CN112592904A

CN112592904A - 17 beta-hydroxysteroid dehydrogenase mutant of mycobacterium and heterologous expression thereof

Info

Publication number: CN112592904A
Application number: CN202011620795.8A
Authority: CN
Inventors: 许正宏; 李会; 刘伟
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2020-12-31
Filing date: 2020-12-31
Publication date: 2021-04-02
Anticipated expiration: 2040-12-31
Also published as: CN112592904B

Abstract

The invention discloses a 17 beta-hydroxysteroid dehydrogenase mutant of mycobacterium and heterologous expression thereof, belonging to the field of enzyme gene engineering and enzyme engineering. The invention provides an actinomycete 17 beta-hydroxysteroid dehydrogenase 17 beta-HSDy 1 from mycobacterium (Mcbacterium sp.) LY-1 and a gene sequence thereof, which realizes the exogenous expression of 17 beta-HSDy 1 of mycobacterium LY-1 by taking pMA5 as an expression plasmid and taking bacillus subtilis WB600 as an expression host. For softnessThe key T239 site of the enzyme obtained by simulation is mutated into tyrosine, the enzyme activity is improved by 19.2 percent and reaches 9571.77 U.mg^‑1. And (3) measuring an enzyme activity-growth curve of the recombinant bacillus subtilis WB600-pMA5-17 beta HSDy1-T239Y obtained by mutation, wherein the specific enzyme activity of the recombinant bacillus subtilis WB600-pMA5-17 beta HSDy1-T239Y is highest when the fermentation is carried out for 28 hours. The heterologous expression strain can be used for the production of androstene-1, 4-ene-3, 17-dione converted baodanone, and has potential industrial application.

Description

17 beta-hydroxysteroid dehydrogenase mutant of mycobacterium and heterologous expression thereof

Technical Field

The invention belongs to the field of genetic engineering and fermentation engineering, and particularly relates to a 17 beta-hydroxysteroid dehydrogenase mutant derived from mycobacteria and heterologous expression thereof.

Background

Steroid drugs are the second largest class of drugs second to antibiotics, and have wide applications in the medical field, and are commonly used in clinical treatments for allergic diseases, rheumatoid arthritis, contraception, surgical anesthesia, and the like. Common steroid drug intermediates are classified into C19 and C22, and among them, steroid drug intermediates mainly comprising Boldenone (BD), androstene-4-ene-3, 17-dione (AD), androstene-1, 4-ene-3, 17-dione (ADD), 9 α -hydroxyandrosten-4-ene-3, 17-dione (9 α -OH-AD) and testosterone (T) have been increasingly demanded in the medical field market year by year.

Boldan (BD) is an androgen anabolic steroid and Testosterone (TS) derivative that promotes protein synthesis, supports nitrogen retention and stimulates renal erythropoietin release. BD is typically prepared from androstene-4-ene-3, 17-dione (AD) by chemical synthesis. However, the microbial transformation method has the advantages of high stereoselectivity, environmental protection, and the like, and has gradually played a greater role in the application of steroid drug intermediates, and the biosynthesis of BD has become a research hotspot in recent years.

17 beta-hydroxysteroid dehydrogenase (17 beta-HSD), a type of oxidoreductase, can catalyze the interconversion between the hydroxyl group and the keto group of C-17 in steroids, and requires the cofactor NAD (P) H/NAD (P) ++. Many microorganisms can produce BD by a reduction reaction of 17 beta-hydroxysteroid dehydrogenase using androst-1, 4-diene-3, 17-dione (ADD) as a substrate, but their low substrate concentration and conversion rate do not meet the requirements of industrial production. To date, researchers have identified 15 types of 17 β -HSD isozymes, all belonging to the short-chain dehydrogenase/reductase, SDR superfamily, with the exception that 17 β -HSD5 belongs to the aldehyde, ketoreductase, AKR superfamily.

Aiming at the application of the enzyme in the actual transformation of steroid drug intermediates, the identification of more and efficient 17 beta-hydroxysteroid dehydrogenase for the biotransformation of the boldenone is of great significance.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to provide a 17 beta-hydroxysteroid dehydrogenase mutant derived from mycobacteria and heterologous expression thereof, obtains a mutant with high enzyme activity by simulation analysis of an enzyme activity site, and researches relevant properties of the mutant; the transformation from androstene-1, 4-ene-3, 17-dione (ADD) to boldenone is successfully realized by utilizing heterologous expression mutant engineering bacteria.

