Steroid hydroxylase in beauveria bassiana as well as coding gene and application thereof

Abstract

The invention relates to the fields of steroid drugs and biotechnology, and relates to a novel steroid hydroxylase CYP68BE1 from beauveria bassiana, and a coding gene and an application of the novel steroid hydroxylase CYP68BE1. Specifically, the invention provides a novel steroid hydroxylase CYP68BE1 from beauveria bassiana, a nucleic acid molecule encoding the enzyme, a vector containing the nucleic acid molecule, and a recombinant bacterium containing the vector or integrated with the nucleic acid molecule. According to the application disclosed by the invention, through an experiment of transforming steroids by recombinant saccharomycetes or microsomes, the CYP68BE1 enzyme is found to be capable of catalyzing a hydroxylation reaction on C11alpha and / or C6beta and / or C1beta and / or C1alpha of a steroid compound. Wherein the conversion product comprises a plurality of steroid drug intermediates and / or steroid active compounds, and the method has great application potential and economic value.

Description

Technical Field

[0001] The present invention relates to the field of steroidal drugs and biotechnology, and more specifically, to a steroidal hydroxylase obtained from Beauveria bassiana and its encoding gene. The present invention also relates to the use of the steroidal hydroxylase in the preparation of steroidal drug intermediates and steroidal active compounds. Background Art

[0002] Steroidal compounds have important and extensive pharmacological activities, such as anti-infection, anti-tumor, anti-inflammatory, and anti-AIDS. Currently, there are more than 400 steroidal drugs widely used to treat inflammation, cancer, rheumatism, arthritis and other diseases. Hydroxyl is an important functional group on the skeleton of steroidal drugs. The hydroxylation of steroidal nuclei by chemical methods is usually complicated and inefficient. However, compared with chemical synthesis, microbial transformation has the advantages of mild reaction conditions, green environmental protection, and simpler experimental processes. Therefore, the use of microbial transformation methods for hydroxylation of specific positions of steroidal compounds has become a key technology in the field of steroidal pharmaceutical industry, laying the foundation for the industrial production of steroidal drugs and the development of innovative steroidal drugs. The positions of microbial hydroxylation reported so far 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 usually used as biomaterials for steroid microbial catalytic hydroxylation in industry, but due to the relatively complex culture conditions of fungi, some fungi may produce some toxins and harmful substances or cause certain environmental pollution. Therefore, exploring the relevant steroid hydroxylases in fungi, selecting hosts with simple background, easy culture, high efficiency and harmless to the environment (Saccharomyces cerevisiae, Pichia pastoris, etc.) to construct relevant engineering bacteria, and applying them to the production of key intermediates of steroid medicines will surely bring the industrial manufacturing of steroid drugs into a new stage.

[0004] It is reported that Beauveria bassiana can carry out microbial hydroxylation reaction (11α monohydroxylation, 11α, 6β dihydroxylation, 11α, 6β, 17β trihydroxylation) on steroidal compounds, with high conversion rate 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). There are 83 cytochrome P450 monooxygenases (CYP450 genes) in Beauveria bassiana, but the CYP450 related to steroidal hydroxylation has not been identified. Therefore, it is of great significance to explore the CYP450 with high efficiency catalytic hydroxylation ability in Beauveria bassiana and apply it to the production of steroidal drugs or their key intermediates. Summary of the invention

[0005] The technical problem solved by the present invention is to provide a highly efficient steroid hydroxylase in Beauveria bassiana and its amino acid sequence, a nucleic acid molecule encoding the enzyme, and a vector and a recombinant cell containing the nucleic acid molecule, and to provide a production method for single-site and / or multi-site hydroxylation reactions (11 and / or 6 and / or 1 positions) of steroid substances.

[0006] In order to solve the above technical problems, the present invention discovered a steroid hydroxylase CYP68BE1 gene from Beauveria bassiana. The recombinant bacterial cells obtained by heterologous expression have the ability to catalyze hydroxylation reactions at positions 11 and / or 6 and / or 1 of sterol compounds to produce a variety of steroid drugs and / or steroid drug intermediates.

[0007] Therefore, in the first aspect, the present invention provides steroid hydroxylase CYP68BE1, the amino acid sequence of CYP68BE1 is shown in SEQ ID NO: 1, or a sequence with the same function obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO: 1.

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

[0009] The protein provided by the present invention is a protein shown in any one of the following (P1)-(P4):

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

[0011] (P2) a protein having the same function as the amino acid sequence defined in (P1) after one or more amino acid residues are substituted and / or deleted and / or added;

[0012] (P3) a protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology with the amino acid sequence defined in (P1) or (P2) and having the same function;

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

[0014] In a second aspect, the present invention provides a nucleic acid molecule, which is a nucleic acid molecule encoding the CYP68BE1 of the present invention.

