H-18) 13 C-NMR(125MHz,CDCl3)δ: 35.7(C-1), 34.2(C-2), 199.9(C-3), 125.0(C-4), 162.7(C-5), 129.3(C-6), 136.8(C-7), 37.8(C-8), 56.3(C-6). 9), 35.3(C-10), 67.9(C-11), 48.4(C-12), 43.1(C-13), 48.0(C-14), 21.2(C-15), 35.7(C-16), 217.8(C-17), 14.6(C-18), 17.2(C-19).

[0102] 6β,11α-induced polymer-4-activated-3,17-induced(6β,11α-dihydroxyandrost-4-ene-3,17-dione,1b):ESI-MS(-):m / z 317.15[MH] - 100. 1 H-NMR(500MHz,CDCl3)δ:5.86(1H,s,H-4),4.43(1H,dd,J3.0,3.0Hz,H-6α),4. 16(1H,ddd,J10.5,10.5,5.0Hz,H-11β),1.28(3H,s,H-19),1.00(3H,s,H-18) 13 C-NMR(125MHz,CDCl3)δ: 35.7(C-1), 34.4(C-2), 200.5(C-3), 127.3(C-4), 167.4(C-5), 73.0(C-6), 38.9(C-7), 28.1(C-8), 59.2(C-9). ),39.3(C-10),68.7(C-11),43.0(C-12),48.1(C-13),50.1(C-14),21.1(C-15),36.2(C-16),218.4(C-17),14.7(C-18),20.3(C-19).

[0103] 6β,11α-dihydroxytestosterone (1c): ESI-MS(-): m / z 319.25 [M-H]-; 1 1H-NMR (500 MHz, CDCl3) δ: 5.80 (1H, s, H-4), 4.28 (1H, dd, J = 3.0, 3.0 Hz, H-6α), 4.04 (1H, ddd, J = 10.5, 10.5, 5.0 Hz, H-11β), 3.65 (1H, t, J = 8.5 Hz, H-17α), 1.53 (3H, s, H-19), 0.86 (3H, s, H-18); 13 13C-NMR (125 MHz, CDCl3) δ: 35.2 (C-1), 34.4 (C-2), 200.8 (C-3), 127.1 (C-4), 170.0 (C-5), 73.2 (C-6), 37.1 (C-7), 28.5 (C-8), 60.5 (C-9), 38.9 (C-10), 69.0 (C-11), 49.8 (C-12), 43.7 (C-13), 48.5 (C-14), 22.3 (C-15), 30.6 (C-16), 81.1 (C-17), 12.0 (C-18), 20.3 (C-19).

[0104] 11α-hydroxy-17a-oxa-D-homo-androst-4-en-3,17-dione (1d): ESI-MS(-): m / z 317.15 [M-H]-; 1 1H-NMR (125 MHz, CDCl3) δ: 5.78 (1H, s, H-4), 3.78 (1H, ddd, J = 10.0, 10.0, 4.5 Hz, H-11β), 1.39 (3H, s, H-18), 1.33 (3H, s, H-19); 13 13C-NMR (500 MHz, CDCl3) δ: 37.4 (C-1), 34.6 (C-2), 198.5 (C-3), 125.5 (C-4), 161.1 (C-5), 33 .1 (C-6), 28.4 (C-7), 37.4 (C-8), 58.2 (C-9), 39.7 (C-10), 68.7 (C-11), 49.9 (C-12), 82.6 (C-13), 45.2 (C-14), 19.9 (C-15), 28.4 (C-16), 170.9 (C-17), 21.2 (C-18), 18.2 (C-19).

