A c11 alpha-hydroxylase mutant with improved catalytic activity, methods and uses thereof

Abstract

The application belongs to the technical field of genetic engineering and enzyme engineering, and discloses a C11 alpha-hydroxylase mutant D118V with improved catalytic activity, wherein the amino acid sequence is shown as SEQ ID NO. 4, and the corresponding nucleotide sequence is shown as SEQ ID NO. 5. The C11 alpha-hydroxylase from Aspergillus ochraceus is subjected to site-directed saturation mutation, and the C11 alpha-hydroxylase mutant D118V for improving the yield of 11 alpha, 17 alpha-dihydroxy progesterone is obtained by using a Saccharomyces cerevisiae expression platform. When the concentration of the substrate 17 alpha-hydroxy progesterone is 2 g / L, the concentration of 11 alpha, 17 alpha-dihydroxy progesterone after 36 h of conversion is 1.19 g / L, and the production intensity is 758.15 mg / (L·d), which is increased by 16.67% and 172.77% compared with the wild-type CYP68J5, thereby providing an application case and basic data support for the modification of steroid C11 alpha-hydroxylase.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and enzyme engineering technology, and in particular to a C11α-hydroxylase mutant with enhanced catalytic activity, a method thereof, and its application. Background Technology

[0002] Steroid compounds are structurally diverse and numerous, possessing unique physiological and pharmacological activities, making them indispensable in clinical treatment. Steroid drugs are widely used for anti-inflammatory, antitoxic, and anti-allergic purposes, and are also used to treat cancer, control fertility, and address hormonal imbalances. Introducing functional groups at specific sites on the steroid skeleton is crucial for drug efficacy; commonly used methods include chemical synthesis, microbial transformation, and semi-synthesis. Compared to chemical synthesis, microbial transformation offers advantages such as high specificity, fewer synthetic steps, shorter production cycles, fewer byproducts, and environmental friendliness. Furthermore, it exhibits high stereoselectivity and regioselectivity, enabling the introduction of specific functional groups to inert sites on the steroid drug core that are difficult or impossible to achieve through chemical synthesis. In the production of steroid hormone drugs, microbial transformation has become a key technology in the synthetic routes of many steroid drugs or pharmaceutical intermediates.

[0003] 11α,17α-Dihydroxyprogesterone is an intermediate in the preparation of steroidal hormone drugs through bio-fermentation, using 17α-hydroxyprogesterone as a raw material. It can be used to prepare various corticosteroid drugs with effects such as enhancing bodily functions, improving immunity, and anticoagulation, such as dexamethasone, betamethasone, hydrocortisone, prednisone acetate, and hydrocortisone acetate. Currently, industrially, 11α,17α-dihydroxyprogesterone is mainly produced by converting 17α-hydroxyprogesterone using *Aspergillus ochraceus*. However, *Aspergillus ochraceus* suffers from problems such as pigment and toxin accumulation in the later stages of conversion, which not only significantly increases the cost of separation and purification but also seriously affects product quality. Furthermore, the genetic background of *Aspergillus ochraceus* is unclear, and molecular manipulation tools are immature. In contrast, *Saccharomyces cerevisiae*, as a eukaryotic model microorganism, is renowned for its natural advantages, such as rapid growth, clear genetic background, mature gene manipulation, and safe use.

[0004] Steroidal 11α-hydroxylases belong to the cytochrome P450 enzyme superfamily. Their structure mainly consists of a cytoplasmic domain, a transmembrane domain, and a heme prosthetic group. The heme group is stacked between I and L helices in a relatively large pocket, linked to the protein backbone via coordination with thioferric thiolates bound to proximal cysteine ​​residues. It is surrounded by hydrophobic amino acid residues to accommodate hydrophobic substrates. Steroidal hydroxylases are membrane-bound proteins with strong hydrophobicity, tending to polymerize in water rather than crystallize, making isolation and purification very difficult. In recent years, researchers have heterologously expressed and functionally identified 11α-hydroxylase genes from different sources in *Escherichia coli*, *Saccharomyces cerevisiae*, and *Pichia pastoris*, but reports on key amino acid sites and molecular modifications of hydroxylases are limited. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a C11α-hydroxylase mutant with enhanced catalytic activity, a method thereof, and its application.

