Engineering strain for overexpressing cytochrome P450 enzyme gene or / and redox chaperonin gene thereof, method and application

Abstract

The invention belongs to the technical field of bioengineering, and discloses an engineering strain applied to biosynthesis of 16 alpha-hydroxyprednisolone, and the engineering strain comprises a gene for coding cytochrome P450 enzyme and a redox chaperonin gene thereof. The cytochrome P450 enzyme gene, the redox chaperonin gene and the combination of the cytochrome P450 enzyme gene and the redox chaperonin gene are screened through an overexpression technology. Compared with a wild strain, the initial production rate of a target product of the obtained excellent engineering strain is improved by 12.17%-161.96%, the maximum yield is improved by 0.37%-38.08%, meanwhile, the fermentation period is shortened, the production intensity is improved by 82.33%-147.64%, and theoretical guidance and application cases are provided for preparation of 16 alpha-hydroxyprednisolone.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular to an engineering strain overexpressing a cytochrome P450 enzyme gene or / and a redox chaperone protein gene thereof, a construction method thereof, and an application thereof in synthesizing 16α-hydroxyprednisolone. Background Art

[0002] Steroid hormone drugs are a class of hormone drugs containing steroid structures, which are widely used in the treatment of various diseases such as asthma, rheumatoid arthritis, and anti-tumor. The structure of steroid compounds is very complex, and their pharmacological activity is highly dependent on substituents at specific positions. 16α-hydroxylation is an important reaction in the synthesis of corticosteroids. By introducing a hydroxyl group at the C16 position, it can not only effectively reduce the side effects of the drug, but also maintain or even enhance its anti-inflammatory effect and glucose metabolism regulation function.

[0003] Cytochrome P450 enzyme is a key enzyme that catalyzes steroid hydroxylation reactions. It belongs to the heme-thiolate protein superfamily and is widely present in the biological world. As an external monooxygenase, most cytochrome P450 enzymes need to obtain redox equivalents from external NAD(P)H through an electron transfer process, which usually depends on the interaction of one or more redox chaperone proteins. According to the different electron transport chains composed of cytochrome P450 enzymes and different redox chaperone proteins, the P450 system is mainly divided into three categories: the class I P450 system mainly exists in prokaryotes and eukaryotic mitochondria, and is composed of three independently existing proteins - cytochrome P450 enzyme, ferredoxin and ferredoxin reductase, and its electron transfer pathway is NADPH→ferredoxin reductase→ferredoxin→cytochrome P450 enzyme; the class II P450 system mainly exists in eukaryotes and is composed of two components - cytochrome P450 Enzyme and NADPH-cytochrome P450 reductase, the latter contains two cofactors, FAD and FMN, and its electron transfer pathway is NADPH→NADPH-cytochrome P450 reductase→cytochrome P450 enzyme; the characteristic of the Class III P450 system is that the cytochrome P450 enzyme and the reductase are naturally fused into a single protein. This "catalytically autonomous" P450 enzyme can complete the electron transfer process within a single protein, and its electron transfer pathway is NADPH→reductase domain within a single protein→cytochrome P450 enzyme domain.

[0004] 16α-Hydroxyprednisolone is an important adrenal cortical hormone compound. As a key intermediate, it is widely used in the synthesis of a variety of nephron drugs, such as triamcinolone acetonide, budesonide, ciclesonide and desonide. At present, the six-step chemical synthesis method starting from prednisolone is mainly used at home and abroad to produce 16α-hydroxyprednisolone. However, this method has the problems of complex reaction operation, harsh conditions, and the need to use a large amount of organic solvents. Compared with the chemical synthesis method, the biosynthesis method of using whole cells or enzymes to catalyze prednisolone to produce 16α-hydroxyprednisolone (see the reaction equation below) has the advantages of fewer reaction steps, high selectivity, and more environmental friendliness. However, there are still problems such as low molar yield (up to only 34.2%) and unclear key catalytic enzyme information. The present invention provides a method for overexpressing genes encoding cytochrome P450 enzymes and their redox partner proteins to improve the synthetic ability of strains, providing a new way for the efficient and green production of 16α-hydroxyprednisolone.

[0005] Summary of the invention

[0006] The purpose of the present invention is to overcome the deficiencies in the prior art and to provide an engineered strain overexpressing a cytochrome P450 enzyme gene or / and its redox chaperone protein gene, a construction method thereof and an application thereof in the synthesis of 16α-hydroxyprednisolone.

[0007] The technical solution adopted by the present invention to solve its technical problem is:

[0008] An engineered strain used in the biosynthesis of 16α-hydroxyprednisolone, the engineered strain comprising a gene encoding a cytochrome P450 enzyme.

[0009] Further, the gene encoding the cytochrome P450 enzyme is: 3629 or 6606 or 6792 from Streptomyces roseochromogenes DSM 40463; the nucleotide sequence of 3629 is as shown in SEQ ID No.1 or a nucleotide sequence with at least 70% identity to the nucleotide sequence shown therein; the nucleotide sequence of 6606 is as shown in SEQ ID No.2 or a nucleotide sequence with at least 70% identity to the nucleotide sequence shown therein; the nucleotide sequence of 6792 is as shown in SEQ ID No.3 or a nucleotide sequence with at least 70% identity to the nucleotide sequence shown therein;

[0010] Alternatively, the cytochrome P450 enzyme is: 3629 or 6606 or 6792; the amino acid sequence of 3629 is as shown in SEQ ID No.12 or an amino acid sequence that is at least 70% identical to the amino acid sequence shown therein; the amino acid sequence of 6606 is as shown in SEQ ID No.13 or an amino acid sequence that is at least 70% identical to the amino acid sequence shown therein; the amino acid sequence of 6792 is as shown in SEQ ID No.14 or an amino acid sequence that is at least 70% identical to the amino acid sequence shown therein.

[0011] Furthermore, the engineered strain also includes a gene encoding a redox chaperone protein.

[0012] Further, the gene encoding the redox chaperone protein includes a gene encoding ferredoxin and a gene encoding ferredoxin reductase;

[0013] The gene encoding ferredoxin is: 2182 or 2776 or 5437 or 6156 or 7088 from Streptomyces roseochromogenes DSM 40463; the nucleotide sequence of 2182 is as shown in SEQ ID No.4 or a nucleotide sequence having at least 70% identity with the nucleotide sequence shown therein; the nucleotide sequence of 2776 is as shown in SEQ ID No.5 or a nucleotide sequence having at least 70% identity with the nucleotide sequence shown therein; the nucleotide sequence of 5437 is as shown in SEQ ID No.6 or a nucleotide sequence having at least 70% identity with the nucleotide sequence shown therein; the nucleotide sequence of 6156 is as shown in SEQ ID No.7 or a nucleotide sequence having at least 70% identity with the nucleotide sequence shown therein; the nucleotide sequence of 7088 is as shown in SEQ ID No.8 or a nucleotide sequence having at least 70% identity with the nucleotide sequence shown therein;

[0014] The gene encoding ferredoxin reductase is: 0169, 0220 or 6675 from Streptomyces roseochromogenes DSM 40463; the nucleotide sequence of 0169 is shown as SEQ ID No.9 or a nucleotide sequence with at least 70% identity to the nucleotide sequence shown therein; the nucleotide sequence of 0220 is shown as SEQ ID No.10 or a nucleotide sequence with at least 70% identity to the nucleotide sequence shown therein; the nucleotide sequence of 6675 is shown as SEQ ID No.11 or a nucleotide sequence with at least 70% identity to the nucleotide sequence shown therein;

[0015] Alternatively, the redox chaperone protein comprises ferredoxin and ferredoxin reductase, and the ferredoxin is: 2182 or 2776 or 5437 or 6156 or 7088; the amino acid sequence of 2182 is as shown in SEQ ID No.15 or an amino acid sequence having at least 70% identity to the amino acid sequence shown therein; the amino acid sequence of 2776 is as shown in SEQ ID No.16 or an amino acid sequence having at least 70% identity to the amino acid sequence shown therein; the amino acid sequence of 5437 is as shown in SEQ ID No.17 or an amino acid sequence having at least 70% identity to the amino acid sequence shown therein; the amino acid sequence of 6156 is as shown in SEQ ID No.18 or an amino acid sequence having at least 70% identity to the amino acid sequence shown therein; the amino acid sequence of 7088 is as shown in SEQ ID No.19 or an amino acid sequence having at least 70% identity to the amino acid sequence shown therein;

[0016] The ferredoxin reductase is: 0169 or 0220 or 6675; the amino acid sequence of 0169 is as shown in SEQ ID No. 20 or an amino acid sequence that is at least 70% identical to the amino acid sequence shown therein; the amino acid sequence of 0220 is as shown in SEQ ID No. 21 or an amino acid sequence that is at least 70% identical to the amino acid sequence shown therein; the amino acid sequence of 6675 is as shown in SEQ ID No. 22 or an amino acid sequence that is at least 70% identical to the amino acid sequence shown therein.

[0017] A method for constructing the engineered strain as described above comprises the following steps:

[0018] Genes encoding cytochrome P450 enzymes, or genes encoding ferredoxin and genes encoding ferredoxin reductase are obtained by PCR amplification; expression cassettes of the above-mentioned cytochrome P450 enzymes, ferredoxin or ferredoxin reductase encoding genes are respectively constructed by genetic engineering means, and are connected to expression vectors individually or in series, and are transferred to Escherichia coli for replication; recombinant plasmids are extracted and respectively transformed into methylation-deficient Escherichia coli competent ET12567 / pUZ8002; and the verified correct recombinant plasmids are respectively introduced into host cells through conjugation transfer experiments to construct corresponding overexpression engineering strains.

[0019] Further, the host cell is Streptomyces roseochromogenes DSM 40463;

[0020] Alternatively, the expression vector is pIJ8660.

[0021] Application of the engineered strain as described above in the synthesis of 16α-hydroxyprednisolone.

[0022] The method for producing 16α-hydroxyprednisolone using the engineered strain as described above comprises the following steps:

[0023] Streak the engineered strain on a solid culture medium and culture it at 28°C for 5-7 days until spores grow. Take the strain that has good growth and dig out 3 square blocks of bacteria, each 1 cm 2 , inoculated into a 250mL Erlenmeyer flask containing 20mL of seed culture medium, and cultured at 28°C and 200r / min for 16-32 hours to obtain seed solution; inoculated the seed solution into a 250mL Erlenmeyer flask containing 20-40mL of fermentation culture medium at an inoculum amount of 5-20%, and cultured at 28°C and 200r / min for 24 hours, and then added with anhydrous ethanol to dissolve the substrate, so that the final concentration of anhydrous ethanol is 8g / L, and the final concentration of the substrate is 0.5g / L, and 16α-hydroxyprednisolone is obtained after conversion for 24-216 hours.

[0024] Furthermore, the substrate is prednisolone.

[0025] Furthermore, the solid culture medium formula is: glucose 12g / L, yeast extract 6g / L, malt extract 30g / L, calcium carbonate 2g / L, agar 20g / L, and the solvent is water;

[0026] The seed culture medium formula is: 4g / L glucose, 4g / L yeast extract, 10g / L malt extract, and the solvent is water;

[0027] The pH of the fermentation medium is 5.5-8.5, and the formula is: 12 g / L glucose, 6 g / L yeast extract, 30 g / L malt extract, and the solvent is water;

[0028] In order to maintain the stability of the plasmid in the engineered strain, an apramycin antibiotic solution with a final concentration of 50 μg / mL needs to be added to the above solid culture medium, seed culture medium and fermentation medium.

[0029] The advantages and positive effects achieved by the present invention are:

[0030] 1. The present invention adopts overexpression technology to screen cytochrome P450 enzyme genes and their redox partner protein genes, and express them in a host after combining the two. Compared with the chemical method in industrial production, this biosynthesis method has the advantages of fewer reaction steps, high selectivity, and more environmental friendliness, and provides a feasible technical solution and application case for the green synthesis of 16α-hydroxyprednisolone.