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

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

[0017] In the present invention, the nucleic acid molecule encoding steroid hydroxylase CYP68BE1 of the present invention (eg, a DNA sequence encoding an optional spacer sequence) can be obtained by chemical synthesis.

[0018] Furthermore, the nucleic acid molecule may be a DNA molecule as shown in any one of the following (S1)-(S3):

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

[0020] (S2) a DNA molecule that hybridizes with the DNA molecule defined in (S1) under stringent conditions and encodes a protein as shown in any one of (P1) to (P4) described in the first aspect above;

[0021] (S3) A DNA molecule having a homology of more than 99%, more than 95%, more than 90%, more than 85% or more than 80% with the DNA sequence defined in any one of (S1) and (S2) and encoding a protein shown in any one of (P1)-(P4) described in the first aspect above.

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

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

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

[0025] An expression vector, shuttle vector or integration vector containing the CYP68BE1 encoding DNA sequence of the present invention and appropriate transcription / translation control signals is constructed using methods well known to those skilled in the art, including in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc.

[0026] For example, a variety of yeast cell expression vectors well known to those skilled in the art may 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 and pESC-TRP. For example, Escherichia coli cell expression vectors well known to those skilled in the art may 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, which contains the vector described in the third aspect of the present invention, or the nucleic acid molecule described in the second aspect of the present invention is integrated into its genome.

[0028] According to some embodiments of the present invention, the recombinant cell is a eukaryotic cell or a prokaryotic cell. Preferably, the recombinant cell is a Saccharomyces cerevisiae cell, a Pichia pastoris cell or an Escherichia coli cell. The yeast may be Saccharomyces cerevisiae (such as BY4742 strain), Yarrowia lipolytica (such as PO1G strain), Schizosaccharomyces pombe (such as CICC1762 strain) or Pichia pastoris (such as GS115 strain), etc.

[0029] In a preferred embodiment, the recombinant cell is a yeast cell, preferably a Saccharomyces cerevisiae cell.

[0030] The appropriate method for introducing the vector or nucleic acid molecule into the cell can be selected according to the type of recombinant cell. These methods are well known to those skilled in the art. For example, the vector can be transformed into a Saccharomyces cerevisiae cell by methods known in the art, or the nucleic acid molecule fragment can be integrated into the genome of Saccharomyces cerevisiae by homologous recombination. Among them, the method for transforming Saccharomyces cerevisiae can use various transformation methods well known to those skilled in the art, such as electrotransformation, lithium acetate chemical transformation, etc.

[0031] In a fifth aspect, the present invention provides a method for hydroxylating a steroid drug and / or a steroid drug intermediate, the method comprising: carrying out a hydroxylation reaction of a steroid compound in the presence of hydroxylase CYP68BE1.

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

[0033] According to some embodiments of the present invention, the method can be in the form of a whole-cell catalytic method or an enzymatic catalytic method, preferably a whole-cell catalytic method.

[0034] In a specific embodiment of the present invention, a whole-cell catalytic method is specifically used. Further, an engineered bacterium containing the cyp68be1 gene is fermented and cultured, and a steroid compound is added as a substrate to carry out a catalytic reaction. The conditions of the catalytic reaction are specifically 28°C, 200-250rpm (such as 220rpm), and shaking culture for 1-4 days (such as 2 days); the substrate in the reaction system is, and its concentration is 10mg-200mg (such as 150mg / L).

[0035] In a sixth aspect, the present invention provides use of CYP68BE1 for producing hydrocortisone and / or eplerenone and / or strophanthin and / or trenbolone.

[0036] Beneficial technical effects:

[0037] The steroid hydroxylase CYP68BE1 in Beauveria bassiana provided by the present invention can catalyze the hydroxylation reaction of steroid precursor compounds at C11 and / or C6 and / or C1 positions, thereby generating steroid drugs and / or steroid drug intermediates. In addition, CYP68BE1 can also participate in the construction of metabolic pathways of steroid drugs and / or steroid drug intermediates as a basic element, and develop new resources for producing steroid drugs and / or steroid drug intermediates through synthetic biology technology, which has great economic value and good application potential.

[0038] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[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 detected by the yeast recombinant strain SC-1 catalyzing 4AD is shown. Figure 3 A shows the HPLC profiles of 4AD catalyzed by the control group SC-5 (S. cerevisiae INVSc1) and the experimental group yeast recombinant bacteria SC-1. Figure 3 B shows the product structure and conversion route of 4AD catalyzed by yeast recombinant bacteria SC-1.

