Enzyme for preparing c7-hydroxylated steroid compound and gene, recombinant vector, engineered bacteria and application thereof

Abstract

This invention discloses an enzyme for preparing C7-hydroxylated steroidal compounds, its gene, recombinant vector, engineered bacteria, and applications, belonging to the field of synthetic biotechnology. It addresses the problems of low expression levels and low conversion rates in the enzymatic conversion of BA to 7-OH-DBA. The amino acid sequence of the enzyme is shown in SEQ ID NO: 2. This invention also provides the gene sequence of this enzyme and a recombinant genetically engineered bacterium expressing the C7-hydroxylase. The enzyme of this invention is modified from an α-ketoglutarate-dependent dioxygenase. Compared with the wild type, the modified enzyme obtained in this invention can be successfully applied to the conversion of BA, achieving a conversion rate of 75% to produce 7-OH-DBA. This invention achieves enzymatic catalytic C7-hydroxylation of BA, which, compared with traditional chemical synthesis methods, is simpler, greener, and more economical. The synthesis of 7-OH-DBA will provide strong support for the development of new processes for steroidal drug production.

Description

Technical Field

[0001] This invention belongs to the field of synthetic biology technology, specifically relating to an enzyme for preparing C7-hydroxylated steroidal compounds, its gene, recombinant vector, engineered bacteria, and applications. Background Technology

[0002] Steroid drugs primarily function as hormones in vivo, and their demand is second only to antibiotics in the treatment and prevention of diseases, making them crucial. The biosynthesis of these drugs essentially involves using microbially derived enzymes to precisely modify specific positions of steroid substrates, thereby obtaining new compounds with targeted pharmacological activities. Studies have shown that the activity of steroid compounds is closely related to their core structure; even minor changes in the oxidation state of the core, the type and number of side chain functional groups, or stereoconfiguration can significantly affect their biological activity. For example, introducing unsaturation (…) at the C1,2-position of hydrocortisone acetate (HA)… Prednisolone acetate (PA) can be obtained, with its anti-inflammatory activity increased three to four times; while the introduction of a hydroxyl group at the C7-position of dehydroepiandrosterone (DHEA) produces... This indicates that it exhibits pharmacological activity in inhibiting the proliferation of B lymphocytes and tumors.

[0003] In industrial production, traditional chemical methods for site-specific modification of steroid compounds often face problems such as low conversion rates, numerous byproducts, and environmental pollution. In contrast, enzymatic conversion reactions offer milder conditions, higher efficiency, and excellent regio and stereoselectivity, effectively overcoming bottlenecks in certain steps that are difficult to synthesize chemically, and are gradually becoming the mainstream production strategy.

[0004] 7-OH-DBA (i.e., 7,22-hydroxy-23,24-norchosteroid-1,4-dien-3-one) is a key precursor for the synthesis of high-end steroidal drugs such as ursodeoxycholic acid. Ursodeoxycholic acid is widely used clinically as a first-line drug for the treatment of cholestatic liver disease. Currently, 7-OH-DBA mainly relies on cumbersome and costly chemical synthesis routes; therefore, developing its biosynthetic process is of great value.

[0005] PtmO6 was initially reported to catalyze the hydroxylation of diterpenoids (such as ent-kauranol and ent-atiserene) at the C-7β position, but when used with 22-hydroxy-23,24-norcholest-4-en-3-one (BA), it showed only weak C1,2-dehydrogenation activity and low conversion. Summary of the Invention

[0006] The technical problem to be solved by this invention is: to solve the problem of low conversion rate in the enzymatic preparation of 7-OH-DBA, this invention provides an enzyme for preparing C7-hydroxylated steroidal compounds, its gene, recombinant vector, engineered bacteria and applications. By using the enzyme of this invention to catalyze the production of 7-OH-DBA from BA, the conversion rate is greatly improved.

[0007] To achieve the purpose of the invention, the present invention employs the following technical means:

[0008] This invention provides a gene encoding an enzyme for preparing C7-hydroxylated steroidal compounds.

[0009] An enzyme for preparing C7-hydroxylated steroidal compounds is also provided, encoded by the aforementioned gene. The amino acid sequence of the enzyme is shown in SEQ ID NO: 2.