[0105] 6β,11α-Dihydroxyandrost-4-ene-3,17-dione (1β,11α-dihydroxyandrost-4-ene-3,17-dione, 1e): HR-ESI-MS(+): m / z 319.1896 [M+H] + ; 1 H-NMR(500MHz, CDCl3)δ:5.85(1H,s,H-4),4.19(1H,dd,J=10.5,5.0Hz,H-1α),4 .18(1H,ddd,J=10.5,10.5,5.0Hz,H-11β),1.32(3H,s,H-19),0.93(3H,s,H-18); 13 C-NMR (125MHz, CDCl3) δ: 72.3 (C-1), 42.7 (C-2), 198.2 (C-3), 125.5 (C-4), 167.6 (C-5), 33.7 (C-6), 30.9 (C-7), 34.7 (C-8), 60.1 (C-9 ),46.6(C-10),67.1(C-11),42.2(C-12),47.6(C-13),49.7(C-14),22.2(C-15),35.9(C-16),218.4(C-17),14.4(C-18),12.5(C-19).

[0106] 11α-hydroxyandrost-4-ene-3,17-dione (1f): ESI-MS(-): m / z 301.15[MH]-; 1 H-NMR(500MHz, CDCl3)δ:5.77(1H,s,H-4),4.09(1H,ddd,J=10.5,10.5,5.0Hz,H-11β),1.36(3H,s,H-19),0.95(3H,s,H-18); 13 C-NMR(125MHz,CD3Cl)δ:37.4(C-1),33.4(C-2),200.3(C-3),124.8(C-4),170.2(C-5),34.2(C-6),30.2(C-7),34.6(C-8),59.2(C-9 ),40.0(C-10),68.7(C-11),42.9(C-12),48.1(C-13),50.0(C-14),21.7(C-15),35.8(C-16),218.8(C-17),14.7(C-18),18.4(C-19).

[0107] 1β,11α-dihydroxytestosterone (1 g): HR-ESI-MS (+): m / z 321.2051 [M+H] + ; 1 H-NMR(500MHz, CDCl3)δ:5.82(1H,s,H-4),4.16(1H,dd,J=10.0,5.0Hz,H-1α),4.15(1H,ddd,J= 10.5,10.5,5.0Hz,H-11β),3.71(1H,t,J=7.0Hz,H-17α),1.32(3H,s,H-19),0.81(3H,s,H-18); 13 C-NMR (125MHz, CDCl3) δ: 72.5 (C-1), 42.7 (C-2), 198.5 (C-3), 125.2 (C-4), 168.4 (C-5), 33.9 (C-6), 31.7 (C-7), 35.4 (C-8), 60.1 (C-9 ),46.6(C-10),67.3(C-11),47.6(C-12),43.2(C-13),49.5(C-14),23.7(C-15),30.7(C-16),81.2(C-17),11.9(C-18),12.5(C-19).

[0108] 11α-hydroxytestosterone (1h): ESI-MS (-): m / z 303.30 [MH]-; 1 H-NMR (500MHz, CDCl3); 1 H-NMR (500MHz, CDCl3) δ: 5.73 (1H, s, H-4), 4.04 (1H, ddd, J = 10.5, 10.5, 5.0Hz ,H-11β),3.69(1H,t,J=7.0Hz,H-17α),1.36(3H,s,H-19),0.95(3H,s,H-18).

[0109] Using recombinant bacteria SC-1 to catalyze 4AD, 11α-hydroxylation intermediate 1f is generated, which can be used as an important intermediate for the industrial synthesis of hydrocortisone; intermediate 1f and product 1a can both be used as important intermediates for the industrial synthesis of eplerenone; 11α,1β-hydroxylation intermediate 1g is generated, which can be used as an important intermediate for the synthesis of strophanthidin G. Figure 4 A schematic diagram is shown showing the synthesis of hydrocortisone, eplerenone, and strophanthidin G using recombinant bacteria SC-1 (solid arrows indicate catalysis by recombinant bacteria SC-1 / microsomal protein, and dashed arrows indicate chemical catalysis).

[0110] Example 5. Saccharomyces cerevisiae engineered strain SC-1 catalyzes testosterone (2) (whole-cell catalysis)

[0111] Using testosterone (2) as a substrate, the reaction was carried out using the same whole-cell catalytic method as in Example 4.