[0031] 2. The present invention provides a method for improving the ability of the strain to synthesize 16α-hydroxyprednisolone by overexpressing any of the following genes in the wild type of Streptomyces roseochromogenes: (1) a gene encoding a cytochrome P450 enzyme; or (2) a gene encoding a cytochrome P450 enzyme and a gene encoding a redox chaperone protein. The yield of 16α-hydroxyprednisolone is significantly increased, providing a basis for the industrial biosynthesis of 16α-hydroxyprednisolone.

[0032] 3. The Streptomyces roseochromogenes DSM 40463 chassis cells selected in the present invention have good 16α-hydroxyprednisolone production potential.

[0033] 4. The present invention constructs 18 engineered strains, and the initial production rate of 16α-hydroxyprednisolone is increased by 12.17% to 161.96% compared with the wild type, and the maximum yield is increased by 0.37% to 38.08%. At the same time, the fermentation cycle is shortened, and the production intensity is increased by 82.33% to 147.64%, which has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 It is a liquid phase detection diagram of the standard substance and the conversion sample of prednisolone and 16α-hydroxyprednisolone in the present invention;

[0035] Figure 2 It is a synthesis curve diagram of the target product before and after the reaction conditions of wild-type Streptomyces roseochromogenes transforming prednisolone to generate 16α-hydroxyprednisolone are optimized in the present invention;

[0036] Figure 3 The figure is a genome PCR result diagram of the gene overexpression engineering strain encoding cytochrome P450 enzyme or / and its redox partner protein of Streptomyces roseochromogenes in the present invention; wherein, M is 10000DNAMarker, and 1 to 18 are Sr3629, Sr6606, Sr6792, Sr3629-1, Sr3629-2, Sr3629-3, Sr3629-4, Sr3629-5, Sr3629-6, Sr3629-7, Sr3629-8, Sr3629-9, Sr3629-10, Sr3629-11, Sr3629-12, Sr3629-13, Sr3629-14, Sr3629-15;

[0037] Figure 4 The initial production rate and maximum yield of the engineered strain overexpressing the gene encoding cytochrome P450 enzyme or / and its redox chaperone protein of Streptomyces roseochromogenes and the wild-type strain in the present invention when transforming prednisolone to generate 16α-hydroxyprednisolone in a 0.5 g / L transformation system;

[0038] Figure 5This is a production intensity diagram of the engineered strain overexpressing the gene encoding cytochrome P450 enzyme or / and its redox chaperone protein of Streptomyces roseochromogenes in the present invention and the wild type in converting prednisolone to 16α-hydroxyprednisolone under a 0.5 g / L transformation system. DETAILED DESCRIPTION

[0039] The present invention will be further described below in conjunction with the embodiments. The following embodiments are descriptive rather than restrictive, and the protection scope of the present invention cannot be limited by the following embodiments.

[0040] The various experimental operations involved in the specific embodiments are all routine techniques in the art. For parts not specially annotated in this document, ordinary technicians in the art can implement them by referring to various commonly used reference books, scientific and technological literature or related instructions, manuals, etc. before the filing date of this invention.

[0041] An engineered strain used in the biosynthesis of 16α-hydroxyprednisolone, the engineered strain comprising a gene encoding a cytochrome P450 enzyme.

[0042] Further, the gene encoding the cytochrome P450 enzyme is: 3629 or 6606 or 6792 from Streptomyces roseochromogenes DSM 40463; the nucleotide sequence of 3629 is as shown in SEQ ID No.1 or a nucleotide sequence with at least 70% identity to the nucleotide sequence shown therein; the nucleotide sequence of 6606 is as shown in SEQ ID No.2 or a nucleotide sequence with at least 70% identity to the nucleotide sequence shown therein; the nucleotide sequence of 6792 is as shown in SEQ ID No.3 or a nucleotide sequence with at least 70% identity to the nucleotide sequence shown therein;

[0043] Alternatively, the cytochrome P450 enzyme is: 3629 or 6606 or 6792; the amino acid sequence of 3629 is as shown in SEQ ID No.12 or an amino acid sequence that is at least 70% identical to the amino acid sequence shown therein; the amino acid sequence of 6606 is as shown in SEQ ID No.13 or an amino acid sequence that is at least 70% identical to the amino acid sequence shown therein; the amino acid sequence of 6792 is as shown in SEQ ID No.14 or an amino acid sequence that is at least 70% identical to the amino acid sequence shown therein.

[0044] Furthermore, the engineered strain also includes a gene encoding a redox chaperone protein.

[0045] Further, the gene encoding the redox chaperone protein includes a gene encoding ferredoxin and a gene encoding ferredoxin reductase;

[0046] The gene encoding ferredoxin is: 2182 or 2776 or 5437 or 6156 or 7088 from Streptomyces roseochromogenes DSM 40463; the nucleotide sequence of 2182 is as shown in SEQ ID No.4 or a nucleotide sequence having at least 70% identity with the nucleotide sequence shown therein; the nucleotide sequence of 2776 is as shown in SEQ ID No.5 or a nucleotide sequence having at least 70% identity with the nucleotide sequence shown therein; the nucleotide sequence of 5437 is as shown in SEQ ID No.6 or a nucleotide sequence having at least 70% identity with the nucleotide sequence shown therein; the nucleotide sequence of 6156 is as shown in SEQ ID No.7 or a nucleotide sequence having at least 70% identity with the nucleotide sequence shown therein; the nucleotide sequence of 7088 is as shown in SEQ ID No.8 or a nucleotide sequence having at least 70% identity with the nucleotide sequence shown therein;

[0047] The gene encoding ferredoxin reductase is: 0169, 0220 or 6675 from Streptomyces roseochromogenes DSM 40463; the nucleotide sequence of 0169 is shown as SEQ ID No.9 or a nucleotide sequence with at least 70% identity to the nucleotide sequence shown therein; the nucleotide sequence of 0220 is shown as SEQ ID No.10 or a nucleotide sequence with at least 70% identity to the nucleotide sequence shown therein; the nucleotide sequence of 6675 is shown as SEQ ID No.11 or a nucleotide sequence with at least 70% identity to the nucleotide sequence shown therein;

[0048] Alternatively, the redox chaperone protein comprises ferredoxin and ferredoxin reductase, and the ferredoxin is: 2182 or 2776 or 5437 or 6156 or 7088; the amino acid sequence of 2182 is as shown in SEQ ID No.15 or an amino acid sequence having at least 70% identity to the amino acid sequence shown therein; the amino acid sequence of 2776 is as shown in SEQ ID No.16 or an amino acid sequence having at least 70% identity to the amino acid sequence shown therein; the amino acid sequence of 5437 is as shown in SEQ ID No.17 or an amino acid sequence having at least 70% identity to the amino acid sequence shown therein; the amino acid sequence of 6156 is as shown in SEQ ID No.18 or an amino acid sequence having at least 70% identity to the amino acid sequence shown therein; the amino acid sequence of 7088 is as shown in SEQ ID No.19 or an amino acid sequence having at least 70% identity to the amino acid sequence shown therein;

[0049] The ferredoxin reductase is: 0169 or 0220 or 6675; the amino acid sequence of 0169 is as shown in SEQ ID No. 20 or an amino acid sequence that is at least 70% identical to the amino acid sequence shown therein; the amino acid sequence of 0220 is as shown in SEQ ID No. 21 or an amino acid sequence that is at least 70% identical to the amino acid sequence shown therein; the amino acid sequence of 6675 is as shown in SEQ ID No. 22 or an amino acid sequence that is at least 70% identical to the amino acid sequence shown therein.

[0050] A method for constructing the engineered strain as described above comprises the following steps:

[0051] Genes encoding cytochrome P450 enzymes, or genes encoding ferredoxin and genes encoding ferredoxin reductase are obtained by PCR amplification; expression cassettes of the above-mentioned cytochrome P450 enzymes, ferredoxin or ferredoxin reductase encoding genes are respectively constructed by genetic engineering means, and are connected to expression vectors individually or in series, and are transferred to Escherichia coli for replication; recombinant plasmids are extracted and respectively transformed into methylation-deficient Escherichia coli competent ET12567 / pUZ8002; and the verified correct recombinant plasmids are respectively introduced into host cells through conjugation transfer experiments to construct corresponding overexpression engineering strains.

[0052] Further, the host cell is Streptomyces roseochromogenes DSM 40463;

[0053] Alternatively, the expression vector is pIJ8660.

[0054] Application of the engineered strain as described above in the synthesis of 16α-hydroxyprednisolone.

[0055] The method for producing 16α-hydroxyprednisolone using the engineered strain as described above comprises the following steps:

[0056] Streak the engineered strain on a solid culture medium and culture it at 28°C for 5-7 days until spores grow. Take the strain that has good growth and dig out 3 square blocks of bacteria, each 1 cm 2 , inoculated into a 250mL Erlenmeyer flask containing 20mL of seed culture medium, and cultured at 28°C and 200r / min for 16-32 hours to obtain seed solution; inoculated the seed solution into a 250mL Erlenmeyer flask containing 20-40mL of fermentation culture medium at an inoculum amount of 5-20%, and cultured at 28°C and 200r / min for 24 hours, and then added with anhydrous ethanol to dissolve the substrate, so that the final concentration of anhydrous ethanol is 8g / L, and the final concentration of the substrate is 0.5g / L, and 16α-hydroxyprednisolone is obtained after conversion for 24-216 hours.

[0057] Furthermore, the substrate is prednisolone.

[0058] Furthermore, the solid culture medium formula is: glucose 12g / L, yeast extract 6g / L, malt extract 30g / L, calcium carbonate 2g / L, agar 20g / L, and the solvent is water;

[0059] The seed culture medium formula is: 4g / L glucose, 4g / L yeast extract, 10g / L malt extract, and the solvent is water;

[0060] The pH of the fermentation medium is 5.5-8.5, and the formula is: 12 g / L glucose, 6 g / L yeast extract, 30 g / L malt extract, and the solvent is water;

[0061] In order to maintain the stability of the plasmid in the engineered strain, an apramycin antibiotic solution with a final concentration of 50 μg / mL needs to be added to the above solid culture medium, seed culture medium and fermentation medium.

[0062] The Streptomyces roseochromogenes DSM 40463 in the present invention is a strain known in the art and can be obtained from the German Microbiological Culture Collection DSMZ (DSM No.: 40463). The biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0063] Example 1: Analysis of transformation performance of wild-type Streptomyces roseochromogenes under different conditions

[0064] (1) Strain activation and seed culture

[0065] The wild-type strain (Streptomyces roseochromogenes DSM 40463) was inoculated on a solid culture medium and cultured at 28°C for 5-7 days until a large number of spores grew on the plate. Three square fungus blocks (each about 1 cm 2 ) and inoculated into a 250 mL Erlenmeyer flask containing 20 mL of seed culture medium. The culture was shaken at 28°C and 200 r / min for 16, 24, and 32 hours, respectively (Table 1), to obtain sufficient seed solution.

[0066] (2) Fermentation culture and substrate conversion

[0067] The seed solution in (1) was taken and transferred to 250 mL Erlenmeyer flasks containing 20, 30, and 40 mL of fermentation medium at an inoculum of 5%, 10%, and 20%, respectively, with the initial pH values ​​being 5.5, 7.0, and 8.5, respectively (Table 1). After shaking culture at 28°C and 200 r / min for 24 hours, anhydrous ethanol-assisted substrate was added (the final concentrations of anhydrous ethanol and substrate were 8 g / L and 0.5 g / L, respectively, and the substrate was prednisolone). Afterwards, shaking culture was continued at 28°C and 200 r / min to start the biotransformation process.