[0006] The technical solution adopted by this invention to solve its technical problem is:

[0007] A C11α-hydroxylase mutant D118V with enhanced catalytic activity has the amino acid sequence shown in SEQ ID NO.4 and the corresponding nucleotide sequence shown in SEQ ID NO.5.

[0008] Preferably, the C11α-hydroxylase is derived from Aspergillus ochraceus.

[0009] The gene encoding the C11α-hydroxylase mutant D118V as described above.

[0010] Recombinant vectors carrying the genes described above.

[0011] Furthermore, the expression vectors include, but are not limited to, pYES2.

[0012] Genetically engineered bacteria that express the mutants described above, carry the genes described above, or contain the recombinant vectors described above.

[0013] The genetically engineered bacteria described above use host cells including prokaryotes and eukaryotes, wherein the prokaryotes are preferably Escherichia coli; the eukaryotes are preferably Saccharomyces cerevisiae or Pichia pastoris; more preferably, the yeast is Saccharomyces cerevisiae.

[0014] A method for improving the catalytic activity of C11α-hydroxylase, wherein the amino acid sequence is mutated based on the sequence shown in SEQ ID NO.1: the amino acid at position 118 is mutated from aspartic acid to valine.

[0015] A method for producing a target product using genetically engineered bacteria as described above, the production method comprising the following steps:

[0016] The engineered bacteria were inoculated into Erlenmeyer flasks containing YEPD liquid medium and cultured with shaking at 28-35℃ and 180-250 r / min for 18-24 h. Then, substrate was added, followed by methanol (1.0%-5.0% by volume) to aid dissolution, and galactose solution (1.0%-5.0% by volume) at a 20% mass concentration was added for induction. This was repeated every 18-24 h. After complete transformation at 28-35℃ and 180-250 r / min, a transformation solution containing the target product was obtained. This solution was extracted with ethyl acetate to obtain a supernatant containing the target product.

[0017] Furthermore, the substrate is 17α-hydroxyprogesterone, and the final concentration range is 0.2-5.0 g / L.

[0018] The application of the mutant D118V described above, or the recombinant vector described above, or the genetically engineered bacteria described above in the C11α hydroxylation reaction. Preferably, the hydroxylation reaction converts 17α-hydroxyprogesterone to 11α,17α-dihydroxyprogesterone.

[0019] The advantages and positive effects of this invention are as follows:

[0020] 1. This invention involves site-directed saturation mutagenesis of C11α-hydroxylase CYP68J5 derived from *Aspergillus ochraceus*. Through steroidal transformation experiments using recombinant *Saccharomyces cerevisiae* strains expressing the mutant, a C11α-hydroxylase gene mutant, D118V, with enhanced catalytic activity was obtained. HPLC analysis of the transformation results showed that at a substrate concentration of 17α-hydroxyprogesterone of 2 g / L, mutant D118V produced 1.19 g / L of 11α,17α-dihydroxyprogesterone, with a production intensity of 758.15 mg / (L·d), representing increases of 16.67% and 172.77% respectively compared to wild-type CYP68J5. This invention provides application examples and fundamental data support for the modification of steroidal C11α-hydroxylases.

[0021] 2. The starting gene of this invention is the C11α-hydroxylase CYP68J5 of Aspergillus ochraceus. In order to further improve the production of 11α,17α-dihydroxyprogesterone, site-directed saturation mutagenesis was used to obtain a CYP68J5 mutant with increased enzyme activity, as well as a corresponding high-yielding strain of 11α,17α-dihydroxyprogesterone. This is of great significance for realizing the industrial microbial production of 11α,17α-dihydroxyprogesterone.

[0022] 3. This invention improves the catalytic activity of C11α hydroxylase by modifying it using site-directed saturation mutagenesis, providing important methodological guidance for the modification of steroidal C11α hydroxylase. Attached Figure Description

[0023] Figure 1 This diagram illustrates the C11α hydroxylation reaction of 17α-hydroxyprogesterone to 11α,17α-dihydroxyprogesterone in this invention.

[0024] Figure 2 This is a flowchart illustrating the construction process of the pYES2-cyp68j5 expression vector in this invention.