[0112] HPLC-MS results (see) Figure 5 A) shows that, compared with the control group (S. cerevisiae INVSc1), the experimental group (S. cerevisiae INVSc1-CYP68BE1) produced three hydroxylated products (1c, 1g, and 1h), with 1c and 1h being the main products. The product structures and transformation pathways are shown in [Figure 1]. Figure 5 B. Therefore, it can be concluded that the CYP68BE1 protein can catalyze monohydroxylation or dihydroxylation of the substrate testosterone at the 11α and / or 6β and / or 1β positions, and is a hydroxylase at the 11α and / or 6β and / or 1β positions of the steroidal substance testosterone.

[0113] Example 6. Saccharomyces cerevisiae engineered strain SC-1 catalyzes 1,4-androstenedione (3) (whole-cell catalysis)

[0114] The reaction was carried out using 1,4-androstenedione (3) as a substrate, with the same whole-cell catalytic method as in Example 4.

[0115] HPLC-MS results (see) Figure 6 A) showed that, compared with the control group (S. cerevisiae INVSc1), the experimental group (S. cerevisiae INVSc1-CYP68BE1) exhibited two hydroxylated products (3a and 3b). These two products were separated by semi-preparative HPLC. 1 H-NMR, 13 C-NMR confirmed their structures; the product structures and transformation pathways are shown in [see attached diagram]. Figure 6 B. The spectral data of the product are as follows. Therefore, it can be concluded that the CYP68BE1 protein can catalyze the monohydroxylation reaction at the 11α position of the substrate 1,4-Androstenedione, and is an 11α-hydroxylase of the steroidal substance 1,4-Androstenedione.

[0116] The NMR signal assignments for compounds 3a and 3b are as follows:

[0117] 11α-hydroxyandrosta-1,4-diene-3,17-dione (3a): ESI-MS(+): m / z 301.15 [M+H] + ; 1 H-NMR (500MHz, CDCl3) δ: 7.78 (1H, d, J = 10.0Hz, H-1), 6.17 (1H, d, J = 10.0Hz, H-2), 6.12 (1H ,s,H-4),4.13(1H,ddd,J=10.5,10.5,5.0Hz,H-11β),1.33(3H,s,H-19),0.95(3H,s,H-18); 13 C-NMR (125MHz, CDCl3) δ: 158.7 (C-1), 125.1 (C-2), 186.8 (C-3), 124.8 (C-4), 167.6 (C-5), 32.2 (C-6), 32.9 (C-7), 34.0 (C-8), 60.5 (C- 9),43.9(C-10),67.8(C-11),42.3(C-12),47.9(C-13),49.5(C-14),21.9(C-15),35.8(C-16),218.3(C-17),14.6(C-18),18.7(C-19).

[0118] 11α,17β-dihydroxyandrost-1,4-dien-3-one (3b): ESI-MS(+): m / z 303.15 [M+H] + ; 1 H-NMR (500MHz, CDCl3) δ: 7.77 (1H, d, J = 10.0 Hz, H-1), 6.16 (1H, d, J = 10.0 Hz, H-2), 6.12 (1H, s, H-4), 4.13 (1H,ddd,J=10.5,5.0,5.0Hz,H-11β),3.68(1H,t,J=8.5Hz,H-17α),1.32(3H,s,H-19),0.84(3H,s,H-18); 13C-NMR (125MHz, CDCl3) δ: 158.8 (C-1), 125.1 (C-2), 186.8 (C-3), 124.6 (C-4), 167.9 (C-5), 33.0 (C-6), 33.1 (C-7), 34.7 (C-8), 60.6 (C- 9),44.0(C-10),68.1(C-11),43.4(C-12),47.9(C-13),49.5(C-14),23.4(C-15),30.6(C-16),80.8(C-17),12.2(C-18),18.7(C-19).

[0119] Example 7. Progesterone (4) catalysis by engineered Saccharomyces cerevisiae SC-1 (whole-cell catalysis)

[0120] Using progesterone (4) as a substrate, the reaction was carried out using the same whole-cell catalytic method as in Example 4.