[0068] (3) Target product extraction

[0069] 600 μL of the conversion solution after 216 hours of cultivation was collected, and an equal volume of ethyl acetate was added and mixed well, and then ultrasonic extraction was performed for at least 10 minutes. Then, the two phases were separated by centrifugation at 12,000 r / min for 10 minutes. The upper organic phase (ethyl acetate layer) was collected, which contained the target product to be tested.

[0070] (4) Detection of 16α-hydroxyprednisolone

[0071] The detection method of 16α-hydroxyprednisolone is as described in Example 2. The specific results of the orthogonal test are shown in Table 2. Under different conditions, the conversion was performed for 216 hours, and the production range of 16α-hydroxyprednisolone was 39.87-129.63 mg / L. Through range analysis, the order of influence of various factors on the synthesis of 16α-hydroxyprednisolone was determined to be: liquid volume> inoculation volume> seed age> pH. Based on the k value of the range analysis, the optimal fermentation conditions were determined as follows: seed age 24h, inoculation volume 10%, pH 8.5, liquid volume 40mL / 250mL conical flask. Figure 2 As shown in the figure, under the above optimal conditions, the yield of 16α-hydroxyprednisolone was the highest after 216 hours of conversion, reaching 144.63 mg / L, which was 97.47% higher than that before optimization. Therefore, subsequent conversion experiments were carried out under this optimized condition to ensure the optimal yield of 16α-hydroxyprednisolone.

[0072] The solid culture medium formula is: glucose 12g / L, yeast extract 6g / L, malt extract 30g / L, calcium carbonate 2g / L, agar 20g / L, and the solvent is water;

[0073] The seed culture medium formula is: 4g / L glucose, 4g / L yeast extract, 10g / L malt extract, and the solvent is water;

[0074] The pH of the fermentation medium is 5.5-8.5, and the formula is: 12 g / L glucose, 6 g / L yeast extract, 30 g / L malt extract, and the solvent is water.

[0075] Table 1 Orthogonal test factor level table

[0076]

[0077] Table 2 Orthogonal experimental design and results

[0078]

[0079]

[0080] Example 2: Establishment of a liquid phase detection method for 16α-hydroxyprednisolone

[0081] Weigh 0.05g of prednisolone and 16α-hydroxyprednisolone standards respectively, and add 1mL of mobile phase (specific composition and proportion are described below) to dissolve each. Pipette 200μL of the sample to be tested into a centrifuge tube, evaporate naturally in a fume hood, and add 1mL of mobile phase for re-dissolution. Place the above sample and standard solution in an ultrasonic cleaner for 20 minutes to ensure full dissolution. Centrifuge the solution at 13,000r / min for 10 minutes, and take 300μL of the supernatant for liquid chromatography detection. The specific conditions are as follows:

[0082] Liquid chromatograph: Agilent Technologies 1260 Infinity Series

[0083] UV detector: Agilent Technologies 1200Infinity Series

[0084] Chromatographic column: Agela C18 column (250 mm × 4.6 mm, 5 μm);

[0085] Mobile phase: acetonitrile: water = 30:70 (v / v, volume ratio), containing 1% formic acid;

[0086] Detection wavelength: 245nm;

[0087] like Figure 1 As shown in the figure, two clearly separated peaks appeared in the conversion sample at 6.617 minutes and 11.274 minutes, and their retention times were basically consistent with the peak times of prednisolone and 16α-hydroxyprednisolone standards. This result shows that the adopted method can effectively identify and quantify the residual starting substrate in the conversion sample, prednisolone, and the generated target product, 16α-hydroxyprednisolone.

[0088] Example 3: Construction of an engineered strain of Streptomyces roseochromogenes that overexpresses genes encoding cytochrome P450 enzymes

[0089] (1) Acquisition of target genes 3629 / 6606 / 6792

[0090] Using the genome of Streptomyces roseochromogenes DSM 40463 as a template, primers 3629-F and 3629-R, 6606-F and 6606-R, 6792-F and 6792-R were used to amplify fragments 3629, 6606 and 6792 on the genome of Streptomyces roseochromogenes, respectively.

[0091] 3629-F: CCAAAGGAGGCGGACATCATATGAGCATCACAGGTGGTACGTC

[0092] 3629-R:TCGATGTGATATCCAAGATCTTCACAGGGCCACGCCCAG

[0093] 6606-F: CCAAAGGAGGCGGACATCATATGGTCGACAACACCCTCCC

[0094] 6606-R:TCGATGTGATATCCAAGATCTTCAGCCCCTGCCCGCCGC

[0095] 6792-F:CCAAAGGAGGCGGACATCATATGGAGAACACCTCGGTCCG

[0096] 6792-R:TCGATGTGATATCCAAGATCTTCACCATGTGACGGGCAGG

[0097] PCR reaction system:

[0098]

[0099] PCR amplification conditions: 95°C for 3 min, 95°C for 15 s, T m -5℃ for 15s, 72℃ for 1.5min, 72℃ for 5min, for a total of 35 cycles, and keep warm at 4℃.

[0100] The PCR product was recovered by gel extraction kit to obtain the target fragment 3629 / 6606 / 6792.

[0101] (2) Linearization of vector pIJ8660

[0102] The vector was double-digested with restriction endonucleases Nde I and BglⅡ to achieve linearization. The reaction system is as shown in Table 3:

[0103] Table 3 Enzyme digestion reaction system

[0104] Element Volume (μL) N i 1 BglⅡ 1 10×CutsmartBuffer 2 Plasmids 5 <![CDATA[ddH 2 The]]> 11 Total volume 20

[0105] Incubate at 37°C for 2 hours.

[0106] Take 3 μL of the digested product and perform 1% agarose gel electrophoresis at 120 V constant voltage. If a band appears and the size is correct, use the Cycle-Pure Kit for purification and recovery, or cut the gel and use the Gel Extraction Kit to recover the linearized vector.

[0107] (3) Construction of pIJ8660-ermE*p-3629 / 6606 / 6792 recombinant plasmid

[0108] The above PCR product and the digested vector were connected by seamless cloning (see Table 4 for the connection system), and the connection product was obtained by reacting at 50°C for 15 minutes. Next, the E. coli DH5α competent cells were taken out and placed on ice, and the above connection product was added to the clean bench, and the mixture was gently pipetted and mixed, and placed on ice for 30 minutes. Subsequently, the above mixture was heat-shocked at 42°C for 90 seconds, and then quickly transferred to ice for 2 minutes. 800μL LB liquid culture medium was added, and the culture was revived at 37°C and 200r / min for 50 minutes. Centrifuge at 8,000r / min for 5 minutes, remove 750μL supernatant, and gently pipet and mix the remaining bacterial solution. Finally, the mixed bacterial solution was spread on LB solid culture medium containing 50μg / mL apramycin and cultured overnight at 37°C. The plasmids of the positive transformants grown on the above LB solid medium were extracted and verified by PCR, restriction digestion and sequencing to obtain the recombinant plasmid pIJ8660-ermE*p-3629 / 6606 / 6792.

[0109] Table 4 Seamless cloning connection system

[0110] Element Volume (μL) 2 × ClonExpressMix x+y Linearized vector x Insert y

[0111] x = optimal cloning vector usage / linearized vector concentration

[0112] y = optimal amount of insert fragment / concentration of insert fragment

[0113] Optimal amount of cloning vector used = [0.02 × number of base pairs of cloning vector] ng (0.03 pmol)

[0114] Optimal amount of insert fragment = [0.04 × number of base pairs of insert fragment] ng (0.06 pmol)

[0115] (4) Preparation of donor bacterial suspension

[0116] Take out the competent cells of E. coli ET12567 / pUZ8002 and place them on ice. Add the recombinant plasmid pIJ8660-ermE*p-3629 / 6606 / 6792 to the clean bench, mix them by gently pipetting, and place them on ice for 30 minutes. Subsequently, heat shock the mixture at 42°C for 90 seconds, and then quickly transfer it to ice for 2 minutes. Add 800μL LB liquid culture medium and resuscitate and culture at 37°C and 200r / min for 50 minutes. Centrifuge at 8,000r / min for 5 minutes, remove 750μL supernatant, and gently pipette and mix the remaining bacterial solution. Finally, spread the mixed bacterial solution on LB solid culture medium containing 50μg / mL apramycin, 50μg / mL kanamycin, and 25μg / mL chloramphenicol, and culture overnight at 37°C. The grown single colony was transferred to a test tube containing 5 mL LB liquid medium containing the same antibiotics, and cultured overnight at 37°C and 200 r / min. The above culture was transferred to a 250 mL Erlenmeyer flask containing 25 mL LB liquid medium containing the same antibiotics at a 1% inoculum, and cultured at 37°C and 200 r / min until the OD 600 The culture solution was centrifuged at 5,000 r / min for 3 minutes and the cells were collected. Then 1.5 mL of LB liquid medium was added to resuspend the cells to prepare a bacterial suspension, and the cells were centrifuged at 5,000 r / min for 3 minutes and the cells were collected. This step was repeated three times. Finally, 1 mL of LB liquid medium was used to resuspend the cells to prepare a donor bacterial suspension (OD 600 is 0.4-0.6).

[0117] (5) Preparation of Recipient Bacterial Suspension

[0118] Under sterile conditions, the wild-type strain (Streptomyces roseochromogenes DSM 40463) was inoculated on a solid medium and cultured at 28°C for 5-7 days until a large number of spores grew on the plate. Add an appropriate amount of sterile water to the plate and use a sterile inoculation loop to scrape the spores to ensure that the spores are fully suspended in the water. The resulting spore suspension was transferred to a pre-sterilized triangular flask with three layers of gauze covering the bottle mouth and glass beads placed at the bottom of the bottle (glass beads help improve the spore dispersion effect). Shake thoroughly to ensure that the spores are evenly dispersed, and finally prepare a concentration of 1×10 6 ~1×10 7 / mL spore suspension. Take 1mL of the above spore suspension and centrifuge at 5,000r / min for 3 minutes to collect spores. Add 1mL LB liquid medium and centrifuge at 5,000r / min for 3 minutes to collect spores. Repeat this step twice. Resuspend the spores in 1mL LB liquid medium. Heat shock the prepared spore suspension in a 50℃ water bath for 10 minutes, then cool to room temperature to prepare a receptor bacterial suspension (concentration of 1×10 6 ~1×107 / mL).

[0119] The solid culture medium formula is: 12 g / L glucose, 6 g / L yeast extract, 30 g / L malt extract, 2 g / L calcium carbonate, 20 g / L agar, and the solvent is water.

[0120] (6) Co-cultivation and screening

[0121] The donor bacterial suspension and the recipient bacterial suspension were fully mixed. After the mixture was centrifuged at 5,000r / min for 1 minute, part of the supernatant was discarded, and then the bacteria and the remaining supernatant were fully mixed, and then the mixture was spread on the solid culture medium and cultured in a constant temperature incubator at 28°C. After 16 hours, antibiotic solutions with a final concentration of 50μg / mL apramycin and 25μg / mL nalidixic acid were evenly applied to the surface of the solid culture medium in a clean bench. After it was naturally dried, it was continued to be statically cultured at 28°C for 5-7 days until a single colony of Streptomyces conjugated with the target plasmid grew.

[0122] The solid culture medium formula is: 12 g / L glucose, 6 g / L yeast extract, 30 g / L malt extract, 2 g / L calcium carbonate, 20 g / L agar, and the solvent is water.

[0123] (7) Verification of binders

[0124] Pick the grown conjugates and inoculate them on solid culture medium. Pick the single clone grown on the above solid culture medium, extract the genome and perform PCR verification. Figure 3 As shown, the primer pair amplified a band size of 8689 bp, proving that the recombinant plasmid pIJ8660-ermE*p-3629 / 6606 / 6792 has been successfully integrated into the attB site in the genome. Three engineered strains were successfully screened and named Sr3629, Sr6606 and Sr6792 respectively.

[0125] The solid culture medium formula is: 12 g / L glucose, 6 g / L yeast extract, 30 g / L malt extract, 2 g / L calcium carbonate, 20 g / L agar, and the solvent is water.