[0025] Figure 3 This diagram shows the distribution of the three sites D118, F216, and M488 in the CYP68J5-substrate docking structure of this invention.

[0026] Figure 4 This is an agarose gel electrophoresis image of the reverse PCR amplification vector in this invention; where M: marker; 2: pYES2-cyp68j5;

[0027] Figure 5 This is an activity analysis diagram of a series of mutants obtained based on saturation mutations at three sites, D118, F216, and M488, in this invention.

[0028] Figure 6 This is a TLC diagram showing the results of 24 hours after transformation of 17α-hydroxyprogesterone with recombinant Saccharomyces cerevisiae strains expressing CYP68J5 and its mutant D118V in this invention; where 1: 17α-hydroxyprogesterone standard; 2: 11α,17α-dihydroxyprogesterone standard; 3: control strain S.INSc1 / pYES2-cyp68j5; 4: S.INSc1 / pYES2-cyp68j5_D118V;

[0029] Figure 7 This is an HPLC liquid chromatography chromatogram of the recombinant Saccharomyces cerevisiae strain expressing CYP68J5 and its mutant D118V transformed with 17α-hydroxyprogesterone in this invention.

[0030] Figure 8The product generation curves and production intensity diagrams of Saccharomyces cerevisiae expressing CYP68J5 and its mutants D118V, M488W, F216W, and M488L at a substrate concentration of 0.5 g / L (a, b) are shown.

[0031] Figure 9 The product generation curves and production intensity diagrams of Saccharomyces cerevisiae expressing CYP68J5 and its mutant D118V at a substrate concentration of 2.0 g / L (a, b) are shown in this invention. Detailed Implementation

[0032] The present invention will be further described below with reference to the embodiments. The following embodiments are descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.

[0033] The various experimental operations involved in the specific embodiments are all conventional techniques in the field. For parts not specifically annotated in this document, those skilled in the art can refer to various commonly used reference books, scientific and technological documents or related instructions and manuals prior to the filing date of this invention to carry out the operations.

[0034] A C11α-hydroxylase mutant D118V with enhanced catalytic activity has the amino acid sequence shown in SEQ ID NO.4 and the corresponding nucleotide sequence shown in SEQ ID NO.5.

[0035] Preferably, the C11α-hydroxylase is derived from Aspergillus ochraceus.

[0036] The gene encoding the C11α-hydroxylase mutant D118V as described above.

[0037] Recombinant vectors carrying the genes described above.

[0038] Preferably, the expression vectors include, but are not limited to, pYES2.

[0039] Genetically engineered bacteria that express the mutants described above, carry the genes described above, or contain the recombinant vectors described above.

[0040] The genetically engineered bacteria described above use host cells including prokaryotes and eukaryotes, wherein the prokaryotes are preferably Escherichia coli; the eukaryotes are preferably Saccharomyces cerevisiae or Pichia pastoris; more preferably, the yeast is Saccharomyces cerevisiae.

[0041] A method for improving the catalytic activity of C11α-hydroxylase, wherein the amino acid sequence is mutated based on the sequence shown in SEQ ID NO.1: the amino acid at position 118 is mutated from aspartic acid to valine.

[0042] A method for producing a target product using genetically engineered bacteria as described above, the method comprising the following steps:

[0043] The engineered bacteria were inoculated into Erlenmeyer flasks containing YEPD liquid medium and cultured with shaking at 28-35℃ and 180-250 r / min for 18-24 h. Then, substrate was added, followed by methanol (1.0%-5.0% by volume) to aid dissolution, and galactose solution (1.0%-5.0% by volume) at a 20% mass concentration was added for induction. This was repeated every 18-24 h. After complete transformation at 28-35℃ and 180-250 r / min, a transformation solution containing the target product was obtained. This solution was extracted with ethyl acetate to obtain a supernatant containing the target product.

[0044] Preferably, the substrate is 17α-hydroxyprogesterone, and the final concentration range is 0.2-5.0 g / L.

[0045] The application of the mutant D118V described above, or the recombinant vector described above, or the genetically engineered bacteria described above in the C11α hydroxylation reaction. Preferably, the hydroxylation reaction converts 17α-hydroxyprogesterone to 11α,17α-dihydroxyprogesterone.