[0010] A recombinant vector containing the gene is also provided.

[0011] A recombinant genetically engineered bacterium is also provided, which is obtained by transformation of the recombinant vector.

[0012] The application of the enzyme in catalyzing the C7-hydroxylation reaction of steroidal compounds.

[0013] The steroidal compound is 22-hydroxy-23,24-norcholest-4-en-3-one.

[0014] The C7-hydroxylation reaction was used to synthesize 7,22-hydroxy-23,24-norcholesterol-1,4-dien-3-one.

[0015] The application of the enzyme in catalyzing the C7-hydroxylation of the substrate 22-hydroxy-23,24-norcholest-4-en-3-one (BA) to generate 7,22-hydroxy-23,24-norcholest-1,4-dien-3-one (7-OH-DBA).

[0016] The enzyme is provided in the form of crude enzyme solution.

[0017] The reaction conditions for the C7-hydroxylation reaction are: pH 8.0, temperature 30℃, reaction time 2-6 days, and substrate BA concentration 2 g / L.

[0018] The beneficial effects obtained by this invention are as follows:

[0019] Fundamental Enhancement of Enzyme Catalytic Performance: This invention achieves a high-activity mutant, PtmO6-0, by site-directed mutagenesis and codon optimization at five key sites (C29S, H98Y, C116S, C145S, Q190E), on the wild-type PtmO6 enzyme. This mutant successfully alters the enzyme's regioselectivity, transforming it from exhibiting only weak C1,2-dehydrogenation activity in the wild type to possessing potent C7-hydroxylation activity for the steroid substrate BA. In shake-flask processes, its conversion rate reaches over 75%. Compared to the application of the wild-type PtmO6 enzyme in diterpenoids, the modified PtmO6-0 of this invention can be successfully applied to the conversion of 22-hydroxy-23,24-norcholesterol-4-en-3-one (BA).

[0020] High expression and catalytic economy: The gene sequence encoding PtmO6-0 was optimized with E. coli preferred codons, achieving highly efficient soluble expression in the E. coli system. Furthermore, PtmO6-0 acts as a Fe(II) / Oxygenases that are dependent on host bacteria can catalyze reactions that rely on cofactors provided by the host bacteria themselves. and Fe 2+ This process can be performed without the need for additional expensive auxiliary factors, significantly reducing the production cost for industrial applications.

[0021] This invention marks the first successful enzymatic synthesis of 7-OH-DBA. This bioprocess eliminates the need for complex chemical synthesis steps and can be completed in one step within a fermentation or enzymatic conversion system, thus offering unique advantages such as simple process, high space-time yield, and environmental friendliness.

[0022] The PtmO6-0 enzyme catalytic process of this invention has advantages such as high efficiency, specificity and mild conditions. It is not only suitable for the efficient synthesis of 7-OH-DBA from BA, but also has the potential to transform and produce other high-value C7-hydroxylated steroidal drug intermediates, providing a new key enzyme tool and direction for the green biomanufacturing of steroidal drugs. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the reaction catalyzed by PtmO6-0 enzyme to convert BA to 7-OH-DBA.

[0024] Figure 2 This is an image showing the agarose gel electrophoresis results of the recombinant plasmid pSZD-PtmO6-0 and the engineered bacteria. The image shows the agarose gel electrophoresis results; the gel band size should be approximately 1000 bp. M represents the DNA marker; 1 shows the PCR verification result of the pSZD-PtmO6-0 colony; 2 shows the PCR verification result of the DH5α-pSZD-PtmO6-0 colony; and 3 shows the PCR verification result of the BL21-pSZD-PtmO6-0 colony.

[0025] Figure 3 This is an SDS-PAGE analysis of the soluble expression of PtmO6-0 enzyme under different induction conditions. The figure shows the SDS-PAGE electrophoresis analysis of PtmO6-0 enzyme expression. M is the protein marker; 1 is blank; 2 is supernatant after 5 minutes of lysis; 3 is precipitate after 5 minutes of lysis; 4 is supernatant after 10 minutes of lysis; 5 is precipitate after 10 minutes of lysis; 6 is supernatant after 20 minutes of lysis; 7 is precipitate after 20 minutes of lysis; 8 is blank; 9 is supernatant after 5 minutes of lysis; 10 is precipitate after 5 minutes of lysis; 11 is supernatant after 10 minutes of lysis; 12 is precipitate after 10 minutes of lysis; Induction conditions for 1-7: 18°C, 0.4 mM IPTG, induction for 14 h; Induction conditions for 8-12: 18°C, 0.6 mM IPTG, induction for 14 h.