[0121] HPLC-MS results (see) Figure 7 A) showed that, compared with the control group (S. cerevisiae INVSc1), the experimental group (S. cerevisiae INVSc1-CYP68BE1) exhibited three hydroxylated products (4a, 4b, and 4c). These three products were semi-preparatively separated by HPLC. 1 H-NMR, 13 C-NMR confirmed their structures; the product structures and transformation pathways are shown in [see attached diagram]. Figure 7 B. The spectral data of the product are as follows. Therefore, it can be concluded that the CYP68BE1 protein can catalyze the monohydroxylation or dihydroxylation of the substrate progesterone at the 11α and / or 6β positions, and is a hydroxylase at the 11α and / or 6β positions of the steroid progesterone.

[0122] The NMR signals of compounds 4a-4c are assigned as follows:

[0123] 6β,11α-dihydroxyprogesterone (4a): ESI-MS (-): m / z 345.20 [MH]-; 11H-NMR (500 MHz, CDCl3) δ: 5.85 (1H, s, H-4), 4.38 (1H, dd, J = 3.0, 3.0 Hz, H-6α), 4.12 (1H, ddd, J = 10.5, 10.5, 5.0 Hz, H-11β), 2.17 (3H, s, H-21), 1.54 (3H, s, H-19), 0.76 (3H, s, H-18); 13 13C-NMR (125 MHz, CDCl3) δ: 37.5 (C-1), 34.5 (C-2), 200.8 (C-3), 127.2 (C-4), 167.9 (C-5), 73.2 (C-6), 39.0 (C-7), 28.4 (C-8), 59.0 (C-9), 39.3 (C-10), 69.0 (C-11), 50.5 (C-12), 44.3 (C-13), 55.3 (C-14), 24.2 (C-15), 23.0 (C-16), 63.2 (C-17), 14.4 (C-18), 20.3 (C-19), 208.8 (C-20), 31.4 (C-21).

[0124] 11α-Hydroxyprogesterone-6-one (4b): ESI-MS(+): m / z 345.20 [M + H] + ; 1 1H-NMR (500 MHz, CDCl3) δ: 6.24 (1H, s, H-4), 4.13 (1H, ddd, J = 10.5, 10.5, 5.0 Hz, H-11β), 2.18 (3H, s, H-21), 1.34 (3H, s, H-19), 0.73 (3H, s, H-18); 13 13C-NMR (125 MHz, CDCl3) δ: 37.7 (C-1), 34.2 (C-2), 199.8 (C-3), 121.7 (C-4), 160.4 (C-⑤), 200.3 (C-6), 46.1 (C-7), 33.1 (C-8), 56.5 (C-9), 41.3 (C-10), 68.1 (C-11), 50.0 (C-12), 43.8 (C-13), 55.6 (C-14), 23.0 (C-15), 24.1 (C-16), 62.8 (C-17), 14.3 (C-18), 18.8 (C-19), 208.3 (C-20), 31.3 (C-21).

[0125] 11α-hydroxyprogesterone (4c): ESI-MS (+): m / z 333.15 [M+H] + ; 1 H-NMR(500MHz,CDCl3)δ:5.75(1H,s,H-4),4.13(1H,ddd,J=10.5,10.5,5.0Hz,H-11β),2.15(3H,s,H-21),1.33(3H,s,H-19),0.71(3H,s,H-18); 13 C-NMR(125MHz, CDCl3)δ:37.5(C-1),34.9(C-2),200.3(C-3),124.6(C-4 ),171.0(C-5),34.2(C-6),31.5(C-7),33.6(C-8),59.0(C-9),39.5(C-10 ),68.9(C-11),50.4(C-12),44.2(C-13),55.3(C-14),24.2(C-15),22.9( C-16),63.1(C-17),14.5(C-18),18.3(C-19),208.9(C-20),31.4(C-21).

[0126] Example 8. Saccharitomyces SC-1 catalyzing estrone (5) (whole-cell catalysis)

[0127] The reaction was carried out using estrone (5) as a substrate and the same whole-cell catalytic method as in Example 4.