[0046] Specifically, the relevant preparation and testing methods are as follows:

[0047] The culture medium used in this invention can be as follows:

[0048] (1) LB medium (g / L): tryptone 10, yeast extract 5, sodium chloride 10, pH 7.5, ddH2O to a final volume of 1L. Add 20g of agar powder to the solid medium.

[0049] (2) YEPD medium (g / L): 20g peptone, 10g yeast extract, and ddH2O to a final volume of 900mL. Sterilize at 121℃ for 20min, then add 100mL of 20% glucose and 20g agar powder to the solid medium.

[0050] (3) Ura auxotrophic medium (g / L): YNB (yeast nitrogen source) 6.7, add ddH2O to make up to 900mL. Sterilize at 121℃ for 20min, then add 100mL of 20% glucose and 0.78g of DO Supplement-Ura. Add 20g of agar powder to the solid medium.

[0051] The strains involved in this invention are: Saccharomyces cerevisiae INVSc1, Aspergillus ochraceus CICC 41473, and Escherichia coli DH5α.

[0052] Example 1: Construction of a recombinant Saccharomyces cerevisiae strain expressing CYP68J5

[0053] 1.1 Construction of recombinant plasmid pYES2-cyp68j5

[0054] The diagram shows the C11α hydroxylation reaction of 17α-hydroxyprogesterone to 11α,17α-dihydroxyprogesterone. Figure 1 As shown, the C11α hydroxylase CYP68J5 derived from *Aspergillus ochraceus* catalyzes the production of 11α,17α-dihydroxyprogesterone from 17α-hydroxyprogesterone. The nucleotide sequence obtained after codon optimization is shown in SEQ ID NO.3, synthesized by Suzhou Genewiz. This fragment was constructed on plasmid pYES2, and the recombinant plasmid pYES2-cyp68j5 was constructed as shown in [image / description missing]. Figure 2 .

[0055] 1.2 Construction of recombinant strains

[0056] The recombinant plasmid was transformed into competent cells of Saccharomyces cerevisiae using the lithium acetate transformation method. The specific steps were as follows: (1) The yeast strain INVSC1 was cultured in YEPD liquid until the early exponential phase (OD200). 660 (0.6-0.8), take 1×10 7(1) Centrifuge the cells at 13,500 r / min for 1 min and discard the supernatant; (2) Resuspend the cells in 1 mL of sterile water, centrifuge at 13,500 r / min for 1 min and discard the supernatant; (3) Resuspend the cells in 1 mL of 100 mM LiOAc, let stand for 15 min, centrifuge at 13,500 r / min for 1 min and discard the supernatant; (4) Add 7.2 μL of 1 M LiOAc, 10 μL of salmon sperm DNA with a mass concentration of 2 mg / mL, 1000 ng of recombinant plasmid, and 48 μL of 50% PEG3350 to the cells and gently pipette to mix. Incubate at 30℃ for 1 hour, then heat shock at 42℃ for 30 minutes, centrifuge at 13,500 r / min for 1 minute, and discard the supernatant; (5) Resuspend the cells in 1 mL of sterile water, centrifuge at 13,500 r / min for 1 minute, and discard the supernatant; (6) Resuspend the cells in 300 μL of sterile water, take 200 μL and spread it on one Ura auxotrophic plate, place the plate in a constant temperature of 30℃ for 2-3 days, and single colonies can grow. Pick the single colonies on the plate and inoculate them into a test tube containing Ura auxotrophic liquid medium, shake at 220 r / min, 30℃ overnight, and extract the plasmid using the TIANGEN (CHN) Saccharomyces cerevisiae plasmid extraction kit. Use primers cyp68j5-F and cyp68j5-R for PCR verification, and the correct recombinant strain can be preserved.

[0057] The PCR primers are as follows:

[0058] cyp68j5-F:GCCGCCAGTGTGCTGGAATTCATGCCATTTTTTACTGGTTTGTTG

[0059] cyp68j5-R:TACATGATGCGGCCCTCTAGATTAAACAGTCAAAGAAGCCATATCAA

[0060] (1) PCR reaction system

[0061]

[0062] (2) PCR amplification conditions: 95℃ for 30s; 95℃ for 15s; 63.5℃ for 15s; 72℃ for 1min for 32s; 72℃ for 5min; 4℃ for incubation.