[0126] The solid culture medium used needs to be supplemented with apramycin antibiotic solution with a final concentration of 50 μg / mL.

[0127] Example 4: Analysis of transformation performance of engineered Streptomyces roseochromogenes strains Sr3629, Sr6606 and Sr6792

[0128] (1) Strain activation and seed culture

[0129] Strains Sr3629, Sr6606, Sr6792 and wild type (Streptomyces roseochromogenes DSM 40463) were inoculated on solid culture medium and cultured at 28°C for 5-7 days until a large number of spores grew on the plate. Three square fungus blocks (each about 1 cm 2 ) and inoculated into a 250 mL Erlenmeyer flask containing 20 mL of seed culture medium. Cultured at 28°C and 200 r / min for 24 hours to obtain sufficient seed solution.

[0130] (2) Fermentation culture and substrate conversion

[0131] Take the seed solution in (1) and transfer it to a 250 mL Erlenmeyer flask containing 40 mL of fermentation medium at an inoculum of 10%, with an initial pH of 8.5. After shaking culture at 28° C. and 200 r / min for 24 hours, add the substrate solubilized by anhydrous ethanol (the final concentrations of anhydrous ethanol and substrate are 8 g / L and 0.5 g / L, respectively, and the substrate is prednisolone). After that, continue shaking culture at 28° C. and 200 r / min to start the biotransformation process.

[0132] The solid culture medium formula is: glucose 12g / L, yeast extract 6g / L, malt extract 30g / L, calcium carbonate 2g / L, agar 20g / L, and the solvent is water;

[0133] The seed culture medium formula is: 4g / L glucose, 4g / L yeast extract, 10g / L malt extract, and the solvent is water;

[0134] The formula of the fermentation medium is: 12 g / L of glucose, 6 g / L of yeast extract, 30 g / L of malt extract, and the solvent is water.

[0135] The solid culture medium, seed culture medium and fermentation culture medium used by the engineered strains Sr3629, Sr6606 and Sr6792 all need to be supplemented with an apramycin antibiotic solution with a final concentration of 50 μg / mL.

[0136] (3) Target product extraction

[0137] Collect 600 μL of the conversion solution after 120 hours of cultivation, add an equal volume of ethyl acetate, mix well, and then perform ultrasonic extraction for at least 10 minutes. Then, centrifuge at 12,000 r / min for 10 minutes to separate the two phases. Collect the upper organic phase (ethyl acetate layer), which contains the target product to be tested.

[0138] (4) Detection of 16α-hydroxyprednisolone

[0139] The detection method of 16α-hydroxyprednisolone is as described in Example 2. Figure 4As shown, the initial production rates of 16α-hydroxyprednisolone of the engineered strains Sr3629, Sr6606 and Sr6792 were 70.71 mg / L / d, 67.51 mg / L / d and 54.00 mg / L / d, respectively, which were 46.88%, 40.24% and 12.17% higher than those of the wild type (48.14 mg / L / d); the yields reached the maximum at 120 hours of transformation, which were 159.75 mg / L, 146.53 mg / L and 147.06 mg / L, respectively, while the wild type reached the maximum at 216 hours of transformation, which was 145.99 mg / L, and the maximum yields were 9.43%, 0.37% and 0.73% higher than those of the wild type, respectively. Figure 5 As shown, after 120 hours of transformation, the 16α-hydroxyprednisolone production intensities of the engineered strains Sr3629, Sr6606 and Sr6792 were 32.13 mg / L / d, 28.06 mg / L / d and 28.16 mg / L / d, respectively, which were 108.77%, 82.33% and 82.98% higher than the wild type (15.39 mg / L / d), respectively. Among them, Sr3629 showed the best performance in the entire transformation process, with the target product yield, initial production rate and production intensity all higher than other engineered strains and wild-type strains.

[0140] Example 5: Construction of an engineered strain of Streptomyces roseochromogenes that overexpresses genes encoding cytochrome P450 enzymes and their redox chaperone proteins

[0141] (1) Obtaining the target fragment kasO*p-0169 / 0220 / 6675-ermE*p-2182 / 2776 / 5437 / 6156 / 7088

[0142] Using the genome of Streptomyces roseochromogenes DSM 40463 as a template, primers 0169-F and 0169-R, 0220-F and 0220-R, 6675-F and 6675-R, 2182-F and 2182-R, 2776-F and 2776-R, 5437-F and 5437-R, 6156-F and 6156-R, 7088-F and 7088-R were used to amplify fragments 0169, 0220, 6675, 2181, 2776, 5437, 6156 and 7088 on the genome of Streptomyces roseochromogenes, respectively. Plasmid pIJ8660-ermE*p (see Liu SP, Yuan PH, Wang YY, et al. Generation of the natamycin analogs by gene engineering of natamycin biosynthetic genes in Streptomyces chattanoogensis L10. Microbiol Res. 2015; 173: 25-33.) and plasmid pKC-kasO*p (see Li L, Wei K, Zheng G, et al. CRISPR-Cpf1-Assisted Multiplex Genome Editing and Transcriptional Repression in Streptomyces. Appl Environ Microbiol. 2018; 84(18): e00827-18.) were used as templates, and primers ermE*pF and ermE*pR, kasO*pF and kasO*pR were used to amplify the ermE*p on the plasmid (see Liu SP, Yuan PH, WangYY, et al. Generation of the natamycin analogs by gene engineering of natamycin biosynthetic genes in Streptomyces chattanoogensis L10. MicrobiolRes. 2015; 173:25-33.) and kasO*p (see Li L, Wei K, Zheng G, et al. CRISPR-Cpf1-AssistedMultiplex Genome Editing and Transcriptional Repression inStreptomyces.Appl Environ Microbiol.2018;84(18):e00827-18.) fragment.Using kasO*p and 0169 / 0220 / 6675 as templates, kasO*pF and 0169-R / 0220-R / 6675-R as primers, PCR amplification obtained the fragment kasO*p-0169 / 0220 / 6675; using kasO*p-0169 / 0220 / 6675 and ermE*p as templates, kasO*pF and ermE*pR as primers, PCR amplification obtained the fragment kasO*p-0169 / 0220 / 6675. The fragment kasO*p-0169 / 0220 / 6675-ermE*p was obtained; the fragment was amplified by PCR using kasO*p-0169 / 0220 / 6675-ermE*p and 2182 / 2776 / 5437 / 6156 / 7088 as templates and kasO*pF and 2182-R / 2776-R / 5437-R / 6156-R / 7088-R as primers.

[0143] kasO*p-0169 / 0220 / 6675-ermE*p-2182 / 2776 / 5437 / 6156 / 7088.

[0144] 0169-F: GTTGTTGTAAAGTCGTGGCCAATGACCTACGCCATCACC

[0145] 0169-R:GATCGACCGCGTGCACCTTCAGCTCGGCGTCTTCCAGCG

[0146] 0220-F: GTTGTTGTAAAGTCGTGGCCAATGACACGCCCCGTCCGC

[0147] 0220-R: GATCGACCGCGTGCACCTTCAGGCGCCGGAGGCGTCCAG

[0148] 6675-F: GTTGTTGTAAAGTCGTGGCCAGTGCTGCGAGTCGCCGTC

[0149] 6675-R: GATCGACCGCGTGCACCTTCAGTGCTCTCGCCCCGG

[0150] 2182-F:AAAGGAGGCGGACATCATGTGACCTACGTCATCGCGCAG

[0151] 2182-R: CCGCCGGTACCCGGTCTAGATTACTGGTTCTGCGGCGG

[0152] 2776-F:AAAGGAGGCGGACATCATATGCAGATCACCGTGGACATG

[0153] 2776-R:CCGCCGGTACCCGGTCTAGATCACGAGGCGGGGCCAC

[0154] 5437-F:AAAGGAGGCGGACATCATATGGGCGACCGCTGGCACGTG

[0155] 5437-R:CCGCCGGTACCCGGTCTAGATCACTCCTCCGGCGGGAA

[0156] 6156-F:AAAGGAGGCGGACATCATATGACCGTGCAGCAGGACGCG

[0157] 6156-R:CCGCCGGTACCCGGTCTAGATCAGGCCGCGTCCGGCCC

[0158] 7088-F:AAAGGAGGCGGACATCATATGGGCGTCCAGGTCGACAGG

[0159] 7088-R:CCGCCGGTACCCGGTCTAGATCAGTCGGCGGACAGGGC

[0160] ermE*pF:AGGTGCACGCGGTCGATCTT

[0161] ermE*pR:ATGATGTCCCGCCTCCTTTGG

[0162] asO*pF:TGTGAAGATCTTGGATATCTGTTCACATTCGAACCGT

[0163] asO*pR:TGGCCACGACTTTACAACAC

[0164] PCR indicators:

[0165]

[0166] PCR speed:95°3min,95°15s,T m -5°C15s,72°1.5min,72°5min,35°C,4°C.

[0167] The PCR product was recovered by gel extraction kit to obtain the target fragment kasO*p-0169 / 0220 / 6675-ermE*p-2182 / 2776 / 5437 / 6156 / 7088.

[0168] (2) Linearization of vector pIJ8660-ermE*p-3629 (constructed in this patent, wherein the vector pIJ8660-ermE*p is shown in Liu SP, Yuan PH, Wang YY, et al. Generation of the natamycin analogs by gene engineering of natamycin biosynthetic genes in Streptomyces chattanoogensis L10. Microbiol Res. 2015; 173: 25-33.)

[0169] The vector was double-digested with restriction endonucleases EcoRⅤ and XbaⅠ to achieve linearization. The reaction system is shown in Table 5.

[0170] Table 5 Enzyme digestion reaction system

[0171] Element Volume (μL) EcoRⅤ 1 XbaⅠ 1 10×CutsmartBuffer 2 Plasmids 5 <![CDATA[ddH 2 The]]> 11 Total volume 20

[0172] Incubate at 37°C for 2 hours.

[0173] Take 3 μL of the digested product and perform 1% agarose gel electrophoresis at 120 V constant voltage. If a band appears and the size is correct, use the Cycle-Pure Kit for purification and recovery, or cut the gel and use the Gel Extraction Kit to recover the linearized vector.

[0174] (3) Obtaining the recombinant plasmid pIJ8660-ermE*p-3629-kasO*p-0169 / 0220 / 6675-ermE*p-2182 / 2776 / 5437 / 6156 / 7088

[0175] The above PCR products and the vector after enzyme digestion were seamlessly cloned (see Table 4 for the connection system), and the connection products were obtained by reaction at 50°C for 15 minutes. Next, the competent cells of E. coli DH5α were taken out and placed on ice, and the above connection products were added to the clean bench, and the mixture was gently pipetted and mixed, and placed on ice for 30 minutes. Subsequently, the above mixture was heat-shocked at 42°C for 90 seconds, and then quickly transferred to ice for 2 minutes. 800μL LB liquid culture medium was added, and the culture was revived at 37°C and 200r / min for 50 minutes. Centrifuge at 8,000r / min for 5 minutes, remove 750μL supernatant, and gently pipet and mix the remaining bacterial solution. Finally, the mixed bacterial solution was spread on LB solid culture medium containing 50μg / mL apramycin and cultured overnight at 37°C. The plasmids of the positive transformants grown on the above LB solid medium were extracted and verified by PCR, restriction digestion and sequencing to obtain the recombinant plasmid pIJ8660-ermE*p-3629-kasO*p-0169 / 0220 / 6675-ermE*p-2182 / 2776 / 5437 / 6156 / 7088.