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

[0043] Figure 5 The graph shows the reaction product of Testosterone catalyzed by the recombinant yeast SC-1 detected by LC-MS. Figure 5 A shows the HPLC spectrum of Testosterone catalyzed by the control group SC-5 (S. cerevisiae INVSc1) and the experimental group yeast recombinant bacteria SC-1. Figure 5 B shows the product structure and conversion route of testosterone catalyzed by the yeast recombinant strain SC-1.

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

[0045] Figure 7 The graph shows the reaction product of Progesterone catalyzed by the yeast recombinant strain SC-1 detected by LC-MS. Figure 7 A shows the HPLC profiles of Progesterone catalyzed by the control group SC-5 (S. cerevisiae INVSc1) and the experimental group yeast recombinant bacteria SC-1. Figure 7 B shows the product structure and conversion route of Progesterone catalyzed by the yeast recombinant strain SC-1.

[0046] Figure 8 The graph shows the reaction product of Estrone catalyzed by the yeast recombinant bacteria SC-1 detected by LC-MS. Figure 8 A shows the HPLC spectra of Estrone catalyzed by the control group SC-5 (S. cerevisiae INVSc1) and the experimental group yeast recombinant bacteria SC-1. Figure 8 B shows the product structure and conversion route of Estrone catalyzed by the yeast recombinant bacteria SC-1.

[0047] Fig. 9 The LC-MS spectrum shows the reaction product of Estra-4,9-diene-3,17-dione detected by the yeast recombinant strain SC-1. Fig. 9 A shows the HPLC spectra of Estra-4,9-diene-3,17-dione catalyzed by the control group SC-5 (S.cerevisiae INVSc1) and the experimental group yeast recombinant bacteria SC-1. Fig. 9 B shows the product structure and conversion route of Estra-4,9-diene-3,17-dione catalyzed by the yeast recombinant strain SC-1.

[0048] Fig.10The CYP68BE1 protein is used in the synthesis of hydrocortisone. The SC-1 recombinant bacteria catalyzes Estra-4,9-diene-3,17-dione to generate 11α-hydroxylated intermediate 6c, which can be used as an important intermediate for the industrial synthesis of trenbolone (solid arrows indicate catalysis by SC-1 recombinant bacteria, and dotted arrows indicate chemical catalysis).

[0049] Fig.11 The LC-MS spectra of the reaction products of sterol substrates 1-6 detected by Saccharomyces cerevisiae microsomes containing CYP68BE1 are shown. Fig.11 The substrate in A is 4AD; Fig.11 The substrate in B is testosterone; Fig.11 The substrate in C is 1,4-Androstenedione; Fig.11 The substrate in D is progesterone; Fig.11 The substrate in E is Estrone; Fig.11 The substrate in F is Estra-4,9-diene-3,17-dione. DETAILED DESCRIPTION

[0050] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments of the present invention. Those skilled in the art should understand that these embodiments are only for illustrative purposes and do not limit the scope of the present invention. The protection scope of the present invention is only limited 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 belong to the protection scope of the present invention.

[0051] In addition, it should be noted that, unless otherwise specified, the various materials and reagents used in the following examples are commonly used materials and reagents in the art and can be obtained through conventional commercial channels; the 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 is divided into two steps:

[0054] 1) Extraction of total RNA from Beauveria bassiana (total RNA was extracted using 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 50 mL YM (yeast powder-maltose) culture medium;

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

[0057] (3) Take several 1.5 mL RNase-free Eppendorf tubes and place them in an Eppendorf tube rack. Add 500 μL of RB buffer to each tube, and then add 10 μL of β-mercaptoethanol (4°C refrigerator).

[0058] (4) Pour a small amount of liquid nitrogen into a mortar (wrapped in tin foil and placed in an oven at 180°C for more than 3 hours to remove RNase) to cool it down, and use the handle of a spoon to pick up the bacteria and crush them in the mortar. After adding liquid nitrogen, grind quickly, and repeat 4-5 times until it is ground into a fine powder. 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 2 mL collection tube, transfer the lysate to the column, and centrifuge at 14,000 rpm for 5 min.

[0060] (6) Transfer the clarified lysate in the collection tube to a new Eppendorf 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 the HiBind RNAcolumn (yellow) into a 2 mL collection tube, transfer 700 μL of sample (including the formed precipitate) into the column, centrifuge at 12,000 rpm for 1 min, discard the waste liquid and return to the collection tube, and repeat until all samples are transferred to the column;

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

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

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

[0067] 2) Reverse transcription of cDNA and fragment amplification using cDNA as template (the reverse transcription experiment uses the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix Kit).