[0063] Example 2: Construction of saturated mutant recombinant plasmid of hydroxylase CYP68J5

[0064] Based on the docking behavior of CYP68J5 with its substrate, three candidate key amino acid sites were selected: aspartic acid at position 118, phenylalanine at position 216, and methionine at position 488. Their distribution in the protein's three-dimensional structure is as follows: Figure 3Using plasmid pYES2-cyp68j5 as a template, inverse PCR was performed for amplification. Degenerate primers were designed using the degenerate codon NNK. All primers were synthesized by Genewiz Biotechnology Services and purified using tPAGE.

[0065] The PCR primers for saturation mutation are as follows:

[0066] D118-F NNK TCTCATGGTTATATTCCTGGTTTTG

[0067] D118-RATCAGTAGTTGGAGTTTCAAAATCCATTC

[0068] F216-F NNK GGTGTTGGTGATAAATTGAG

[0069] F216-RAGCCAAAGCAGCATATTGAGAAGAAG

[0070] M488-F NNK ACTTATTTGGCTGATCCAAACAC

[0071] M488-RACCAATATCAATGGTTGTGGTTTAAAAC

[0072] In the above degenerate primers, N represents any one of ATCG, and K represents any one of G and T.

[0073] (1) PCR reaction system

[0074]

[0075] (2) Reverse PCR amplification conditions: 94℃ for 2 min, 98℃ for 10 s, T m -5℃ for 30 s, 68℃ for 7.4 min, for a total of 20 cycles, followed by incubation at 4℃. Reverse PCR amplification yielded an open circular plasmid containing the mutation site and a partially methylated template plasmid. Agarose gel electrophoresis detected a product fragment approximately 7.4 kb in length. Results are as follows: Figure 4 As shown, the recombinant plasmid reverse PCR amplification bands are consistent with the theoretical size.

[0076] (3) Construction of mutant recombinant plasmids: PCR products were digested with Dpn I restriction endonuclease at 37℃ for 4 h to remove unmutated template plasmids. Following the instructions of the KOD-Plus mutation kit, T4 polynucleotide kinase and ligase were used to perform self-circularization ligation of the digested products (16℃, 16 h). The ligated plasmids were transformed into *E. coli* DH5α competent cells. An appropriate amount of the recovered bacterial culture was evenly spread on LB solid medium containing ampicillin and incubated overnight at 37℃. Transformants were selected for colony PCR verification. Plasmids from correctly verified positive transformants were extracted and sent for sequencing. Plasmids with correct mutations were preserved for subsequent experiments.

[0077] Example 3: Construction and functional detection of a recombinant Saccharomyces cerevisiae strain expressing the CYP68J5 mutant

[0078] 3.1 Construction of a recombinant Saccharomyces cerevisiae strain expressing the CYP68J5 mutant

[0079] The verified recombinant plasmid was transformed into competent *Saccharomyces cerevisiae* cells using the lithium acetate method described in Example 1. The cells were cultured on Ura auxotrophic plates at 30°C for 2-3 days until single colonies appeared. A single colony was picked from the plate and inoculated into a test tube containing Ura auxotrophic liquid medium. The culture was incubated overnight at 30°C with shaking at 220 rpm. The bacterial culture was then subjected to culture PCR. Once the recombinant strain was verified, it was ready for preservation.

[0080] 3.2 Functional detection of recombinant Saccharomyces cerevisiae strain expressing CYP68J5 mutant

[0081] After the recombinant Saccharomyces cerevisiae strains expressing the CYP68J5 mutant were successfully constructed, the transformation performance of different mutants for 17α-hydroxyprogesterone was investigated by steroid substrate transformation experiments.

[0082] (1) Cultivation of brewing yeast

[0083] Patch single colony: Using a disposable sterile inoculation loop, pick a small amount of bacteria from a single colony grown on a YEPD plate and spread it evenly on fresh solid medium to form a patch of approximately 1 cm. 2 The smeared squares are incubated upside down in a 30℃ constant temperature incubator for 1-2 days, and the growth of the bacteria within the square area can be observed.