[0128] HPLC-MS results (see) Figure 8 A) shows that, compared with the control group (S. cerevisiae INVSc1), the experimental group (S. cerevisiae INVSc1-CYP68BE1) exhibited four hydroxylated products (5a, 5b, 5c, and 5d). ESI-MS results indicate that 5a and 5d are likely monohydroxylated products of the substrate, 5d is likely a dihydroxylated product of the substrate, and 5c is likely a reduction product of a monohydroxylated product. Three of these products (5a, 5b, and 5c) were separated using HPLC semi-preparative separation. 1 H-NMR, 13 C-NMR confirmed their structures, and product 5b was a new compound. The product structure and transformation route diagram are shown below. Figure 8B. The product spectral data are as follows. Therefore, it can be concluded that the CYP68BE1 protein can catalyze the monohydroxylation or dihydroxylation of the substrate Estrone at the 11α and / or 6β positions, and is an 11α and / or 6β-hydroxylase of the steroid Estrone.

[0129] The NMR signals of compounds 5a-5c are assigned as follows:

[0130] 11α-hydroxyestrone (5a): HR-ESI-MS (+): m / z 287.1638 [M+H] + ; 1 H-NMR(500MHz,CD3OD)δ:7.09(1H,d,J=8.5Hz,H-1),6.54(1H,dd,J=3.0,8.5Hz,H-2), 6.49(1H,d,J=3.0Hz,H-4), 4.40(1H,ddd,J=10,8.0,6.5Hz,H-11β), 0.95(3H,s,H-18); 13 C-NMR(125MHz,CD3OD)δ:126.1(C-1),112.5(C-2),154.7(C-3),114.6(C-4),137.4(C-5),29.4(C-6),27.3(C-7),38.8(C-8),56 .5(C-9),130.3(C-10),69.1(C-11),44.1(C-12),45.6(C-13),50.7(C-14),25.9(C-15),31.5(C-16),218.0(C-17),14.6(C-18).

[0131] 6β-hydroxy-9-dehydroestrone (5b): HR-ESI-MS(-): m / z 283.1339 [MH]-; 1 H-NMR (500MHz, CD3OD) δ: 7.48 (1H, d, J = 8.5 Hz, H-1), 6.72 (1H, d, J = 3.0 Hz, H-2), 6.69 (1H, dd, J = 3. 0,8.5Hz,H-4),6.15(1H,t,J=3.0Hz,H-11),4.71(1H,dd,J=3.0,3.0Hz,H-6α),0.98(3H,s,H-18); 13C-NMR(125MHz,CD3OD)δ:126.4(C-1),116.9(C-2),157.9(C-3),117.3(C-4),139.0(C-5),68.1(C-6),37.0(C-7),37.1(C-8),127 .0(C-9),136.8(C-10),117.8(C-11),32.9(C-12),47.7(C-13),50.8(C-14),23.4(C-15),35.0(C-16),224.3(C-17),14.8(C-18).

[0132] 6β-hydroxyestrone (5c): ESI-MS(-): m / z 285.10[MH]-; 1 H-NMR (500MHz, CD3OD) δ: 7.16 (1H, d, J = 8.5 Hz, H-1), 6.80 (1H, d, J = 3.0 Hz, H-2), 6. 70(1H,dd,J=3.0,8.5Hz,H-4),4.69(1H,dd,J=2.5,4Hz,H-6α),0.95(3H,s,H-18); 13 C-NMR(125MHz,CD3OD)δ:127.2(C-1),117.6(C-2),156.6(C-3),116.1(C-4),140.1(C-5),68.2(C-6),26.8(C-7),33.9(C-8),45 .4(C-9),132.1(C-10),36.7(C-11),36.2(C-12),47.9(C-13),51.2(C-14),22.4(C-15),32.8(C-16),223.7(C-17),14.7(C-18).