The invention obtains the key activity T239 site of 17 beta-hydroxysteroid dehydrogenase through software simulation, and the site is mutated into tyrosine, so that the enzyme activity is improved by 19.2 percent. The mutant is heterologously expressed in the bacillus subtilis, and the related properties of the mutant are researched for guiding the subsequent industrial application. The mutant heterologous expression strain is utilized to successfully realize the conversion from androstene-1, 4-ene-3, 17-dione (ADD) to boldenone, and lays a foundation for further industrial application.

The first purpose of the invention is to provide a 17 beta-hydroxysteroid dehydrogenase mutant, wherein the 17 beta-hydroxysteroid dehydrogenase mutant is obtained by replacing threonine at the 239 th position of 17 beta-hydroxysteroid dehydrogenase with tyrosine, the amino acid sequence of which is shown as SEQ ID NO. 2.

Further, when threonine at position 239 of 17 β -hydroxysteroid dehydrogenase whose amino acid sequence is shown in SEQ ID No.2 is replaced with tyrosine, the amino acid sequence of the mutant is shown in SEQ ID No. 4.

The gene of the 17 beta-hydroxysteroid dehydrogenase is derived from Mycobacterium sp LY-1 CGMCC No. 13031.

It is a second object of the present invention to provide a gene encoding the mutant. The nucleotide sequence is any one of the following:

a) a base sequence shown as SEQ ID N0: 3;

b) encodes a protein consisting of the amino acid sequence shown as SEQ ID N0:4 or a nonsense mutant sequence thereof.

The third purpose of the invention is to provide an expression plasmid carrying the gene. Furthermore, the plasmid takes pMA5 as a vector. The recombinant expression plasmid is pMA5-17 beta HSDy 1-T239Y.

The fourth purpose of the invention is to provide a recombinant engineering strain for expressing the mutant, wherein the engineering strain integrates the gene of the 17 beta-hydroxysteroid dehydrogenase mutant or contains the recombinant expression plasmid.

Furthermore, the recombinant engineering strain takes bacillus subtilis WB600 as an expression host. The obtained engineering strain is Bacillus subtilis WB600-pMA5-17 beta HSDy 1-T239Y.

The fifth purpose of the invention is to provide the recombinant engineering strain or/and the crude enzyme solution secreted and expressed by the engineering strain, and the application of the crude enzyme solution in microbial transformation of androstene-1, 4-ene-3, 17-dione (ADD) to generate the boldenone.

Furthermore, the prepared boldenone takes androstene-1, 4-ene-3, 17-dione as a substrate to generate the boldenone.

Further, the reaction conditions of the crude enzyme solution are as follows: the temperature is 30 ℃, the pH is 6, and the fermentation time is 30 h.

Further, the engineering strain or/and crude enzyme solution secreted and expressed by the engineering strain are transferred into a culture medium containing 3g/L androstene-1, 4-ene-3, 17-dione at 30-37 ℃ and 220 r.min^-1And (5) converting for 24-30h to obtain the boldenone.

The sixth purpose of the invention is to provide the boldenone prepared by the application of the engineering strain.

Has the advantages that: the invention discloses 17 beta-hydroxysteroid dehydrogenase 17 beta-HSDy 1 by annotating the gene of mycobacterium LY-1. The active center amino acid of the 17 beta-hydroxysteroid dehydrogenase is determined through molecular docking simulation, and the site-directed mutation is carried out on the key amino acid residue, so that the enzyme activity of the 17 beta-hydroxysteroid dehydrogenase is improved. The enzyme activity of the 17 beta-hydroxysteroid dehydrogenase mutant modified by the invention is improved by 19.2 percent and reaches 9571.77 U.mg^-1. Modified genetically engineered bacteriumSuccessfully realizes the conversion from androstene-1, 4-ene-3, 17-dione (ADD) to boldenone, improves the production efficiency and has potential value of industrial application.

Description of the drawings:

FIG. 1 shows a comparison of the enzymatic activities of different 17 β -hydroxysteroid dehydrogenase mutants heterologously expressed in Bacillus subtilis;

FIG. 2 is an enzyme activity-growth curve of recombinant Bacillus subtilis WB600-pMA 5-17. beta. HSDy 1-T239Y;

FIG. 3 shows the optimum temperature of crude enzyme solution of 17 β -hydroxysteroid dehydrogenase mutant;

FIG. 4 shows the optimum pH of a crude enzyme solution of a 17. beta. -hydroxysteroid dehydrogenase mutant.

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

Examples

The present invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the specific material ratios, process conditions and results thereof described in the examples are illustrative only and should not be taken as limiting the invention as detailed in the claims.