[0084] (2) Substrate transformation experiment

[0085] Using a sterile disposable inoculation loop, scrape a loopful of bacterial cells from a YEPD plate into a 250 mL Erlenmeyer flask containing 30 mL of YEPD liquid medium. Incubate at 28°C and 200 rpm for 18 h with shaking. Then add 17α-hydroxyprogesterone (0.3 g / L) as a substrate, followed by 1.0% methanol (v / v) for dissolution, and then 1.0% 20% galactose solution (v / v) for induction. Repeat this process every 18 h. Transform at 28°C and 200 rpm for 84 h, sampling every 12 h. For sampling, add 1.0 mL of fermentation broth to a 2 mL centrifuge tube and add 1.0 mL of ethyl acetate to terminate the transformation. Sonicate for 20 min using an ultrasonic cleaner, centrifuge at 13000 rpm for 10 min, and the supernatant is the test sample.

[0086] (3) Detection methods for steroidal compounds

[0087] Thin-layer chromatography (TLC): Using a pipette, spot the sample to be tested onto a GF254 silica gel plate with a spot volume of 2 μL. The spot diameter should not exceed 2 mm, and the spacing between spots should be at least 0.6 cm. The distance between the spot and the edge of the silica gel plate should be at least 1 cm. Then, place the silica gel plate sample into a chromatography tank containing the developing solvent. The developing solvent ratio is: n-hexane:acetone:ethyl acetate = 1.4:1:0.5 (v / v / v). The chromatography is complete when the distance between the solvent front and the edge of the silica gel plate is approximately 1 cm. Remove the silica gel plate and let it dry. Finally, observe the spots under a 254 nm UV lamp.

[0088] High-performance liquid chromatography (HPLC): Pipette 200 μL of the sample into a centrifuge tube, allow it to air dry in a fume hood, then add 1 mL of mobile phase for redissolution. Sonicate for 20 min using an ultrasonic cleaner, centrifuge at 13000 rpm for 10 min, and collect 300 μL of the supernatant for HPLC detection. HPLC conditions are as follows:

[0089] Liquid Chromatograph: Agilent Technologies 1260 Infinity Series

[0090] UV detector: Agilent Technologies 1200 Infinity Series

[0091] Chromatographic column: Agela C18 column (250mm × 4.6mm, 5μm);

[0092] Mobile phase: Methanol: Water = 80:20 (v / v);

[0093] Detection wavelength: 254nm;

[0094] Flow rate: 1 mL / min;

[0095] Column temperature: 35℃;

[0096] Injection volume: 10 μL.

[0097] Depend on Figure 5 It can be seen that among the 54 mutants, 4 mutants with good C11α hydroxylation activity were screened. Among them, mutant D118V had the best transformation performance, followed by M488W, while mutants F216W and M488L had weak transformation ability. The transformation performance of the remaining mutants was significantly reduced or even completely lost.

[0098] Example 4: Transformation capacity of recombinant Saccharomyces cerevisiae strains expressing CYP68J5 and its mutants D118V, M488W, F216W, and M488L at a substrate concentration of 0.5 g / L.

[0099] (1) The cultivation of brewing yeast is shown in Example 3.

[0100] (2) Substrate transformation experiment

[0101] Using a sterile disposable inoculation loop, scrape a loopful of bacterial cells from a YEPD plate into a 250 mL Erlenmeyer flask containing 50 mL of YEPD liquid medium. Incubate at 30°C and 210 rpm for 20 h with shaking. Then add 17α-hydroxyprogesterone (0.5 g / L) as a substrate, followed by 1.5% methanol (v / v) for dissolution, and then 1.5% 20% galactose solution (v / v) for induction. Repeat this process every 20 h. Transform at 30°C and 210 rpm for 84 h, sampling every 12 h. For sampling, add 1.0 mL of fermentation broth to a 2 mL centrifuge tube and add 1.0 mL of ethyl acetate to terminate the transformation. Sonicate for 20 min using an ultrasonic cleaner, centrifuge at 13000 rpm for 10 min, and the supernatant is the test sample.

[0102] (3) The detection method for steroidal compounds is described in Example 3.