[0133] Example 9. Catalysis of estradiol-4,9-diene-3,17-dione (6) by engineered Saccharomyces cerevisiae SC-1 (whole-cell catalysis)

[0134] The reaction was carried out using estradiol-4,9-diene-3,17-dione (6) as a substrate, and the reaction was carried out using the same whole-cell catalytic method as in Example 4.

[0135] HPLC-MS results (see) Figure 9A) showed that, compared with the control group (S. cerevisiae INVSc1), the experimental group (S. cerevisiae INVSc1-CYP68BE1) exhibited four hydroxylated products (6a, 6b, 6c, and 6d). The four products were separated by semi-preparative HPLC. 1 H-NMR, 13 C-NMR and 2D-NMR confirmed their structures. Products 6b and 6d are new compounds. The product structures and transformation routes are shown in [reference needed]. Figure 9 B. The spectral data of the product are as follows. Therefore, it can be concluded that the CYP68BE1 protein can catalyze the monohydroxylation reaction at the 11α / β and / or 1α positions of the substrate Estra-4,9-diene-3,17-dione, and is an 11α / β and / or 1α-hydroxylase for the steroid Estra-4,9-diene-3,17-dione.

[0136] The NMR signals of compounds 6a-6d are assigned as follows:

[0137] 11α-hydroxyestra-4,9-diene-3,17-dione (6a): HR-ESI-MS (+): m / z 287.1637 [M+H] + ; 1 H-NMR (500MHz, CDCl3) δ: 5.79 (1H, s, H-4), 4.83 (1H, dd, J = 9.0, 6.0 Hz, H-11β), 1.00 (3H, s, H-18); 13 C-NMR (125MHz, CD3OD) δ: 36.1 (C-1), 37.5 (C-2), 199.9 (C-3), 124.5 (C-4), 156.7 (C-5), 30.8 (C-6), 26.4 (C-7), 38.5 (C-8), 145. 8(C-9),128.7(C-10),69.6(C-11),41.6(C-12),47.3(C-13),48.8(C-14),22.0(C-15),26.5(C-16),218.6(C-17),15.3(C-18).

[0138] 1α-hydroxyestra-4,9-diene-3,17-dione (6b): HR-ESI-MS(+): m / z 287.1638 [M+H] + ; 1H-NMR (500MHz, CDCl3) δ: 5.79 (1H, s, H-4), 5.10 (1H, dd, J = 3.0, 3.0 Hz, H-1β), 1.04 (3H, s, H-18); 13 C-NMR (125MHz, CDCl3) δ: 64.8 (C-1), 45.5 (C-2), 197.2 (C-3), 122.4 (C-4), 152.9 (C-5), 29.6 (C-6), 25.0 (C-7), 38.4 (C-8), 150. 7(C-9),129.2(C-10),25.6(C-11),36.0(C-12),47.5(C-13),49.8(C-14),22.4(C-15),32.0(C-16),219.5(C-17),13.3(C-18).

[0139] 11β,17β-dihydroxyestra-4,9-diene-3-one (6c): HR-ESI-MS(+): m / z 289.1794 [M+H] + ; 1 H-NMR (500MHz, CDCl3) δ: 5.78 (1H, s, H-4), 5.04 (1H, dd, J = 2.0, 5.0 Hz, H-11α), 3.65 (1H, t, J = 8.0 Hz, H-17α), 1.09 (3H, s, H-18); 13 C-NMR(125MHz,CD3OD)δ:36.0(C-1),37.2(C-2),199.4(C-3),124.4(C-4),157.0(C-5),30.8(C-6),27.1(C-7),39.1(C-8),145 .4(C-9),129.6(C-10),66.2(C-11),42.2(C-12),43.9(C-13),50.4(C-14),23.5(C-15),30.5(C-16),82.2(C-17),12.3(C-18).