[0026] Figure 4 This is a TLC chromatogram of the conversion products of wild-type PtmO6 and mutant PtmO6-0 to BA. The figure shows the TLC (thin-layer chromatography) analysis of the PtmO6-0 enzyme conversion of BA to 7-OH-DBA. In the figure, 1: BA standard; 2: wild-type PtmO6 to BA conversion reaction solution; 3: PtmO6-0 to BA conversion reaction solution.

[0027] Figure 5 This is an HPLC quantitative analysis chromatogram of the products from the conversion of wild-type PtmO6 and PtmO6-0 to BA. The figure shows the HPLC analysis of the reactions of wild-type PtmO6 enzyme conversion to BA to produce 22-hydroxy-23,24-norcholesterol-1,4-dien-3-one (DBA) and PtmO6-0 enzyme conversion to BA to produce 7-OH-DBA. a: HPLC analysis chromatogram of BA standard; b: HPLC analysis chromatogram of wild-type PtmO6 conversion to BA; c: HPLC analysis chromatogram of PtmO6-0 conversion to BA reaction solution.

[0028] Figure 6 This is the LC-MS chromatogram of the product 7-OH-DBA. Detailed Implementation

[0029] The following examples will further illustrate this point. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0030] This invention uses the PtmO6 gene sequence (SEQ ID NO:1) of the PTM-PTN dual-producing bacterium S. platensis CB00739 as a template. By introducing five site-directed mutations (C29S, H98Y, C116S, C145S, and Q190E) and optimizing the preferred codons of Escherichia coli, the optimized gene sequence PtmO6-0 was obtained. The amino acid sequence it encodes is SEQ ID NO: 2. This gene was synthesized by Shanghai Sangon Biotech Co., Ltd.

[0031] This invention describes the cloning, expression, enzyme structure analysis, and transformation reaction of the PtmO6-0 enzyme gene. An expression vector for *E. coli* was constructed, and the cloned PtmO6-0 gene was introduced into *E. coli* via the pSZD expression vector for induced expression. The enzyme's specificity and selectivity were then characterized.

[0032] Example 1

[0033] This example illustrates the construction of the recombinant PtmO6-0 enzyme plasmid and the expression vector.

[0034] Gene Design and Synthesis: The PtmO6 sequence (SEQ ID NO: 1) from the PTM-PTN dual-producing strain *S. platensis* CB00739 was downloaded from GeneBank (KJ189771.3). The gene and structural design were optimized, and the DNA sequence was optimized using codons preferred by *E. coli* to ensure efficient expression in *E. coli*. Five sites were combined with mutations: C29S, H98Y, C116S, C145S, and Q190E. The modified enzyme gene was named PtmO6-0. This sequence was synthesized by Shanghai Sangon Biotech Co., Ltd., with BamHI and KpnI restriction enzyme sites added before and after the gene, respectively.

[0035] Double digestion of plasmid and gene fragment: The synthesized PtmO6-0 gene fragment and pSZD empty vector plasmid were double-digested using BamHI and KpnI restriction endonucleases, respectively. The digestion system (50 μL) is shown in Table 1, and the details are as follows:

[0036] 10×Buffer: 5 μL;

[0037] PtmO6-0 gene fragment / pSZD: approximately 1000 ng each;

[0038] BamHI: 1 μL;

[0039] KpnI: 1 μL;

[0040] Add ddH2O to 50 μL.

[0041] Reaction conditions: 37℃, incubation for 3 hours.