[0103] The transformation performance of wild-type strain CYP68J5 and screened mutants D118V, M488W, F216W, and M488L on 17α-hydroxyprogesterone was investigated using substrate transformation experiments. TLC was performed on samples transformed for 24 hours (see [link to TLC]). Figure 6 ) and HPLC (see Figure 7Analysis (showing only results for superior mutants) revealed that the product concentration of mutant D118V was significantly higher than that of wild-type CYP68J5. Further analysis showed that at a substrate concentration of 0.5 g / L, wild-type CYP68J5 reached its maximum product concentration of 434.50 mg / L at 72 hours, while mutant D118V reached its maximum product concentration of 452.56 mg / L at 24 hours, a higher level than wild-type CYP68J5. Figure 8 a) Importantly, the time to reach maximum product concentration for the mutant D118V was 66.70% shorter than that for the wild-type CYP68J5. For example... Figure 8 As shown in b, the production intensity of mutant D118V is 431.66 mg / (L·d), which is 212.46% higher than that of wild-type CYP68J5 (138.15 mg / (L·d)). The product concentration and growth intensity of the other three mutants, M488W, F216W, and M488L, are all lower than those of wild-type CYP68J5.

[0104] Example 5: Transformation capacity of recombinant Saccharomyces cerevisiae strains expressing CYP68J5 and its mutant D118V at a substrate concentration of 2.0 g / L.

[0105] (1) The cultivation of brewing yeast is shown in Example 3.

[0106] (2) Substrate transformation experiment

[0107] Using a sterile disposable inoculation loop, scrape a loopful of bacterial cells from a YEPD plate into a 250 mL Erlenmeyer flask containing 50 mL of YEPD liquid medium. Incubate at 32°C and 220 rpm for 24 h with shaking. Then add 17α-hydroxyprogesterone (17α-hydroxyprogesterone) to a final concentration of 2.0 g / L, followed by 4.0% methanol (v / v) for dissolution. Induction is then initiated with 4.0% 20% galactose solution (v / v), repeated every 24 h. Transformation is carried out at 32°C and 220 rpm for 84 h, with samples taken every 12 h. For sampling, add 1.0 mL of fermentation broth to a 2 mL centrifuge tube and add 1.0 mL of ethyl acetate to terminate the transformation. Sonicate for 20 min using an ultrasonic cleaner, centrifuge at 13000 rpm for 10 min, and the supernatant is the test sample.

[0108] (3) The detection method for steroidal compounds is described in Example 3.

[0109] To further investigate the transformation performance of the superior mutant D118V at high substrate concentrations, other transformation conditions were kept constant, but the concentration of 17α-hydroxyprogesterone was increased to 2.0 g / L. Figure 9As shown in Figure a, increasing substrate concentration prolonged the time to peak product concentration but also increased the maximum product yield. The mutant D118V reached its maximum product concentration of 1.19 g / L at 36 h, a 16.67% increase compared to the wild-type CYP68J5 (1.02 g / L). Its production intensity (758.15 mg / (L·d)) was 172.77% higher than that of the wild-type CYP68J5 (277.94 mg / (L·d)). Figure 9 (b) This enhancement effect is similar to that at a substrate concentration of 0.5 g / L, but the mutant can synthesize more products at higher substrate concentrations.

[0110] The relevant sequences used in this invention are as follows:

[0111] CYP68J5 amino acid-SEQ ID NO.1

[0112] MPFFTGLLAIYHSLILDNPVQTLSTIVVLAAAYWLATLQPSDLPELNPAKPFEFTNRRRVHEFVENSKSLLARGRELHGHEPYRLMSEWGSLIVLPPECADELRNDPRMDFETPTTDDSHGYIPGFDA LNADPNLTKVVTKYLTKALNKLTAPISHEASIAMKAVLGDDPDWREIYPARDLLQLVARMSTRVFLGEEMCNNQDWIQTSSQYAALAFGVGDKLRIYPRMIRPIVHWFMPSCWELRRSLRRCRQILTPY IHKRKSLKGTTDEQGKPLMFDDSIEWFERELGPNHDAVLKQVTLSIVAIHTTSDLLLQAMSDLAQNPKVLQAVREEVVRVLSTEGLSKVSLHSLKLMDSALKESQRLRPTLLGSFRRQATNDIKLKSG FVIKKGTRVVIDSTHMWNPEYYTDPLQYDGYRYFNKRQTPGEDKNALLVSTSANHMGFGHGVHACPGRFFASNEIKIALCHIILNYEWRLPDGFKPQPLNIGMTYLADPNTRMLIRPRKAEIDMASLTV