[0176] (4) Preparation of donor bacterial suspension

[0177] Take out the competent cells of E. coli ET12567 / pUZ8002 and place them on ice. Add the recombinant plasmid pIJ8660-ermE*p-3629-kasO*p-0169 / 0220 / 6675-ermE*p-2182 / 2776 / 5437 / 6156 / 7088 in the clean bench, mix by gently pipetting, and place on ice for 30 minutes. Subsequently, heat shock the above mixture at 42°C for 90 seconds, and then quickly transfer it to ice for 2 minutes. Add 800μL LB liquid culture medium and resuscitate and culture at 37°C and 200r / min for 50 minutes. Centrifuge at 8,000r / min for 5 minutes, remove 750μL supernatant, and gently pipette to mix the remaining bacterial solution. Finally, spread the mixed bacterial solution on LB solid culture medium containing 50μg / mL apramycin and culture overnight at 37°C. Transfer the grown single colony to a test tube containing 5 mL LB liquid medium containing the same antibiotics and culture overnight at 37°C and 200 r / min. Transfer the above culture to a 250 mL Erlenmeyer flask containing 25 mL LB liquid medium containing the same antibiotics at a 1% inoculum. Culture at 37°C and 200 r / min with shaking until OD 600 The culture solution was centrifuged at 5,000 r / min for 3 minutes and then the cells were collected. Then 1.5 mL of LB liquid medium was added to resuspend the cells and centrifuged at 5,000 r / min for 3 minutes and then the cells were collected. This step was repeated three times. Finally, 1 mL of LB liquid medium was used to resuspend the cells and prepare a donor bacterial suspension (OD 600 is 0.4-0.6).

[0178] (5) Preparation of Recipient Bacterial Suspension

[0179] The recipient bacterial suspension was prepared as described in Example 3.

[0180] (6) Co-cultivation and screening

[0181] Co-cultivation and screening were as described in Example 3.

[0182] (7) Verification of binders

[0183] Pick the grown conjugates and inoculate them on solid culture medium. Pick the single clone grown on the above solid culture medium, extract the genome and perform PCR verification, such as Figure 4 As shown, the primer pair amplified a band size of 10657 bp, proving that the recombinant plasmid pIJ8660-ermE*p-3629-kasO*p-0169 / 0220 / 6675-ermE*p-2182 / 2776 / 5437 / 6156 / 7088 has been successfully integrated into the attB site of the genome. Fifteen engineered strains were successfully screened and named They are Sr3629-1, Sr3629-2, Sr3629-3, Sr3629-4, Sr3629-5, Sr3629-6, Sr3629-7, Sr3629-8, Sr3629-9, Sr3629-10, Sr3629-11, Sr3629-12, Sr3629-13, Sr3629-14 and Sr3629-15.

[0184] The solid culture medium formula is: 12 g / L glucose, 6 g / L yeast extract, 30 g / L malt extract, 2 g / L calcium carbonate, 20 g / L agar, and the solvent is water.

[0185] The solid culture medium used needs to be supplemented with apramycin antibiotic solution with a final concentration of 50 μg / mL.

[0186] Example 6: Analysis of transformation performance of engineered Streptomyces roseochromogenes strains Sr3629-1-15

[0187] (1) Strain activation and seed culture

[0188] Inoculate strains Sr3629-1-15 and wild type on solid culture medium and culture at 28°C for 5-7 days until a large number of spores grow on the plate. 2 ) and inoculated into a 250 mL Erlenmeyer flask containing 20 mL of seed culture medium. Cultured at 28°C and 200 r / min for 24 hours to obtain sufficient seed solution.

[0189] The solid culture medium formula is: glucose 12g / L, yeast extract 6g / L, malt extract 30g / L, calcium carbonate 2g / L, agar 20g / L, and the solvent is water;

[0190] The seed culture medium formula is: 4g / L glucose, 4g / L yeast extract, 10g / L malt extract, and the solvent is water;

[0191] The solid culture medium and seed culture medium used for the engineered strains Sr36291-15 both need to be supplemented with an apramycin antibiotic solution with a final concentration of 50 μg / mL.

[0192] (2) Fermentation culture and substrate conversion

[0193] Fermentation culture and substrate conversion are as described in Example 4.

[0194] (3) Target product extraction

[0195] The target product was extracted as described in Example 4.

[0196] (4) Detection of 16α-hydroxyprednisolone

[0197] The detection method of 16α-hydroxyprednisolone is as described in Example 2. Figure 4As shown, the initial production rates of 16α-hydroxyprednisolone of the engineered strains Sr3629-1 to 15 were 119.87 mg / L / d, 112.63 mg / L / d, 103.41 mg / L / d, 113.73 mg / L / d, 93.05 mg / L / d, 97.70 mg / L / d, 103.48 mg / L / d, 109.85 mg / L / d, 100.58 mg / L / d, 115.60 mg / L / d, and 83.74 mg / L / d, respectively. L / d, 100.47mg / L / d, 126.11mg / L / d, 101.69mg / L / d and 109.07mg / L / d, which were 148.99%, 133.96%, 114.80%, 136.24%, 93.28%, 102.95%, 114.95%, 128.18%, 108.93%, 140.13%, 73.94%, 108.70%, 16 respectively, compared with the wild type (48.14mg / L / d). The yields reached the maximum after 120 hours of conversion, which were 181.78mg / L, 160.02mg / L, 178.89mg / L, 184.06mg / L, 180.26mg / L, 150.98mg / L, 186.21mg / L, 177.6mg / L, 167.84mg / L, 174.96mg / L, 181.16mg / L, 179.76mg / L, 201 .59mg / L, 168.68mg / L and 151.13mg / L, while the wild type reached the maximum at 145.99mg / L at 216 hours of transformation. The maximum yield was 24.52%, 9.61%, 22.54%, 26.08%, 23.47%, 3.42%, 27.55%, 21.65%, 14.97%, 19.84%, 24.09%, 23.13%, 38.08%, 15.54% and 3.52% higher than that of the wild type. Figure 5As shown, after 120 hours of transformation, the 16α-hydroxyprednisolone production intensities of the engineered strains Sr3629-1 to 15 were 34.46 mg / L / d, 30.64 mg / L / d, 34.26 mg / L / d, 35.24 mg / L / d, 34.52 mg / L / d, 28.91 mg / L / d, 35.66 mg / L / d, 34.01 mg / L / d, 32.14 mg / L / d, 33.50 mg / L / d, 34.69 mg / L / d, and 34.42 The results showed that the expression of 100 mg / L / d of Sr3629-13 strain was 123.94%, 99.10%, 122.58%, 129.01%, 124.29%, 87.86%, 131.69%, 120.98%, 108.84%, 117.69%, 125.41%, 123.67%, 147.64%, 109.88% and 88.04% higher than that of the wild type (15.39 mg / L / d). In particular, the Sr3629-13 strain showed the best performance in the whole transformation process.

[0198] The sequences used in the present invention are as follows:

[0199] Cytochrome P450 enzyme 3629 DNA-SEQ ID NO.1

[0200] ATGAGCATCACAGGTGGTACGTCCAGGACCCCCGATGGTCGGCGCGCGGTCATCGGCCT

[0201] GCTCCGCAGACTCAATTCGCCCGAGGGACAGGCCGAGCCGTACGGAATACTCCGGGAG

[0202] CTGCGGACGATGGGGTGACGTCGTCCGCGCTCCGTGGAACGGATACTTCGTCACCGGATT

[0203] CGACGCCTGCAGCCAGGTGCTGCGCGGCCGCAACTGGCAGGCCCCCGACTTCGCCTGG

[0204] CAGGAGCGGCAGGACGACGCCAAGCGGTGGGACGCCCTCGCCACCCGGGAGATGACG

[0205] AGCACCCTGGCCCGGCTCAACGCTCCCGAACACACCTGCCAGCGCCGCTCGCTGGGCA

[0206] ACCTCTTCGACCGCTCCACCATCGAACGCCTCACCCCCGACGTCGAGCGCCACGCCGAC

[0207] CGGCTCCTGGACGAGCTGGCTGACAAGCTCCGCTGGGGTGAGGCCGACTTCGTCTCCA

[0208] CGGTCAGCGAACAGTTGCCGATCCACACCATCGGCTCCTGGCTGGGGCTGCCGCCCGA

[0209] GGACTACCCGCACATCCTGGAGATCACCCACAACCAGGTGTACGCGCAGGAGCTGCTG

[0210] CCCACCAGCAGCGATCTGGCGGTCTCCGCCGAGGCGACCGTGCAGTTGCGCGCCTACTT

[0211] CACCGATCTGGTCGCCCGCCGCCGCGCCGAGCCCCGCCACGACGTGCTGACCGGCTGG

[0212] ATCCACACCTGGGACGCGCTGGAGCCGGACCGCGAGCAGGCGGACGAGATCCTCTACC

[0213] GGCTCACCATGTTCGTCACCATCGCCTCGCTGGAGACCACCGCGACGCTGCTGACGTCG

[0214] ATGGTTTCTCTGCTGTCGGCCGAGCCCGGCCGCTGGGAGTGGCTGCGCTCCTGGCCCGA

[0215] GCACATCGACGCGGCCGTGGACGAGGTGCTGCGCTACGACCCGCCGATCCACATCAAC

[0216] ACCCGGATCGCCGCCGAGGACACCGTCCTGGCCGGCGTCCCCATCGAGAAGGACAGCA

[0217] TGGTCCACGTCCTGTACGGCGCCGCCAACCACGACCCGCGCCGCAACCCCGACCCCGG

[0218] CACCTTCGACATCCTGCGCGGCGGCAGCCACCTCACCTTCGGCGGCGGGGTGCACTACT

[0219] GCCTGGGCGCGGCGCTGGCCAAGCTGGAGGCGCGGACCCTGCTGGCCCGCATGCTGGA

[0220] TCGTTTCCCCGCCCTGCGGACGGTCTCCCCGCCGGTCTACGCCCCCCGGATGGTCTTCCG

[0221] GCGCATCACCTCGCTGGGCGTGGCCCTGTGA

[0222] Cytochrome P450 enzyme 6606 DNA - SEQ ID NO.2

[0223] ATGGTCGACAACACCCTCCCGCTCCTGCTGCGCGGCTACGGCTGGCTGCCCGACCGCAG

[0224] GCGCCGCATCGGAGGAGCGCCCGTGGAGACCCGGCTGCTCGGCAAACGGGCGATCGCC

[0225] CTGCACGGTCCGGCGGCCGTGCGGTTCTTCTACGACGAGCACCACATCCGGCGCACGG

[0226] ACGCCCTGCCCCAGCCCGTGATCGACACCCTCTTCGGCCGGGGTGCTGTCCACGCCCTG

[0227] GACGGCGAGCGCCACGGGGTGCGCAAGGCCCTCTTCACGGGGCTGCTCAAGGAGCCCA

[0228] CCCGGGTACGCGACCTCTCCGAACGGGTCGCCCGGGAGCTGGAGCGGGCACGGGCGGG

[0229] GTGGGCCGCGCGGGGGCGGGTGGTCCTCTTCGACGAGACGGCCGAGGTCCTCACCCGT

[0230] GCGGTGCACGCCTGGGCCGGGGTGCCGCTGGCCGAGGACGCGGTGGCCACCACGGCCC

[0231] GGGACATGATCGCCATGGTGGACGGCTTCGCCTCCGTGGGCCCCCGCCATCTCCGGGCC

[0232] CGGCGGGCCCGCCGGGCCCAGGAGGAACGGCTGGCCCCGCTGGTGGGGACGGCGCGT

[0233] GAGGGGGCCGCGCCGGAGGGCAACGGCTCCGGTCCCACCGTGTTCCGTACGATCGCGG

[0234] CGCACCGGCACGCGGACGGGGCCCTGCTCGATCCGCGCACCGCCGCGGTGGAGGTGCT

[0235] GAACGTGCTGCGGCCGACGGTGGCGATCACCTGGTTCGCCGTCTTCGCCGCCCACGCCC

[0236] TGCACCGCCACCCCGAGCACCGCGAGGTGCTGGCGGGCGGGGACGGCCCCGAATACGC

[0237] CTGGGCGTTCGCGCAGGAGGTGCGCCGGTTCTATCCGTTCGCGCCCTTCGTGGGCGGGC

[0238] TGGCGGCGGAGCGGCTGGAGTGGGCCGGGCGGACGATCGAGCCGGGCACGATGGTGCT

[0239] GCTCGATCTGTACGGGCTGGATCACGACCCGGACCTGTGGCCCGAACCGTACCGCTTCG

[0240] ACCCGACCCGCTTCCTGGGCAGTGCCCCGCCCGTCGACGCGCTGGTCCCCCAGGGAGG

[0241] CGGTCCCGTGGACGGCCACCGGTGCCCCGGTGAGGACATCACGGTCGCGGTCCTGGCC

[0242] GAGGTGGCGAGGTTCCTGGCGCGGACCTCGTACGACGTCGTGGAGCAGGATCTGCGTAT

[0243] CCCGCTGAGCCGGATCCCGACGGCGCCCCGGAGCGGATTCGTGATCGGGAACGTGGCC

[0244] CCGGCCCCGGCCGGAGCCGGCGCGGGGGCGGCGGGCAGGGGCTGA

[0245] Cytochrome P450 enzyme 6792 DNA - SEQ ID NO.3

[0246] ATGGAGAACACCTCGGTCCGACACGCCTCGGTGCAGAACACCTCGGCGCAGAGCACCC

[0247] CGGCGCGGAACGCCCCGGCGCAGGACAAGGAAACCGTACGGAGCTGCCCCTTCGACTT

[0248] CGCGGAGCAACTGGAGTTCGACCCCCAGCTCAAGGAACTGCTCACCGAGGAGCCCGTC

[0249] TCCCGTATCCGTATGGCGTACGGCGAGGGCGAGGCCTGGCTCGTCACCCGGTACGAGGA

[0250] CGTCCGGACGGTCACCACCGACCGGCGCTTCAGCCGCAGCGCCGTCCTCGGCCGGGAC

[0251] TTCCCCCGCATGACCCCGGAACCGATCGTCCAGGCCGAGTCCATCAACCTCATGGACCC

[0252] GCCCGCCAGCAGCCGGCTGCGCAGCCTGGTCGCCAAGAGCTTCACCCCGCGCCGCGTC

[0253] GAGCAGATGCGGCCGGGCACCCAGCGCGTCGTGGACCGGCTGCTCGACGAGATGGAGG

[0254] CCGAGGGCTCACCCGCCGACTTCGTCGCCCGCGTCTCCTCGCCGCTGCCGCTCATCACC

[0255] ATCTGCGAGGCGCTCGACATCCCCGAGACCGACCGCCCCTGGCTCCGCGCCCACGCCAT

[0256] GACGATGATGAGCGTCGGCGCGGCGGGCAAGGAGGACGCGGTACGGGCCAAGGCCGA

[0257] ACTGCGCGGCTACTTCACCGAACTGACCGCCGAGCGCCGCCGCTCCCCGGGCCAGGAC

[0258] CTCATCAGCACCCTGGCCACCGCCCGCGACGGCGACGAACTGCTCGACGACAAGGAAC

[0259] TCGCCGTCATGGCGATGGTCCTGCTCATCACCGGCCAGGACACCACCACCTACGAACTC

[0260] GGCAACATCGCCTACACCCTGCTGACCCGGCCCGACCTGCTCAAGACGCTCCAGGCCG

[0261] AACCCGAGCGGCTGCCCCGCACGATCGACGAGCTGCTGCGGTACATCCCCTTCCGCAAG

[0262] GGCGTCGGCATCCCCCGCATCGCCATGGAGGACGTGGAGCTGGGCGGGGTCACCATCA

[0263] AGGCGGGCGACGTCGTCCATGTGTCGTACCTGACGGCCAACCGGGACGGCGCCAAGTT

[0264] CGACCGCCCCGACGAACTCGACCCCGACCGGCCGTCCATCCCGCACATGACCTTCGGCT

[0265] GGGGCGCCCACCACTGCCTGGGCGCACCGCTCGCCACCATGGAACTGGAGGTCGCCTT

[0266] CACCACGCTGCTGGCCCGCTTCCCGGGCCTGCGTCTCGACGCCGAACCCGAGGACATCC

[0267] GGTGGAACACCACGTCCATCTGGCGTTACCCGCTCGCCCTGCCCGTCACATGGTGA

[0268] Ferredoxin 2182 DNA - SEQ ID NO.4

[0269] GTGACCTACGTCATCGCGCAGCCTTGTGTCGACGTGAAGGACAAGGCCTGCATCGAGG

[0270] AGTGCCCCGTCGACTGCATCTACGAGGGCCAGCGGTCCTTGTACATCCACCCGGACGAA

[0271] TGCGTCGACTGCGGAGCCTGTGAGCCGGTCTGCCCGGTCGAGGCCATCTTCTACGAGGA

[0272] CGACACCCCGGAGGAGTGGAAGGACTACTACAAGGCGAACGTCGAGTTCTTCGACGAC

[0273] CTCGGCTCGCCGGGCGGTGCTTCCAAGCTGGGCCTCATCGAGCGCGACCACTCCTTCAT

[0274] CGCCGGGCTGCCGCCGCAGAACCAGTAA

[0275] Ferredoxin 2776 DNA - SEQ ID NO.5

[0276] ATGCAGATCACCGTGGACATGGACGTCTGCGAGAGCCACGGCCAGTGCGTCTTCGCGG

[0277] CTCCCGAGACCTTCTCCTTCGACGACGACGAGTACCTCCAGTACGACCCGGCGCCGAAC

[0278] GACGCACTGCGCGACAGGATCGAGAAGGCCGCCGCCTCCTGCCCGGTCCGCGCCATCA

[0279] CCGTGGCCCCCGCCTCGTGA

[0280] Ferredoxin 5437 DNA - SEQ ID NO.6

[0281] ATGGGCGACCGCTGGCACGTGGAGGTCGACCGCTCCGTCTGCATCGGCTCCGGCATGTG

[0282] CGTCAACCACGCCCCCGACGCCTTCCGCCTGGACACCGCCCGCCAGTCCCACCCCCGC

[0283] ACCGAGGACCGCGACGCGGGCGAACAGGTCCTGGCGGCGGCGGAGGGCTGCCCGGTG

[0284] GAGGCGATCACGCTGACGCTGGCGGCGTCGGGGGAGGTGGTGTTCCCGCCGGAGGAGT

[0285] GA

[0286] Ferredoxin 6156 DNA - SEQ ID NO.7

[0287] ATGACCGTGCAGCAGGACGCGCCGGGCGACGGTGACGCGCAGGACCTGGAGGTCTGGA

[0288] TCGACCAGGACCTCTGTACGGGCGACGGCATCTGCGCGCAGTACGCGCCGGAGGTCTTC

[0289] GAGCTGGACATCGACGGCCTGGCGTACGTGAAGAGCGCCGAGGACGAGCTGCTGCAGG

[0290] ACCCCGGCGCCACCACCCCGGTGCCGCTGACGCTCCTCCAGGACGTGGTCGATTCGGCC

[0291] AAGGAGTGCCCCGGGGACTGCATCCATGTCCGGCGGGTCAAGGACAGCGTCGAGGTGT

[0292] ACGGGCCGGACGCGGCCTGA

[0293] Ferredoxin 7088 DNA - SEQ ID NO.8

[0294] ATGGGCGTCCAGGTCGACAGGGAACGCTGCGTGGGGGCGGGCATGTGCGCGCTGACCG

[0295] CGCCGGACGTGTTCACGCAGGACGACGACGGCCTCAGCGAGGTGCTCCCGGGCCGCGC

[0296] GGAGACCGCTGGAGGACATCCCTTGGTGGGGGAGGCCGTACGGGCCTGCCCGGTGGGG

[0297] GCGGTGGCCCTGTCCGCCGACTGA

[0298] Ferredoxin reductase 0169 DNA - SEQ ID NO.9

[0299] ATGACCTACGCCATCACCCAGACCTGCTGCTCCGATGCCACCTGCGTTGCCGTCTGCCCC

[0300] GTCAACTGCATCCATCCCACCCCCGAGGAGCGGGCGTTCGGGTCCACGGAGATGCTCTA

[0301] CGTCGACCCGCAGACCTGCATCGACTGCGGGGCCTGCGCCGACGCCTGCCCGGTCGAC

[0302] GCGATCTTCCCGGTGGACAGCCTCTTCGGGGCGCAGAAGGAGTACGCCGCCATCAACG

[0303] CGGCGTACTACGCGGACCGGAAGCCCGAACCCGCCCCCGCCGGGCCGAACTTCCACGC

[0304] CTGGGGCGAGCCCGCCTTCGAGCGCAGCCTGCCCGCCGACTTCGCCCCGATCCGGGTC

[0305] GCGGTCGTCGGCACCGGACCGGCCGGCATGTACGCCGCCGAGGACCTGCTCCTGCACA

[0306] CCAACGCCGAGGTCACCCTCGTCGACCGGCTGCCGGTGGCGGGCGGGCTCGTCCGGTA

[0307] CGGCGTGGCGCCCGACCACCCCGCCACGAAGAAGGTCGGCGAGACCTTCGCCCGGTTC

[0308] CACTCCCACCCCCGGGTACGGATGCACCTGGGCGTCGAGGTCGGCAAGGACATCACCG

[0309] CCCAGGAGCTGGCCGCCCACCACGACGCGGTGATCTACGCGGTCGGCGCCTCCACCGA

[0310] CCGCAGACTCCGCATCCCCGGCGAGGAACTGCCCGGCTCCGCCTCCGCCACCTCGTTCG

[0311] TCGCCTGGTACAACGCCCACCCCGAGGTGGCACCCGATTCGGTCTCCCTGGACGCCGAG

[0312] CGCGTGGTCGTCGTCGGCAACGGCAACGTCGCCCTCGACGTCGCGCGCATCCTCGTCTC

[0313] CGACCCGGAGACGCTGGCCGCCACCGACATCGCCGACCACGCGCTGGCGGCGCTGCGG

[0314] GGGTCCCGGGTGCGGGAGGTGGTGCTGCTGGGCAGGCGCGGCCCCGAGGACGCGGCG

[0315] TGCACCGTCTCCGAACTGCTCGCCCTCAAACATCTGCCGGGCGTCGGCCTCGTCGTCGA

[0316] CGACCACGACCCCAGGACCGGCCCCGCGATCGACGGGGCGGCCCCGGACACGAAGGC

[0317] GGCCCTCCTCCAGGGCGTGGAGCGCAGGGCCGTGGACTGGTCGCGGCCGCCCGCACCG

[0318] GGGCGGCGGATCGTCTTCCGCTTCCACTCCGCCCCGGTGGCGGCGGTGGGGGAGGACG

[0319] CCGTCACGGCCGTACGCGTGACGGGGGACGGGTCCGAGCGGGAGATCCCGGCGGGGCT

[0320] GCTGCTGCGGGCGATCGGCTACCGGGGCGTGGCCGTGCCGGGCCTGCCCTTCGACGAG

[0321] GCGTCCGGGACCGTCCCGCACGAGGGCGGCCGGGTGAGCGGCATGACGGGGACGTACG

[0322] TCGTCGGCTGGATCAAGCGCGGCCCCTCGGGCGGCATCGGCGCCAACCGCACCTGCGC

[0323] CGCCGAGACCGTGGGCACGCTGCTGGCCGACGCCACGGCGGGCGCCCTCGCCTCCCCG