[0140] 1α,17β-dihydroxyestra-4,9-diene-3-one (6d): HR-ESI-MS(+): m / z 289.1795 [M+H] + ; 1H-NMR (500MHz, CDCl3) δ: 5.77 (1H, s, H-4), 5.09 (1H, dd, J = 3.5, 3.5 Hz, H-1β), 3.69 (1H, t, J = 8.0 Hz, H-17α), 0.92 (3H, s, H-18); 13 C-NMR (125MHz, CDCl3) δ: 64.9 (C-1), 45.5 (C-2), 197.5 (C-3), 121.8 (C-4), 153.7 (C-5), 29.6 (C-6), 25.5 (C-7), 39.0 (C-8), 152 .8(C-9),128.4(C-10),26.2(C-11),37.2(C-12),43.0(C-13),49.7(C-14),23.8(C-15),30.7(C-16),81.1(C-17),10.6(C-18).

[0141] The recombinant bacteria SC-1 was used to catalyze estra-4,9-diene-3,17-dione(6) to generate intermediate 6c, which can be used as an important intermediate for the industrial synthesis of trenbolone. Figure 10 A schematic diagram of the synthesis of trenbolone using recombinant bacteria SC-1 is shown (solid arrows indicate catalysis by recombinant bacteria SC-1 / microsomal protein, and dashed arrows indicate chemical catalysis).

[0142] Example 10. CYP68BE1 microsomal protein method catalyzes substrates 1-6

[0143] It consists of the following two steps:

[0144] 1) Extraction of microsomal proteins (all reactions described below were performed on ice at 4°C)

[0145] (1) Fermentation culture: 200 ml of the corresponding selective medium was cultured overnight at 30℃ and 220 rpm. 1% inoculum was then added to 200 ml of the corresponding selective medium. OD was measured after 12 h. 600 When the value reaches 2-3, the bacteria are harvested, washed 3 times with sterile water, transferred to 200ml LYPL medium, and induced at 30℃ and 220rpm for 12h before harvesting.

[0146] (2) Bacterial cell collection: Centrifuge at 7000 rpm for 5 min, discard the supernatant, and wash 3 times with sterile water.

[0147] Weigh the bacterial cells and resuspend them in BufferTEK at a ratio of 1 mL of BufferTEK (0.1 M KCl, 500 mM Tris-HCl, pH 7.4) for every 0.5 g of wet bacteria. Centrifuge at room temperature for 5 min, discard the supernatant, and resuspend in 2 mL of BufferTESB (0.6 M sorbitol, 500 mM Tris-HCl, pH 7.4).

[0148] (3) Cell disruption: Add an equal volume of zirconium beads and disrupt the cells using an automated sample grinder. After disruption, incubate on ice for 60 seconds and repeat 3 times. Centrifuge at 12000g, collect the supernatant, wash the zirconium beads with 5mL Buffer TESB, centrifuge at 12000g, and collect the supernatant.

[0149] (4) Microparticle preparation: Add NaCl (to a final concentration of 0.15M) and PEG4000 (to a final concentration of 0.1g / mL) to the supernatant and incubate on ice for 30 min. Centrifuge at 10000 rpm for 10 min, discard the supernatant, collect the microparticles (white, precipitated on the walls and bottom of the centrifuge tube), add 1 mL of Buffer TEG (20% glycerol, 500mM Tris-HCl, pH 7.4), and freeze at -80℃.

[0150] 2) Microsomal protein method catalyzes substrates 1-6

[0151] The total volume of the catalytic system was 2 mL (0.2 mM substrate; 2.6 mg / mL glucose-6-phosphate; 0.4 μU glucose-6-phosphate-dehydrogenase; 5 mg CYP68BE1-containing microsomes; 75 mL M KH2PO4-K2HPO4 pH 7.4). The reaction was initiated with NADPH at 37 °C and terminated with the addition of ethyl acetate as the corresponding extractant. The ethyl acetate layer was concentrated and evaporated to dryness, then reconstituted with 1 mL of methanol solution. The supernatant was centrifuged and filtered through a 0.22 μm organic filter membrane into a liquid chromatography bottle for HPLC-MS analysis, using the same method as in Example 4. The results showed that the microsomes extracted from engineered bacteria SC-1 could catalyze monohydroxylation or dihydroxylation of substrates 1-6 at the 11α and / or 6β and / or 1α and / or 1β positions. The products and yields differed slightly from those catalyzed by the engineered bacteria. HPLC results are shown in [Figure 4]. Figure 11 .