[0042] Purification and ligation of digestion products: The digested PtmO6-0 gene fragment and pSZD linearized vector were purified using a universal DNA purification and recovery kit. The purified products were mixed with T4 DNA ligase and ligated at 16°C for 4 hours. The ligation system (10 μL) is shown in Table 2, as detailed below:

[0043] 10×Buffer: 5 µL;

[0044] T4 DNA ligase: 1µL;

[0045] PtmO6-0-BamHⅠ / KpnI recovery product: approximately 90 ng;

[0046] pSZD-BamHⅠ / KpnI recovery product: approximately 90 ng;

[0047] ddH2O: Bring the total volume to 10 µL.

[0048] Transformation and positive clone screening:

[0049] The ligation product was transformed into *E. coli* DH5α chemocompetent cells and plated. The plate should show uniformly distributed single colonies; otherwise, it may be contaminated by other microorganisms. Several clear, appropriately sized single colonies were picked from the plate using a pipette tip and used as templates for colony validation PCR. PCR reactions were performed using the vector pre-primer T7-F and post-primer T7-R to verify positive transformants. Simultaneously, the pipette tips containing the picked single colonies were transferred to 5 mL of LB broth containing 5 μL of kanamycin-resistant medium for amplification. The T7-F sequence was: TAATACGACTCACTATAGGG. The T7-R sequence was: GCTAGTATTGCTCAGCGG.

[0050] LB liquid culture medium formulation (1 L): 10 g peptone; 5 g yeast extract; 10 g sodium chloride. The PCR reaction system (20 μL) is shown in Table 3, as detailed below:

[0051] 2 × Taq Master: 10 µL;

[0052] T7-F (10 μM): 1 µL;

[0053] T7-R (10 μM): 1 μL;

[0054] Single colony: trace amount;

[0055] ddH2O: Bring the total volume to 20 µL.

[0056] The PCR amplification procedure was performed according to Table 4. After the PCR reaction was completed, the size of the PCR-amplified bands of each colony was identified by 1% agarose gel electrophoresis. The theoretical size of the amplified bands in this study was expected to be around 1000 bp. Using the control marker bands, the bacterial cultures corresponding to the positive transformants whose band sizes met the expectations were sent to Sangon Biotech Co., Ltd. for gene sequencing. The correctly sequenced bacterial cultures were preserved. A glycerol-to-bacterial culture volume ratio of 3:7 was used, and the mixture was added to a glycerol tube, labeled DH5α-pSZD-PtmO6-0. After amplification, plasmids were extracted using the Tiangen Plasmid Mini-Prep Kit, and the extracted plasmid was labeled pSZD-PtmO6-0. The extracted plasmid was then transformed into E. coli BL21(DE3) competent cells, and positive transformants were selected using the same method, labeled BL21-pSZD-PtmO6-0, and amplified. The strains were then preserved by adding 30% glycerol and storing at -80℃. The agarose gel electrophoresis results of pSZD-PtmO6-0, DH5α-pSZD-PtmO6-0 and BL21-pSZD-PtmO6-0 are as follows: Figure 2 As shown. The gel band size should be around 1000bp. Figure 2 In the images, 1 represents the colony PCR verification result of plasmid pSZD-PtmO6-0; 2 represents the colony PCR verification result of DH5α-pSZD-PtmO6-0; and 3 represents the colony PCR verification result of BL21-pSZD-PtmO6-0. The gel band size is consistent with 1000 bp, indicating successful construction of the expression vector and engineered bacteria.

[0057] Example 2

[0058] This example illustrates the expression of PtmO6-0 enzyme and the preparation of crude enzyme solution.

[0059] 100 μL of the constructed BL21-pSZD-PtmO6-0 glycerol tube culture was added to a test tube containing 3 mL of 0.1% kanamycin-resistant LB liquid medium and incubated at 37°C and 200 rpm for 6 h on a shaker. The culture was then serially expanded to 50 mL Erlenmeyer flasks, and the 50 mL culture was then poured into 1 L of 0.1% kanamycin-resistant LB liquid medium. The culture was incubated at 37°C and 200 rpm until OD... 600When the pH reached 0.6, the temperature was lowered from 37℃ to 20℃, and isopropyl thio-β-D-galactopyranoside (IPTG) was added to a final concentration of 0.4 mM to induce protein expression. After 14 h of shaking culture, the cells were collected. The cells were centrifuged at 6000 rpm for 20 min at 4℃ in a refrigerated centrifuge, and the supernatant was discarded to collect the bacterial sludge. The cells were resuspended in an appropriate amount of 0.1 M Tris-HCl buffer (pH=8.0) and homogenized using a high-pressure homogenizer at 4℃ and 1200 bar for 6 cycles. The cell lysate was collected in a centrifuge cup, precisely balanced, and centrifuged at 12000 rpm for 30 min at 4℃ in a floor-standing refrigerated centrifuge. The supernatant after centrifugation was the crude enzyme solution of PtmO6-0 enzyme, used for fermentation and substrate transformation.