[0113] CYP68J5 DNA-SEQ ID NO.2

[0114]

[0115] CYP68J5 codon-optimized nucleotide sequence - SEQ ID NO.3

[0116]

[0117] D118V amino acid-SEQ ID NO.4

[0118] MPFFTGLLAIYHSLILDNPVQTLSTIVVLAAAYWLATLQPSDLPELNPAKPFEFTNRRRVHEFVENSKSLLARGRELHGHEPYRLMSEWGSLIVLPPECADELRNDPRMDFETPTTDVSHGYIPGFDALNADPNLTKVVTKYLTKALNKLTAPISHEASIAMKAVLGDDPDWREIYPARDLLQLVARMSTRVFLGEEMCNNQDWIQTSSQYAALAFGVGDKLRIYPRMIRPIVHWFMPSCWELRRSLRRCRQILTPYIHKRKSLKGTTDEQGKPLMFDDSIEWFERELGPNHDAVLKQVTLSIVAIHTTSDLLLQAMSDLAQNPKVLQAVREEVVRVLSTEGLSKVSLHSLKLMDSALKESQRLRPTLLGSFRRQATNDIKLKSGFVIKKGTRVVIDSTHMWNPEYYTDPLQYDGYRYFNKRQTPGEDKNALLVSTSANHMGFGHGVHACPGRFFASNEIKIALCHIILNYEWRLPDGFKPQPLNIGMTYLADPNTRMLIRPRKAEIDMASLTV

[0119] D118V DNA-SEQ ID NO.5

[0120]

[0121] Although embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, variations, and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the scope of the invention is not limited to the contents disclosed in the embodiments.

Claims

1. A C11α-hydroxylase mutant D118V with enhanced catalytic activity, characterized in that: Its amino acid sequence is shown in SEQ ID NO.4, and the corresponding nucleotide sequence is shown in SEQ ID NO.

5.

2. The gene encoding the C11α-hydroxylase mutant D118V as described in claim 1.

3. A recombinant vector carrying the gene as described in claim 2.

4. The recombinant vector according to claim 3, characterized in that: Expression vectors include, but are not limited to, pYES2.

5. Genetically engineered bacteria that express the mutant as described in claim 1, or carry the gene as described in claim 2, or contain the recombinant vector as described in claim 3.

6. The genetically engineered bacteria as described in claim 5, characterized in that: Host cells include prokaryotes and eukaryotes.

7. A method for improving the catalytic activity of C11α-hydroxylase, characterized in that: The amino acid sequence was mutated based on the sequence shown in SEQ ID NO.1: the 118th amino acid was mutated from aspartic acid to valine.

8. A method for producing a target product using the genetically engineered bacteria as described in claim 5, characterized in that: The production method includes the following steps: The engineered bacteria were inoculated into Erlenmeyer flasks containing YEPD liquid medium and cultured with shaking at 28-35℃ and 180-250 r / min for 18-24 h. Substrate was then added, followed by the addition of methanol (1.0%-5.0% by volume) to aid dissolution, and then a 20% galactose solution (1.0%-5.0% by volume) was added for induction. This was repeated every 18-24 h. After complete transformation at 28-35℃ and 180-250 r / min, a transformation solution containing the target product was obtained. This solution was extracted with ethyl acetate to obtain a supernatant containing the target product. The substrate is 17α-hydroxyprogesterone, and the target product is 11α,17α-dihydroxyprogesterone.

9. The method according to claim 8, characterized in that: The substrate is 17α-hydroxyprogesterone, and the final concentration range for addition is 0.2-5.0 g / L.

10. The use of the mutant D118V as described in claim 1, or the recombinant vector as described in claim 3, or the genetically engineered bacteria as described in claim 5 or 6 in the C11α hydroxylation reaction using 17α-hydroxyprogesterone as a substrate.

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