[0068] (1) Reverse transcription of cDNA: Take an RNase-free PCR tube and amplify cDNA using the reverse transcription kit. Reaction system:

[0069]

[0070] In order to obtain higher synthesis efficiency, RNA template, primers and RNase-free water were first mixed, incubated at 65℃ for 5min in a PCR instrument, ice bathed for 2min, and then other reaction components were added. Incubated at 42℃ for 30min, heated at 85℃ for 5s to inactivate TransScript RT / RI and gDNARemover. The concentration of cDNA was detected by UV spectrophotometer.

[0071] (2) PCR amplification: The cDNA obtained by reverse transcription was used as a template, and 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 product was detected by 1.0% agarose gel electrophoresis ( Figure 1 ).

[0072] Table 1 Primer sequences

[0073]

[0074] After the PCR reaction, 4 μL of PCR product was connected to 1 μL of cloning vector pEASY-Blunt simple vector. The reaction conditions for connecting the PCR product and the cloning vector were as follows: react at room temperature (20°C-37°C) for 20 minutes. The ligation product was directly transformed into Trans1-T1 competent cells, and a single colony was picked for PCR verification. The transformant with the correct PCR result was selected for sequencing confirmation. The sequencing results showed that the length of the glycosyltransferase gene CYP68BE1 was 1563 bp, which was consistent with the agarose gel electrophoresis results and had a nucleotide sequence as 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 the digestion was complete 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, primers pESC-LEU-CYP68BE1-F1 (SEQ ID NO: 5) and pESC-LEU-CYP68BE1-R1 (SEQ ID NO: 6) were used to PCR amplify a fragment containing the pESC-LEU homology arm, wherein 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 connected to the NheI / XhoI double-digested pESC-LEU vector using homologous recombinase Exnase II (NoviZan). The molar ratio of vector to insert fragment was 1:2, the optimal amount of cloning vector was (0.02×the number of base pairs of cloning vector) ng, and the optimal amount of insert fragment was (0.04×the number of base pairs of insert fragment) ng. The following reaction system was prepared on ice, and the reaction solution was collected to the bottom of the tube by brief centrifugation.

[0081]

[0082] React at 37℃ for 30min, cool on ice, transfer 5μL of recombinant product to 50μL competent cells, and place on ice for 30min. Heat shock at 42℃ water bath for 45s, cool on ice for 2min, rejuvenate for 1h, spread on LB solid plate with corresponding resistance, and culture at 37℃ overnight.

[0083] The next day, colony PCR was performed using universal primers GAL1-F / R. The positive transformants screened by colony PCR were sent for sequencing. The transformants with correct sequencing were cultured and the plasmid pESC-LEU-cyp68be1( Figure 2 ).

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

[0085] (1) Pick out the monoclonal bacteria of Saccharomyces cerevisiae INVSc1 and inoculate them into an appropriate amount of liquid yeast extract peptone dextrose (YPD) medium. Set the shaking incubator at 30°C and 220 rpm for 16-18 h until the OD 600 =0.8-1.0. The YPD medium is composed of 2.1% yeast extract, 2% peptone, and 2% glucose. Glucose is prepared separately, filtered and sterilized, and 1.5% agar is added to the solid medium, and sterilized at high temperature after packaging. When used, glucose solution is added to make the final concentration 2% (each percentage sign represents g / 100mL).

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

[0087] (3) The cells were combined, washed twice with sterile double-distilled water, centrifuged at 4000 rpm for 2 min, and the supernatant was discarded.

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

[0089] (5) Take out 2 mg / ml ssDNA from -20°C, melt it at 100°C for 10 min, and quickly cool it on ice.

[0090] (6) Add in order:

[0091]

[0092] Mix well with a pipette

[0093] (7) Incubate at 30°C for 30 min and heat-shock at 42°C in a water bath for 60 min, inverting and mixing several times.

[0094] (8) Centrifuge at 5000 rpm for 2 min, discard the supernatant, wash twice with sterile double distilled water, centrifuge and discard the supernatant. Add appropriate amount of sterile double distilled water and mix well, apply to the plate (SD-LEU-), seal the edge of the plate with sealing film, and place in a 30°C constant temperature incubator for 2-3 days.