[0152] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these examples without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A hydroxylase CYP68BE1 for hydroxylating steroid compounds at the C11α and / or C6β and / or C1β and / or C1α positions, characterized in that, The amino acid sequence of the hydroxylase CYP68BE1 is shown in SEQ ID NO:

1.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule is a nucleic acid molecule encoding the hydroxylase CYP68BE1 as described in claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that, The sequence of the nucleic acid molecule is shown in SEQ ID NO:

2.

4. An expression carrier, characterized in that, The vector contains the nucleic acid molecule as described in any one of claims 2–3, and the expression vector is a prokaryotic expression vector or a eukaryotic expression vector; the eukaryotic expression vector includes the yeast expression vector PESC series.

5. An engineered bacterium, characterized in that, The engineered bacteria are recombinant microorganisms, and the recombinant bacteria contain the vector as described in claim 4, or the genome of the recombinant bacteria is integrated with the nucleic acid molecules as described in any one of claims 2-3.

6. A hydroxylation method, the method comprising: In the presence of the hydroxylase CYP68BE1 as described in claim 1, a steroidal compound substrate undergoes a hydroxylation reaction, wherein... The steroidal compound substrate is 4-androstenedione or testosterone, which undergoes hydroxylation at the C11α, C11α and C6β, or C11α and C1β positions. The steroidal compound substrate is 1,4-androstenedione, which undergoes a hydroxylation reaction at the C11α position. The steroidal compound substrate is progesterone, which undergoes a hydroxylation reaction at the C11α position, or at both the C11α and C6β positions. The steroidal compound substrate is estrone, which undergoes hydroxylation at C11α, C6β, or C11α and C6β positions, or The substrate of the steroidal compound is estradiol-4,9-diene-3,17-dione, which undergoes hydroxylation at the C1α or C11α / 11β position.

7. The use of the protein of claim 1, the nucleic acid molecule of any one of claims 2-3, the carrier of claim 4, or the engineered bacteria of claim 5 in a hydroxylation reaction as a substrate for a steroid compound, wherein, The steroidal compound substrate is 4-androstenedione or testosterone, which undergoes hydroxylation at the C11α, C11α and C6β, or C11α and C1β positions. The steroidal compound substrate is 1,4-androstenedione, which undergoes a hydroxylation reaction at the C11α position. The steroidal compound substrate is progesterone, which undergoes a hydroxylation reaction at the C11α position, or at both the C11α and C6β positions. The steroidal compound substrate is estrone, which undergoes hydroxylation at C11α, C6β, or C11α and C6β positions, or The substrate of the steroidal compound is estradiol-4,9-diene-3,17-dione, which undergoes hydroxylation at the C1α or C11α / 11β position.

8. A method for producing a steroidal drug and / or a steroidal drug intermediate, comprising whole-cell catalysis or microsomal synthesis; The whole-cell catalytic method includes the following steps: fermenting the engineered bacteria described in claim 5, adding the substrate directly or concentrated bacterial cells before adding the substrate, and carrying out a catalytic reaction. The reaction product contains hydrocortisone and / or eplerenone and / or strophanthidin G and / or trenbolone. The substrate is a substance that can be catalyzed by steroidal 11α and / or C6β and / or C1β hydroxylases to generate hydrocortisone and / or eplerenone and / or strophanthidin G and / or trenbolone. The microsomal method comprises the following steps: preparing microsomes from the engineered bacteria of claim 5, and then catalyzing the substrate in microsomal form to generate hydrocortisone and / or eplerenone and / or strophanthidin G and / or trenbolone; wherein the substrate is a substance capable of being catalyzed by steroidal 11α and / or C6β and / or C1β hydroxylases to generate hydrocortisone and / or eplerenone and / or strophanthidin G and / or trenbolone; in, The substrate is 4-androstenedione or estradiol-4,9-diene-3,17-dione.

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