[0060] After centrifugation, the supernatant and precipitate were collected separately, and protein samples were prepared. SDS-PAGE was used to detect the protein expression of PtmO6-0. The PtmO6-0 enzyme size is 32.8 kDa. Results are as follows: Figure 3 As shown in the figure: 1 is blank; 2 is supernatant after 5 minutes of disruption; 3 is precipitate after 5 minutes of disruption; 4 is supernatant after 10 minutes of disruption; 5 is precipitate after 10 minutes of disruption; 6 is supernatant after 20 minutes of disruption; 7 is precipitate after 20 minutes of disruption; 8 is blank; 9 is supernatant after 5 minutes of disruption; 10 is precipitate after 5 minutes of disruption; 11 is supernatant after 10 minutes of disruption; 12 is precipitate after 10 minutes of disruption; Induction conditions for 1-7: 18°C, 0.4 mM IPTG, induction for 14 h; Induction conditions for 8-12: 18°C, 0.6 mM IPTG, induction for 14 h. Based on the expression results, the induction conditions of 18°C, 0.4 mM IPTG, induction for 14 h, and disruption for 5 minutes were selected as the disruption conditions for preparing the crude enzyme solution.

[0061] Example 3

[0062] This embodiment is a construction example of a shake-flask PtmO6-0 enzyme conversion BA reaction system.

[0063] We used the prepared crude PtmO6-0 enzyme solution to test the enzyme's catalytic activity against the steroid substrate BA, and constructed an in vitro transformation system. All enzymes used in the in vitro transformation system of this study were prepared according to Example 2. The reaction system (10 mL) is shown in Table 5, as detailed below:

[0064] PtmO6-0 protein solution: 5 mL;

[0065] Steroid substrate BA: 20 mg;

[0066] ddH2O: Add to the total volume to 10 mL.

[0067] The reaction was incubated in 50 mL Erlenmeyer flasks at 200 rpm on a shaker at 30°C and pH 8.0 for 2 to 6 days. A protein-free reaction mixture was used as a negative control under the same conditions. Samples were taken every day. 1 mL of the reaction mixture was removed from the Erlenmeyer flask and mixed with an equal volume of ethyl acetate to extract the reaction product. The extract was centrifuged at 12000 rpm for 10 min. At this point, the lipid-soluble steroid substrate and product dissolved in the supernatant. The supernatant was pipetted off, and the ethyl acetate was dried at 65°C to obtain a powder or oily reaction product.

[0068] Example 4

[0069] Detection of products from the PtmO6-0 enzyme conversion of BA

[0070] TLC detection: A Huanghai HSGF254 silica gel plate (2.5 × 7.5 cm) was used to detect substrate consumption and product formation. Ethyl acetate-petroleum ether (volume ratio 2:3) was used as the developing solvent, with the sample dissolved in ethyl acetate. One to two light spots were applied to the bottom of the plate, 1 cm from the end, using a capillary glass tube. If a standard sample was available, it could be applied next to it for reference. After spotting, the bottom of the plate was placed in the developing solvent, but not in contact with the spotting area. Development was complete when the developing solvent had penetrated 2 / 3 of the plate. The plate was then removed and the developing solvent was dried in a fume hood. At this point, some products could be observed under UV254 wavelength. Some steroid products showed no absorption at this wavelength; in such cases, after development, the plate could be sprayed with an ethanol solution containing 20% ​​dilute sulfuric acid, followed by baking at 100°C for 5 to 10 minutes until clear substrate or product spots appeared on the plate. Detection was performed by comparing the spot size and relative migration with the standard.