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

[0096] Example 4. Catalysis of 4-androstenedione (androst-4-en-3,17-dione, 4AD, 1) by engineered yeast SC-1

[0097] Shake flask fermentation catalysis: In solid selection medium (formula: solid yeast screening 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 SC-1 yeast strain was activated in 1% L-valine, dissolved in distilled water, fixed to 500 mL, and sterilized by filtration with a 0.22 μm filter membrane), 2% glucose, 0.005% His, 0.005% Ura, 0.005% Trp, and 1.5% agar; each percentage sign represents g / 100 mL) and the SC-1 yeast strain was cultured in the corresponding liquid selection medium (formula: liquid yeast selection medium SD (Glu + ,LEU-), 10% 10×SD, 2% glucose, 0.005% His, 0.005% Ura, 0.005% Trp; each percentage sign represents g / 100mL) was used to prepare seed solution (28°C, 220rpm, 12h), and 1mL of inoculum was inoculated into 3 bottles containing 100ml of liquid yeast screening medium SD (Glu + ,LEU-) in a 500 ml Erlenmeyer flask at 28°C and 220 rpm shaking until OD 600 =2.5, 5,000 rpm to collect yeast cells, wash them twice with sterile water, and resuspend them in the same volume of liquid yeast screening medium SD (Gal + ,LEU -) (formula: 10% 10×SD, 2% galactose, 0.005% His, 0.005% Ura, 0.005% Trp; each percentage sign represents g / 100mL), after 28°C, 220rpm shaking induction for 6h, add substrate 4AD with a final concentration of 150mg / L to carry out catalytic reaction, and shake culture at 28°C, 220rpm for 2 days. Add 3 times the volume of extractant (such as ethyl acetate), concentrate the ethyl acetate layer under reduced pressure, re-dissolve with 1mL of methanol solution, centrifuge and take the supernatant through a 0.22μm organic filter membrane to a liquid phase bottle for HPLC-MS detection.

[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, chromatographic column: Shim-pack GIST C18 reverse phase column (2.1×100 mm), UV detection wavelength 244 nm. MS conditions:

[0099] HPLC-MS results (see Figure 3 A) shows that compared with the control group (S.cerevisiae INVSc1), the experimental group (S.cerevisiae INVSc1-CYP68BE1) produced 6 hydroxylated products (1a, 1b, 1e, 1f, 1g, 1h), with 1b and 1f as the main products. The above 6 products were separated by HPLC semi-preparative separation. 1 H-NMR, 13 C-NMR and 2D-NMR confirmed their structures. The product structures and transformation routes are shown in Figure 3 B, product spectral data are as follows, among which products 1e and 1g are new compounds. Therefore, it can be concluded that CYP68BE1 protein can catalyze the monohydroxylation or dihydroxylation reaction of the 11α and / or 6β and / or 1β position of the substrate 4AD, and is a 11α and / or 6β and / or 1β position hydroxylase of the steroid substance 4AD.

[0100] Compounds 1a-1h 1 H NMR and 13 The signals of C NMR are assigned as follows:

[0101] 11α-hydroxyandrosta-4,6-diene-3,17-dione (11α-hydroxyandrosta-4,6-diene-3,17-dione, 1a): ESI-MS (+): m / z 301.30 [M+H] + ; 1 H-NMR (500MHz, CDCl 3)δ: 6.23 (1H, dd, J = 9.5, 2.5 Hz, H-7), 6.17 (1H, dd, J = 9.5, 2.5 Hz, H-6), 5.74 (1H, s, H-4), 4.13 (1H, ddd, J = 10.5, 10.5, 5.0 Hz, H-11β), 1.29 (3H, s, H-19), 1.02 (3H, s, H-18); 13 C-NMR (125 MHz, CDCl 3 )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-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-dihydroxyandrost-4-ene-3,17-dione (1b): ESI-MS(-): m / z 317.15 [M-H] - ; 1 H-NMR (500 MHz, CDCl 3 )δ: 5.86 (1H, s, H-4), 4.43 (1H, dd, J = 3.0, 3.0 Hz, H-6α), 4.16 (1H, ddd, J = 10.5, 10.5, 5.0 Hz, H-11β), 1.28 (3H, s, H-19), 1.00 (3H, s, H-18); 13 C-NMR (125 MHz, CDCl 3 )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, CDCl 3 ) 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, CDCl 3 ) 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, CDCl 3 ) 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, CDCl 3 ) 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 / z319.1896[M+H] + ; 1 H-NMR (500MHz, CDCl 3 )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, CDCl 3 )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 (11α-hydroxyandrost-4-ene-3,17-dione, 1f): ESI-MS (-): m / z 301.15 [MH]-; 1 H-NMR (500MHz, CDCl 3 )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, CD 3Cl)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 (1g):HR-ESI-MS(+):m / z321.2051[M+H] + ; 1 H-NMR (500MHz, CDCl 3 )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, CDCl 3 )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, CDCl 3 ); 1 H-NMR (500MHz, CDCl 3)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] The recombinant bacteria SC-1 was used to catalyze 4AD to generate 11α-hydroxylated intermediate 1f, which can be used as an important intermediate for the industrial synthesis of hydrocortisone; intermediate 1f and product 1a, both of which can be used as important intermediates for the industrial synthesis of eplerenone; and 11α,1β-hydroxylated intermediate 1g was generated, which can be used as an important intermediate for the synthesis of strophanthin G. Figure 4 A schematic diagram of the use of recombinant bacteria SC-1 to synthesize hydrocortisone, eplerenone, and strophanthocyanin G is shown (solid arrows indicate catalysis by SC-1 recombinant bacteria / microsomal proteins, and dotted arrows indicate chemical catalysis).