[0324] GCCCATCCGTCCTCGGCCTTCCGCCGCCTGGCCCGCAGTCGTGGCCGGAACGTCGTGGA

[0325] CGCCCGGGGGCTGGCCGCCATCGACCGGGCGGAGGTGGCCCGCGGCCGCGAGCACGG

[0326] CCGCCCGCGCGTCAAGCTCGGCACGGTCCCCGCCCTGGTGGCGGCGGCGAAGGGCCGC

[0327] CGCCGCTGGAAGACGCCGAGCTGA

[0328] Ferredoxin reductase 0220 DNA - SEQ ID NO.10

[0329] ATGACACGCCCCGTCCGCGTCGCCATTGTCGGAGCCGGCCCCGCCGGTATCTACGCTGC

[0330] GGACGCGCTGCTCAAATCCGAGGTCTGCCAGGACCCGGGGGTCTCGATCGACCTGTTCG

[0331] AGCGCATGCCCGCGCCCTTCGGCCTGATCCGTTACGGCGTGGCCCCCGACCACCCCCGG

[0332] ATCAAGGGCATCGTCAAGGCGCTGCACCAGGTGCTGGACAAGCCGCAGATCCGTCTCTT

[0333] CGGCAACGTCAGCTACCCGAACGACATCGACCTGGACGACCTGCGCTCCTTCTACGACG

[0334] CGGTGATCTTCTCCACCGGCGCCGAGGCCGACCGGGCGCTGGACATCCCCGGCATCGAG

[0335] CTGGACGGCTCCTACGGCGCGGCCGAGTTCGTCTCCTGGTACGACGGCCACCCCGAGGT

[0336] CCCGCGCACCTGGCCGCTGACCGCCGAGAAGGTCGCGGTCCTCGGCGTCGGCAACGTG

[0337] GCGCTGGACGTGGCCCGCGTGCTGGCCAAGACGGCGGACGAGCTGCTGCCGACCGAGA

[0338] TCCCGGACAACGTCTACCAGGGCCTCAAGGCGAACAAGGCGCTGGAGGTGCATGTGTT

[0339] CGGGCGGCGTGGCCCGGCGCAGGCCAAGTTCAGCCCGATGGAGCTGCGGGAGCTGGAC

[0340] CACTCCCCCAACATCGAGGTCATCGTCAACCCCGAGGACATCGACTACGACGAGGGCTC

[0341] CATCGCCACCCGGCGGGAGAACAAGCAGGCCAACATGGTCGCGCAGACCCTGGAGAAC

[0342] TGGGCGATCCGCGATGTCGGCGACCGCCCGCACAAGCTCTTCCTGCACTTCTTCGAGTC

[0343] CCCGGTCGAGATCCTCGGCGAGGACGGCAAGGTCACCGCGCTGCGTACCGAGCGCACC

[0344] GAGCTGGACGGCACCGGCAACGTCCGGGGCACCGGGAAGTTCACCGACTGGGAGATG

[0345] CAGAGCGTCTACCGGGCCGTCGGCTACTACTCGGAGGAGCTGCCCAAGCTCCCCTTCGA

[0346] CGTGGCCTCCGGCACGGTCCCGCACGAGGCGGGCCGGGTCATCTCCGGTGACGAGCAC

[0347] ATGGCCTCGGTCTACGTCACCGGCTGGATCAAGCGCGGCCCGATCGGTCTGATCGGCCA

[0348] CACCAAGGGCGATGCCAACGAGACGGTCGCCTGCCTCCTGGAGGACCGCGCCGCCGGC

[0349] CGGCTGCCCGAGCCGACGACGCCCGCCCCGGAGGCCGTGGATGCCTTCCTGGAGAGCC

[0350] GGAACGTCCGCTACACGACGTGGGAGGGCTGGCACAAGCTGGACGCCCACGAGCAGG

[0351] CGCTCGGCGCGGCCCAGGGCCGTGAGCGGATCAAGGTCGTGGAGCGCGAGGGCATGCT

[0352] GGACGCCTCCGGCGCCTGA

[0353] Ferredoxin reductase 6675 DNA - SEQ ID NO.11

[0354] GTGCTGCGAGTCGCCGTCGTCGGTTCCGGTCCCAGCGGGGTCTACACCGCCCAGGCCCT

[0355] GCTGAACCAGTCGCTCGTGCCCGAGGTCCGCGTCCATGTCCTGGACCGGCTGCCCACCC

[0356] CGTACGGCCTGGTGCGGTACGGGGTCGCGCCCGACCACGAGAAGATCAAATCGCTCCA

[0357] GAACAGCCTGCGCGCGGTCCTGGAGGACGAGCGGGTCACCTTCGTCGGCCATGTGCAG

[0358] ATCGGCGGGGCGGACGGCGTCCCTCCGGCCCGTCTCGCGGAGTTGTACCACGCGGTCGT

[0359] CTACTGCGTGGGGGCCGCCGCCGACCGCCCGCTGGCGGTGCCGGGCGAGGAGCTGCCG

[0360] GGGAGCTGCTCCGCCACCCGGTTCGTCTCCTGGTACAGCGCGCATCCGGACTCCGCCCC

[0361] CGACGACTTCGCCCTGCGGGCCCGCTCGGCCGTGGTCATCGGGGTCGGCAACGTGGCG

[0362] GTCGACGTGGCCCGGATCCTGGCGCGCGGTGCCGACGAGCTGCGCGCCACCGACGTCC

[0363] CCCGGGCCGCGCTCGGGGCGCTCGCGGGCAGCCGGGTGCGCGAGGTGCACATCGTGGG

[0364] GCGCCGCGGCCCCTCCCAGGCCCGGTTCACCACCAAGGAACTGCGGGAACTGGGCGCG

[0365] CTGCCCGGCGCCCGGATCGTCGTCGACCCGGCGGAGCTGGCGCGGGACCCGGCGTACG

[0366] CGGCCCCGGACGGGCTGCCGCTGCCGGCGGTGGTCCGGCGGAACCTGGAGGTGCTGCG

[0367] CGGCTGGGCGGTACGGGGAGAGCCCGCGCCGTCGGGGGCGGTGTACGGGCCGTCCGGG

[0368] GCGGCACACGGTCCGTCCGAAACCGCGTACGGGCCATGCGGGGCCGCGCACGATCCCT

[0369] CCGAAACCGCGTACGGGCCGTCCGGGGCCCCGTACGATCCCTCCGAAGCCGGGTACGG

[0370] GCCGTCCGAGGCTGCGTACGACGGCGTACGACGGATCCGGCTGCGGTTCTTCCTGCGCC

[0371] CCGTCGAGTTGCTGGAGCGCGGGGGCAGGGTGGCGGGGGTGCGGTTCGCCCGTACGGT

[0372] CCCCGACGGCGCCGGGGGCGTACGGGACACGGGAACGTACGAGGACATCGAGGCCCA

[0373] GCTGGTGCTGCGGGCGGTCGGCTACCGGGGCGTGGAGCTGCCCGGGCTGCCCTTCGAC

[0374] CCGGTGCGCGGGACGGTGCCGCACACGGCGGGGCGGGTGCTGCGGGACGGGGTGCCC

[0375] TCGCCCGGGGAGTACGTCGCCGGGTGGATCAAGCGCGGACCGACCGGGGTGATCGGGT

[0376] CGAACCGGTCCTGCGCCAAGGAGACCGTCGCCTCGCTGATCGAGGACGCGCCCCTGCT

[0377] CGCCCGGCGGCCCGCCGCCGACGACCCGCTGGCCGTGCTGCGGGCGTGGGGGCAGCGG

[0378] CCGGTGGAGTGGGACGGCTGGCTCTCGATCGAGCGGGCGGAGCAGGCCCTGGGGCGCT

[0379] CGCTGGGGCGGGGGCCGGTGAAGATCCCCGACTGGTCCGGGCTGCTGGCGGCGGCCCG

[0380] GGGCGAGGAGCACTGA

[0381] Cytochrome P450 enzyme 3629 amino acid - SEQ ID NO.12

[0382] MSITGGTSRTPDGRRAVIGLLRRLNSPEGQAEPYGILAELRTMGDVVRAPWNGYFVTGFDA

[0383] CSQVLRGRNWQAPDFAWQERQDDAKRWDALATREMTSTLARLNAPEHTCQRRSLGNLFD

[0384] RSTIERLTPDVERHADRLLDELADKLRWGEADFVSTVSEQLPIHTIGSWLGLPPEDYPHILEI

[0385] THNQVYAQELLPTSSDLAVSAEATVQLRAYFTDLVARRRAEPRHDVLTGWIHTWDALEPDR

[0386] EQADEILYRLTMFVTIASLETTATLLTSMVSLLSAEPGRWEWLRSWPEHIDAAVDEVLRYDP

[0387] PIHINTRIAAEDTVLAGVPIEKDSMVHVLYGAANHDPRRNPDPGTFDILRGGSHLTFGGGVH

[0388] YCLGAALAKLEARTLLARMLDRFPALRTVSPPVYAPRMVFRRITSLGVAL

[0389] Cytochrome P450 enzyme 6606 amino acid - SEQ ID NO.13

[0390] MVDNTLPLLLRGYGWLPDRRRRIGGAPVETRLLGKRAIALHGPAAVRFFYDEHHIRRTDA

[0391] LPQPVIDTLFGRGAVHALDGERHGVRKALFTGLLKEPTRVRDLSERVARELERARAGWAA

[0392] RGRVVLFDETAEVLTRAVHAWAGVPLAEDAVATTARDMIAMVDGFASVGPRHLRARRARR

[0393] AQEERLAPLVGTAREGAAPEGNGSGPTVFRTIAAHRHADGALLDPRTAAVEVLNVLRPTV

[0394] AITWFAVFAAHALHRHPEHREVLAGGDGPEYAWAFAQEVRRFYPFAPFVGGLAAERLEWA

[0395] GRTIEPGTMVLLDLYGLDHDPDLWPEPYRFDPTRFLGSAPPVDALVPQGGGPVDGHRCPG

[0396] EDITVAVLAEVARFLARTSYDVVEQDLRIPLSRIPTAPRSGFVIGNVAPAPAGAGAGAAG

[0397] RG

[0398] Cytochrome P450 enzyme 6792 amino acid - SEQ ID NO.14

[0399] MENTSVRHASVQNTSAQSTPARNAPAQDKETVRSCPFDFAEQLEFDPQLKELLTEEPVSRIR

[0400] MAYGEGEAWLVTRYEDVRTVTTDRRFSRSAVLGRDFPRMTPEPIVQAESINLMDPPASSRLR

[0401] SLVAKSFTPRRVEQMRPGTQRVVDRLLDEMEAEGSPADFVARVSSPLPLITICEALDIPETDRP

[0402] WLRAHAMTMMSVGAAGKEDAVRAKAELRGYFTELTAERRRSPGQDLISTLATARDGDELL

[0403] DDKELAVMAMVLLITGQDTTTYELGNIAYTLLTRPDLLKTLQAEPERLPRTIDELLRYIPFRK

[0404] GVGIPRIAMEDVELGGVTIKAGDVVHVSYLTANRDGAKFDRPDELDPDRPSIPHMTFGWG

[0405] AHHCLGAPLATMELEVAFTTLLARFPGLRLDAEPEDIRWNTTSIWRYPLALPVTW

[0406] Ferredoxin 2 182 amino acid - SEQ ID NO.15

[0407] VTYVIAQPCVDVKDKACIEECPVDCIYEGQRSLYIHPDECVDCGACEPVCPVEAIFYEDDTP

[0408] EEWKDYYKANVEFFDDLGSPGGASKLGLIERDHSFIAGLPPQNQ

[0409] Ferredoxin 2 776 amino acid - SEQ ID NO.16

[0410] MQITVDMDVCESHGQCVFAAPETFSFDDDEYLQYDPAPNDALRDRIEKAAASCPVRAITVA

[0411] PAS

[0412] Ferredoxin 5 437 amino acid - SEQ ID NO.17

[0413] MGDRWHVEVDRSVCIGSGMCVNHAPDAFRLDTARQSHPRTEDRDAGEQVLAAAEGCPVE

[0414] AITLTLAASGEVVFPPEE

[0415] Ferredoxin 6 156 amino acid - SEQ ID NO.18

[0416] MTVQQDAPGDGDAQDLEVWIDQDLCTGDGICAQYAPEVFELDIDGLAYVKSAEDELLQDP

[0417] GATTPVPLTLLQDVVDSAKECPGDCIHVRRVKDSVEVYGPDAA

[0418] Ferredoxin 7088 amino acid - SEQ ID NO.19

[0419] MGVQVDRERCVGAGMCALTAPDVFTQDDDGLSEVLPGRAETAGGHPLVGEAVRACPVGA

[0420] VALSAD

[0421] Ferredoxin reductase 0169 amino acid - SEQ ID NO.20

[0422] MTYAITQTCCSDATCVAVCPVNCIHPTPEERAFGSTEMLYVDPQTCIDCGACADACPVDAIF

[0423] PVDSLFGAQKEYAAINAAYYADRKPEPAPAGPNFHAWGEPAFERSLPADFAPIRVAVVGTGP

[0424] AGMYAAEDLLLHTNAEVTLVDRLPVAGGLVRYGVAPDHPATKKVGETFARFHSHPRVRMH

[0425] LGVEVGKDITAQELAAHHDAVIYAVGASTDRRLRIPGEELPGSASATSFVAWYNAHPEVAPD

[0426] SVSLDAERVVVVGNGNVALDVARILVSDPETLAATDIADHALAALRGSRVREVVLLGRRGP

[0427] EDAACTVSELLALKHLPGVGLVVDDHDPRTGPAIDGAAPDTKAALLQGVERRAVDWSRPP

[0428] APGRRIVFRFHSAPVAAVGEDAVTAVRVTGDGSEREIPAGLLLRAIGYRGVAVPGLPFDEASG

[0429] TVPHEGGRVSGMTGTYVVGWIKRGPSGGIGANRTCAAETVGTLLADATAGALASPAHPSS

[0430] AFRRLARSRGRNVVDARGLAAIDRAEVARGREHGRPRVKLGTVPALVAAAKGRRRWKTPS

[0431] Ferredoxin reductase 0220 amino acid - SEQ ID NO.21

[0432] MTRPVRVAIVGAGPAGIYAADALLKSEVCQDPGVSIDLFERMPAPFGLIRYGVAPDHPRIKGI

[0433] VKALHQVLDKPQIRLFGNVSYPNDIDLDDLRSFYDAVIFSTGAEADRALDIPGIELDGSYGA

[0434] AEFVSWYDGHPEVPRTWPLTAEKVAVLGVGNVALDVARVLAKTADELLPTEIPDNVYQGLK

[0435] ANKALEVHVFGRRGPAQAKFSPMELRELDHSPNIEVIVNPEDIDYDEGSIATRRENKQANM

[0436] VAQTLENWAIRDVGDRPHKLFLHFFESPVEILGEDGKVTALRTERTELDGTGNVRGTGKFT

[0437] DWEMQSVYRAVGYYSEELPKLPFDVASGTVPHEAGRVISGDEHMASVYVTGWIKRGPIGLI

[0438] GHTKGDANETVACLLEDRAAGRLPEPTTPAPEAVDAFLESRNVRYTTWEGWHKLDAHEQA

[0439] LGAAQGRERIKVVEREGMLDASGA

[0440] Ferredoxin reductase 6675 amino acid - SEQ ID NO.22

[0441] VLRVAVVGSGPSGVYTAQALLNQSLVPEVRVHVLDRLPTPYGLVRYGVAPDHEKIKSLQNS

[0442] LRAVLEDERVTFVGHVQIGGADGVPPARLAELYHAVVYCVGAAADRPLAVPGEELPGSCSA

[0443] TRFVSWYSAHPDSAPDDFALRARSAVVIGVGNVAVDVARILARGADELRATDVPRAALGAL

[0444] AGSRVREVHIVGRRGPSQARFTTKELRELGALPGARIVVDPAELARDPAYAAPDGLPLPAVV

[0445] RRNLEVLRGWAVRGEPAPSGAVYGPSGAAHGPSETAYGPCGAAHDPSETAYGPSGAPYDPS

[0446] EAGYGPSEAAYDGVRRIRLRFFLRPVELLERGGRVAGVRFARTVPDGAGGVRDTGTYEDIE

[0447] AQLVLRAVGYRGVELPGLPFDPVRGTVPHTAGRVLRDGVPSPPGEYVAGWIKRGPTGVIGSN