[0071] Test results as follows Figure 4 As shown in the figure, 1: BA standard; 2: wild-type PtmO6 to BA conversion reaction solution; 3: PtmO6-0 to BA conversion reaction solution. It can be clearly seen on the TLC plate that the product of wild-type PtmO6 to BA conversion is DBA, and the product of PtmO6-0 conversion is 7-OH-DBA.

[0072] HPLC detection: The sample must be dissolved in chromatographic grade methanol and filtered through a 0.22 μm filter membrane. The sample loading volume is set to 10 μL. The detector emits 254 nm ultraviolet light and detects the UV absorbance of the sample as it passes through the chromatographic column. A Sunfire C18 reverse-phase column (5 μm, 4.6 × 150 mm, Waters) is used. The mobile phase is methanol:water = 6:4, the column temperature is 30℃, and the flow rate is 1 mL / min. If a standard curve is established for the sample to be analyzed, HPLC can quantify the concentration of the sample. Detection results are as follows: Figure 5As shown in the figure, the horizontal axis represents retention time (min), and the vertical axis represents the response signal intensity. Figure a: HPLC analysis chromatogram of BA standard; b: HPLC analysis chromatogram of wild-type PtmO6 converting BA; c: HPLC analysis chromatogram of PtmO6-0 converting BA reaction solution. From the figures, it can be seen that the BA peak time is approximately 12 min, which can be used as a reference for qualitative and quantitative analysis; peaks of DBA and BA can be detected in sample PtmO6, but the signal of 7-OH-DBA is extremely weak; sample PtmO6-0 simultaneously exhibits characteristic peaks of 7-OH-DBA, DBA, and BA, with clear peak shapes. The 7-OH-DBA peak time is approximately 6 min, and the DBA peak time is approximately 9 min. According to the HPLC detection results, the conversion rate of BA from wild-type PtmO6 is 10.5%, and the conversion rate of BA from PtmO6-0 is 75%, indicating that the modified enzyme has significantly improved substrate conversion activity and specificity.

[0073] LC-MS detection: Sample preparation was the same as for HPLC detection. Analysis was performed using a TSQ Quantis mass spectrometer, and mass spectrometry data were recorded in positive electrospray ionization mode (ESI+). Results are as follows: Figure 6 As shown, the molecular formula of 7-OH-DBA is C 22 H 34 O3, with a molecular weight of 344.24, has a mass-to-charge ratio of 345.2433 in positive electrospray ionization mode, consistent with the product of this invention.

[0074] Those skilled in the art should understand that the above embodiments are merely exemplary embodiments, and various changes, substitutions, and modifications can be made without departing from the spirit and scope of this application.

[0075] Table 1. Double enzyme digestion reaction system (50 µL)

[0076]

[0077] Table 2 Enzyme ligation reaction system (10 µL)

[0078]

[0079] Table 3. Colony PCR reaction system (20 μL)

[0080]

[0081] Table 4 PCR Amplification Program

[0082]

[0083] Table 5. In vitro conversion system of PtmO6-0 enzyme (10 mL)

[0084]

Claims

1. An enzyme for preparing a C7-position hydroxylated steroid compound, characterized by, The amino acid sequence of the enzyme is shown in SEQ ID NO:

2.

2. The use of the enzyme as described in claim 1 in catalyzing the C7-hydroxylation reaction of a steroid compound, wherein the steroid compound is 22-hydroxy-23,24-norcholesterol-4-en-3-one.

3. The application according to claim 2, characterized in that, The C7-hydroxylation reaction was used to synthesize 7,22-hydroxy-23,24-norcholesterol-1,4-dien-3-one.

4. The application according to claim 3, characterized in that, Using the enzyme described above, the substrate 22-hydroxy-23,24-norcholest-4-en-3-one is catalyzed for C7-position hydroxylation to generate 7,22-hydroxy-23,24-norcholest-1,4-dien-3-one.

5. The application according to claim 4, characterized in that, The reaction conditions for the C7-hydroxylation reaction are: pH 8.0, temperature 30℃, and the concentration of the substrate 22-hydroxy-23,24-norchos-4-en-3-one is 2 g / L.

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