[0110] Example 5. Catalysis of testosterone (2) by engineered yeast SC-1 of Saccharomyces cerevisiae (whole cell catalysis method)

[0111] Testosterone (2) was used as a substrate and 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 hydroxylation products (1c, 1g, 1h), with 1c and 1h as the main products. The product structures and transformation roadmap are shown in Figure 5 B. Therefore, it can be concluded that CYP68BE1 protein can catalyze the monohydroxylation or dihydroxylation reaction of the substrate testosterone at the 11α and / or 6β and / or 1β positions, and is a 11α and / or 6β and / or 1β hydroxylase of the steroid testosterone.

[0113] Example 6. Catalysis of 1,4-androstenedione (3) by engineered yeast SC-1 of Saccharomyces cerevisiae (whole cell catalysis method)

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

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

[0116] The NMR signals of compounds 3a and 3b are assigned as follows:

[0117] 11α-hydroxyandrosta-1,4-diene-3,17-dione (11α-hydroxyandrosta-1,4-diene-3,17-dione,3a):ESI-MS(+):m / z 301.15[M+H] + ; 1 H-NMR (500MHz, CDCl 3 )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, CDCl 3 )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 (11α,17β-dihydroxyandrost-1,4-dien-3-one,3b):ESI-MS(+):m / z 303.15[M+H] + ; 1 H-NMR (500MHz, CDCl 3 )δ:7.77(1H,d,J=10.0Hz,H-1),6.16(1H,d,J=10.0Hz,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); 13 C-NMR (125MHz, CDCl 3 )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. Saccharomyces cerevisiae engineered bacteria SC-1 catalyzes progesterone (Progesterone, 4) (whole cell catalysis method)

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

[0121] HPLC-MS results (see Figure 7 A) shows that compared with the control group (S.cerevisiae INVSc1), the experimental group (S.cerevisiae INVSc1-CYP68BE1) showed three hydroxylated products (4a, 4b, 4c). The above three products were separated by HPLC semi-preparative separation. 1 H-NMR, 13 C-NMR confirmed their structures. The product structures and transformation routes are shown in Figure 7B, product spectrum data are as follows. Therefore, it can be concluded that CYP68BE1 protein can catalyze the monohydroxylation or dihydroxylation reaction of the substrate progesterone at the 11α and / or 6β positions, and is a 11α and / or 6β hydroxylase of the steroidal substance 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]-; 1 H-NMR (500MHz, CDCl 3 )δ:5.85(1H,s,H-4),4.38(1H,dd,J=3.0,3.0Hz,H-6α),4.12(1H,ddd,J=10.5, 10.5,5.0Hz,H-11β),2.17(3H,s,H-21),1.54(3H,s,H-19),0.76(3H,s,H-18); 13 C-NMR (125MHz, CDCl 3 )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 H-NMR (500MHz, CDCl 3 )δ:6.24(1H,s,H-4),4.13(1H,ddd,J=10.5,10.5,5.0Hz,H-11β),2.18(3H,s,H-21),1.34(3H,s,H-19),0.73(3H,s,H-18); 13 C-NMR (125MHz, CDCl 3)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, CDCl 3 )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, CDCl 3 )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. Catalysis of Estrone (Estrone, 5) by Saccharomyces cerevisiae engineered bacteria SC-1 (whole cell catalysis method)