[0448] RSCAKETVASLIEDAPLLARRPAADDPLAVLRAWGQRPVEWDGWLSIERAEQALGRSLGRG

[0449] PVKIPDWSGLLAAARGEEH

[0450] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will appreciate that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. An engineered strain for use in the biosynthesis of 16α-hydroxyprednisolone, characterized in that: The engineered strain includes a gene encoding a cytochrome P450 enzyme.

2. The engineered strain according to claim 1, characterized in that: The gene encoding the cytochrome P450 enzyme is: 3629 or 6606 or 6792 from Streptomyces roseochromogenes DSM 40463; the nucleotide sequence of 3629 is as shown in SEQ ID No. 1 or a nucleotide sequence having at least 70% identity with the nucleotide sequence shown therein; the nucleotide sequence of 6606 is as shown in SEQ ID No. 2 or a nucleotide sequence having at least 70% identity with the nucleotide sequence shown therein; the nucleotide sequence of 6792 is as shown in SEQ ID No. 3 or a nucleotide sequence having at least 70% identity with the nucleotide sequence shown therein; Alternatively, the cytochrome P450 enzyme is: 3629 or 6606 or 6792; the amino acid sequence of 3629 is as shown in SEQ ID No.12 or an amino acid sequence that is at least 70% identical to the amino acid sequence shown therein; the amino acid sequence of 6606 is as shown in SEQ ID No.13 or an amino acid sequence that is at least 70% identical to the amino acid sequence shown therein; the amino acid sequence of 6792 is as shown in SEQ ID No.14 or an amino acid sequence that is at least 70% identical to the amino acid sequence shown therein.

3. The engineered strain according to claim 1 or 2, characterized in that: The engineered strain also includes a gene encoding a redox chaperone protein.

4. The engineered strain according to claim 3, characterized in that: The gene encoding the redox chaperone protein includes a gene encoding ferredoxin and a gene encoding ferredoxin reductase; The gene encoding ferredoxin is: 2182 or 2776 or 5437 or 6156 or 7088 from Streptomyces roseochromogenes DSM 40463; the nucleotide sequence of 2182 is as shown in SEQ ID No.4 or a nucleotide sequence having at least 70% identity with the nucleotide sequence shown therein; the nucleotide sequence of 2776 is as shown in SEQ ID No.5 or a nucleotide sequence having at least 70% identity with the nucleotide sequence shown therein; the nucleotide sequence of 5437 is as shown in SEQ ID No.6 or a nucleotide sequence having at least 70% identity with the nucleotide sequence shown therein; the nucleotide sequence of 6156 is as shown in SEQ ID No.7 or a nucleotide sequence having at least 70% identity with the nucleotide sequence shown therein; the nucleotide sequence of 7088 is as shown in SEQ ID No.8 or a nucleotide sequence having at least 70% identity with the nucleotide sequence shown therein; The gene encoding ferredoxin reductase is: 0169, 0220 or 6675 from Streptomyces roseochromogenes DSM 40463; the nucleotide sequence of 0169 is shown as SEQ ID No.9 or a nucleotide sequence with at least 70% identity to the nucleotide sequence shown therein; the nucleotide sequence of 0220 is shown as SEQ ID No.10 or a nucleotide sequence with at least 70% identity to the nucleotide sequence shown therein; the nucleotide sequence of 6675 is shown as SEQ ID No.11 or a nucleotide sequence with at least 70% identity to the nucleotide sequence shown therein; Alternatively, the redox chaperone protein comprises ferredoxin and ferredoxin reductase, and the ferredoxin is: 2182 or 2776 or 5437 or 6156 or 7088; the amino acid sequence of 2182 is as shown in SEQ ID No.15 or an amino acid sequence having at least 70% identity to the amino acid sequence shown therein; the amino acid sequence of 2776 is as shown in SEQ ID No.16 or an amino acid sequence having at least 70% identity to the amino acid sequence shown therein; the amino acid sequence of 5437 is as shown in SEQ ID No.17 or an amino acid sequence having at least 70% identity to the amino acid sequence shown therein; the amino acid sequence of 6156 is as shown in SEQ ID No.18 or an amino acid sequence having at least 70% identity to the amino acid sequence shown therein; the amino acid sequence of 7088 is as shown in SEQ ID No.19 or an amino acid sequence having at least 70% identity to the amino acid sequence shown therein; The ferredoxin reductase is: 0169 or 0220 or 6675; the amino acid sequence of 0169 is as shown in SEQ ID No. 20 or an amino acid sequence that is at least 70% identical to the amino acid sequence shown therein; the amino acid sequence of 0220 is as shown in SEQ ID No. 21 or an amino acid sequence that is at least 70% identical to the amino acid sequence shown therein; the amino acid sequence of 6675 is as shown in SEQ ID No. 22 or an amino acid sequence that is at least 70% identical to the amino acid sequence shown therein.

5. A method for constructing an engineered strain according to any one of claims 1 to 4, characterized in that: The steps include: Genes encoding cytochrome P450 enzymes, or genes encoding ferredoxin and genes encoding ferredoxin reductase are obtained by PCR amplification; expression cassettes of the above-mentioned cytochrome P450 enzymes, ferredoxin or ferredoxin reductase encoding genes are respectively constructed by genetic engineering means, and are connected to expression vectors individually or in series, and are transferred to Escherichia coli for replication; recombinant plasmids are extracted and respectively transformed into methylation-deficient Escherichia coli competent ET12567 / pUZ8002; and the verified correct recombinant plasmids are respectively introduced into host cells through conjugation transfer experiments to construct corresponding overexpression engineering strains.

6. The construction method according to claim 5, characterized in that: The host cell is Streptomyces roseochromogenes DSM40463; Alternatively, the expression vector is pIJ8660.

7. Use of the engineered strain according to any one of claims 1 to 4 in the synthesis of 16α-hydroxyprednisolone.

8. A method for producing 16α-hydroxyprednisolone using the engineered strain according to any one of claims 1 to 4, characterized in that: The steps include: Streak the engineered strain on a solid culture medium and culture it at 28°C for 5-7 days until spores grow. Take the strain that has good growth and dig out 3 square blocks of bacteria, each 1 cm 2 , inoculated into a 250mL Erlenmeyer flask containing 20mL of seed culture medium, and cultured at 28°C and 200r / min for 16-32 hours to obtain seed solution; inoculated the seed solution into a 250mL Erlenmeyer flask containing 20-40mL of fermentation culture medium at an inoculum amount of 5-20%, and cultured at 28°C and 200r / min for 24 hours, and then added with anhydrous ethanol to dissolve the substrate, so that the final concentration of anhydrous ethanol is 8g / L, and the final concentration of the substrate is 0.5g / L, and 16α-hydroxyprednisolone is obtained after conversion for 24-216 hours.

9. The method according to claim 8, characterized in that: The substrate is prednisolone.

10. The method according to claim 8 or 9, characterized in that: The solid culture medium formula is: glucose 12g / L, yeast extract 6g / L, malt extract 30g / L, calcium carbonate 2g / L, agar 20g / L, and the solvent is water; The seed culture medium formula is: 4g / L glucose, 4g / L yeast extract, 10g / L malt extract, and the solvent is water; The pH of the fermentation medium is 5.5-8.5, and the formula is: 12 g / L glucose, 6 g / L yeast extract, 30 g / L malt extract, and the solvent is water; In order to maintain the stability of the plasmid in the engineered strain, an apramycin antibiotic solution with a final concentration of 50 μg / mL needs to be added to the above solid culture medium, seed culture medium and fermentation culture medium.

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