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

[0128] HPLC-MS results (see Figure 8A) shows that compared with the control group (S.cerevisiae INVSc1), the experimental group (S.cerevisiae INVSc1-CYP68BE1) showed 4 hydroxylation products (5a, 5b, 5c, 5d). According to the ESI-MS results, 5a and 5d should be the monohydroxylation products of the substrate, 5d should be the dihydroxylation product of the substrate, and 5c should be the reduction product of a monohydroxylation product. Among them, 3 products (5a, 5b, 5c) were separated by HPLC semi-preparative separation. 1 H-NMR, 13 C-NMR confirmed their structures. Product 5b is a new compound. The product structure and transformation route are shown in Figure 8 B, product spectrum data are as follows. Therefore, it can be concluded that CYP68BE1 protein can catalyze the monohydroxylation or dihydroxylation reaction of the substrate Estrone at the 11α and / or 6β positions, and is a 11α and / or 6β hydroxylase of the steroid substance Estrone.

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

[0130] 11α-Hydroxyestrone (11α-hydroxyestrone, 5a): HR-ESI-MS(+): m / z287.1638[M+H] + ; 1 H-NMR (500MHz, CD 3 OD)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, CD 3 OD)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, CD 3 OD)δ:7.48(1H,d,J=8.5Hz,H-1), 6.72(1H,d,J=3.0Hz,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); 13 C-NMR (125MHz, CD 3 OD)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, CD 3 OD)δ:7.16(1H,d,J=8.5Hz,H-1), 6.80(1H,d,J=3.0Hz,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, CD 3 OD)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 estra-4,9-diene-3,17-dione (Estra-4,9-diene-3,17-dione, 6) by engineered yeast Saccharomyces cerevisiae SC-1 (whole cell catalysis method)

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

[0135] HPLC-MS results (see Fig. 9 A) shows that compared with the control group (S.cerevisiae INVSc1), the experimental group (S.cerevisiae INVSc1-CYP68BE1) showed four hydroxylated products (6a, 6b, 6c, 6d). The four products were separated by HPLC semi-preparative separation. 1 H-NMR, 13 Their structures were confirmed by C-NMR and 2D-NMR. Products 6b and 6d are new compounds. The product structures and transformation routes are shown in Fig. 9 B, product spectrum data are as follows. Therefore, it can be concluded that CYP68BE1 protein can catalyze the monohydroxylation reaction of the substrate Estra-4,9-diene-3,17-dione at the 11α / β and / or 1α positions, and is a 11α / β and / or 1α hydroxylase of the steroid substance 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 (11α-hydroxyestra-4,9-diene-3,17-dione,6a):HR-ESI-MS(+):m / z287.1637[M+H] + ; 1 H-NMR (500MHz, CDCl 3 )δ:5.79(1H,s,H-4),4.83(1H,dd,J=9.0,6.0Hz,H-11β),1.00(3H,s,H-18); 13 C-NMR (125MHz, CD 3OD)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),1 28.7(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 (1α-hydroxyestra-4,9-diene-3,17-dione,6b):HR-ESI-MS(+):m / z 287.1638[M+H] + ; 1 H-NMR (500MHz, CDCl 3 )δ:5.79(1H,s,H-4),5.10(1H,dd,J=3.0,3.0Hz,H-1β),1.04(3H,s,H-18); 13 C-NMR (125MHz, CDCl 3 )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),12 9.2(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 (11β,17β-dihydroxyestra-4,9-diene-3-one,6c):HR-ESI-MS(+):m / z289.1794[M+H] + ; 1 H-NMR (500MHz, CDCl 3 )δ:5.78(1H,s,H-4),5.04(1H,dd,J=2.0,5.0Hz,H-11α),3.65(1H,t,J=8.0Hz,H-17α),1.09(3H,s,H-18); 13 C-NMR (125MHz, CD 3OD)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 / z289.1795[M+H] + ; 1 H-NMR (500MHz, CDCl 3 )δ:5.77(1H,s,H-4),5.09(1H,dd,J=3.5,3.5Hz,H-1β),3.69(1H,t,J=8.0Hz,H-17α),0.92(3H,s,H-18); 13 C-NMR (125MHz, CDCl 3 )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),1 28.4(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] 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. Fig.10 A schematic diagram of the synthesis of trenbolone using the recombinant bacteria SC-1 is shown (the solid arrows indicate the catalysis of the SC-1 recombinant bacteria / microsomal protein, and the dotted arrows indicate chemical catalysis).

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

[0143] It is divided into two steps:

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

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

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

[0147] The bacteria were weighed and resuspended in BufferTEK at a ratio of 1 mL BufferTEK (0.1 M KCl, 500 mM Tris-HCl, pH 7.4) per 0.5 g wet bacteria. The cells were incubated at room temperature for 5 min, centrifuged, the supernatant was discarded, and the cells were resuspended in 2 mL BufferTESB (0.6 M sorbitol, 500 mM Tris-HCl, pH 7.4).

[0148] (3) Cell disruption: Add an equal volume of zirconium beads and use an automatic sample rapid grinder to disrupt the cells. After completion, place the mixture on ice for 60 seconds and repeat three times. Centrifuge at 12,000 g, collect the supernatant, wash the zirconium beads with 5 mL of Buffer TESB, and centrifuge at 12,000 g to collect the supernatant.

[0149] (4) Preparation of microsomes: NaCl (final concentration 0.15 M) and PEG4000 (final concentration 0.1 g / mL) were added to the supernatant and placed on ice for 30 min. The supernatant was discarded and the microsomes (white, precipitated on the wall and bottom of the centrifuge tube) were collected. 1 mL of Buffer TEG (20% glycerol, 500 mM Tris-HCl, pH 7.4) was added and stored at -80°C.

[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 U glucose-6-phosphate-dehydrogenase; 5 mg CYP68BE1-containing microsomes; 75 mM KH 2 PO 4 -K 2 HPO 4pH 7.4); 37°C, 70 rpm slow shaking, the reaction was started by the addition of NADPH, the reaction was terminated by the addition of the corresponding extractant ethyl acetate, the ethyl acetate layer was concentrated and evaporated to dryness, and 1 mL of methanol solution was used for redissolution, centrifuged and the supernatant was passed through a 0.22 μm organic filter membrane to a liquid phase bottle for HPLC-MS detection, and the detection method was the same as in Example 4. The results showed that the microsomes extracted from the engineered bacteria SC-1 could catalyze the monohydroxylation or dihydroxylation reaction of the 11α and / or 6β and / or 1ɑ and / or 1β positions of the substrates 1-6, and the products and yields were slightly different from those catalyzed by the engineered bacteria, and the HPLC detection results were shown in Fig.11 .

[0152] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that numerous changes, modifications, substitutions and variations may be made to the examples without departing from the principles and spirit of the invention, the scope of the invention being defined by the appended claims and their equivalents.

Claims

1. A hydroxylase CYP68BE1 that causes a steroid compound to undergo a hydroxylation reaction at C11α and / or C6β and / or C1β and / or C1α, characterized in that: The amino acid sequence of the hydroxylase CYP68BE1 is shown in SEQ ID NO: 1, or a sequence with the same function obtained by replacing, deleting or adding one or more amino acids to the amino acid sequence 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, or a sequence formed by codon optimization of the sequence of SEQ ID NO: 2, and a complementary sequence thereof.

4. An expression vector, characterized in that: The vector contains the nucleic acid molecule according to 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 is a recombinant microorganism, and the recombinant bacteria contains the vector as described in claim 4, or the nucleic acid molecule as described in any one of claims 2-3 is integrated into the genome of the recombinant bacteria.

6. A hydroxylation method, comprising: The hydroxylation reaction is carried out in the presence of the hydroxylase CYP68BE1 as claimed in claim 1.

7. The method according to claim 6, characterized in that The steroidal compound substrate undergoes hydroxylation reaction at C11α and / or C6β and / or C1β and / or C1α.

8. Use of the protein according to claim 1, the nucleic acid molecule according to any one of claims 2-3, the vector according to claim 4, or the engineered bacteria according to claim 5 in the hydroxylation reaction of 11α and / or 6β and / or 1β and / or C1α positions of steroid compound substrates.

9. A method for producing steroid drugs and / or steroid drug intermediates, which is a whole cell catalytic method or a microsome method; The whole-cell catalytic method comprises the following steps: fermenting and culturing the engineered bacteria according to claim 5, directly adding a substrate or adding the substrate after concentrating the bacteria, and performing a catalytic reaction, wherein the reaction product contains hydrocortisone and / or eplerenone and / or strophanthus glucoside and / or trenbolone; the substrate is a substance that can be catalyzed by steroid 11α and / or C6β and / or C1β and / or C1α hydroxylases to generate hydrocortisone and / or eplerenone and / or strophanthus glucoside and / or trenbolone; The microsome method comprises the following steps: preparing microsomes from the engineered bacteria according to claim 5, and then catalyzing the substrate in the form of microsomes to produce hydrocortisone and / or eplerenone and / or strophanthus glucoside and / or trenbolone; the substrate is a substance that can be catalyzed by steroid 11α and / or C6β and / or C1β and / or C1α hydroxylases to produce hydrocortisone and / or eplerenone and / or strophanthus glucoside and / or trenbolone.

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