(1) Culture medium:

LB liquid medium: 10.0g/L of peptone, 5.0g/L of yeast powder and 5.0g/L of NaCl.

ADD transformation medium: 3g/L of ADD, 20g/L of hydroxypropyl-beta-cyclodextrin, 10.0g/L of peptone, 5.0g/L of yeast powder and 5.0g/L of NaCl.

(2) Enzyme activity determination of 17 beta-hydroxysteroid dehydrogenase and mutant thereof

A2% methanol solution containing 0.3 mmol. multidot.L-1 substrate ADD was mixed with 1mL of 50 mmol. multidot.L-1 potassium phosphate buffer (pH 6.0) containing 1.5 mmol. multidot.L-1 NADPH, and the mixture was incubated at 30 ℃ in a metal bath, and then 500. mu.L of the crude enzyme solution of the disrupted supernatant was added and reacted in a metal bath at the corresponding temperature for 5 minutes, and the change in OD340 was recorded.

Enzyme activity: the amount of enzyme required to consume 1. mu. mol of NADPH per minute for catalyzing the reaction was defined as 1 enzyme activity unit (U). Wherein ε NADPH is 6.22L (mmol. cm) -1.

Example 1: excavation of 17 beta-HSD gene in mycobacteria LY-1

Final sequencing analysis report based on the results of the whole gene annotation for mycobacterium LY-1. The obtained 17 beta-HSD gene has a base sequence shown as SEQ ID N0:1, and a corresponding deduced amino acid sequence of the complete target gene is SEQ ID NO 2 and is named as 17 beta-HSDy 1. The 17 β -HSDy1 sequence was aligned by Blastp to confirm the presence of a DNA sequence encoding the 17 β -HSD enzyme with high sequence identity.

Example 2: extraction of genomic DNA of Mycobacterium LY-1

(1) Strain of Mycobacterium LY-1 in liquid seed Medium (15.0 g.L)^-1Yeast powder, 0.6 g.L^-1(NH₄)₂HPO₄，5.4g·L^-1NaNO₃，2.0g·L^-1Glycerol) for 2-3 days at the temperature of 30 ℃ and at the rotating speed of 120 rpm/min. Well-grown bacterial solution was pipetted 1mL into a 1.5mL EP tube at 12000rpm, centrifuged for 1min, and the medium was discarded completely.

(2) Add 150. mu.L of TE buffer pH 8.0 to the tube to suspend the bacteria thoroughly, and add 8. mu.L of 3 mg/mL^-1The lysozyme is prepared by grinding thallus by using a gun head to fully crack the thallus;

(3) sequentially adding 300 mu L of gelatin Solution and 4 mu L of RNase A into a centrifuge tube, uniformly mixing, and placing in a 55 ℃ metal bath for heat preservation for 10 min; then 4. mu.L of protease K is added, and the mixture is placed in a metal bath at the temperature of 55 ℃ for heat preservation for 30 min.

(4) After adding 300. mu.L of Ext solution and 300. mu.L of PB solution in this order and sufficiently shaking, the mixture was centrifuged at 12000rpm for 5min at room temperature, and the lower layer solution was transferred to a 2mL collection column.

(5) Centrifuging at 8000rpm at room temperature for 1min, pouring the collected liquid into the collecting column, and centrifuging at 8000rpm at room temperature for 1 min.

(6) The collection column was returned to the collection tube and 500. mu.L of Wash solution was added and centrifuged at 8000rpm for 1min at room temperature.

(7) Repeating the step (6) once, putting the collection column back into the collection tube again, centrifuging at 12000rpm for 1min at room temperature, putting the collection column into a sterilized 1.5mL EP tube, placing the tube in an oven to dry for 3-5min, adding 100 μ L of precipitation Buffer in the center of the collection column, standing at room temperature for 2min, and centrifuging at 12000rpm for 1min at room temperature. The liquid in the centrifuge tube is the genome DNA.

Example 3: cloning of 17 beta-HSDy 1 gene of mycobacterium LY-1 and construction of plasmid pMA5-17 beta-HSDy 1

Primers were designed based on the sequencing of the complete genome of Mycobacterium LY-1 from the obtained 17 β -HSDy1 gene sequence.

Primer P1: aaagtgaaatcagggggatccTCATCGGGGTGCCATCCT

Primer P2: atttcgacctctagaacgcgtGTGCACCACCACCACCACCACAAGTTAGAGAACTCCATCGCA

And (3) amplifying the coding gene of the 17 beta-HSD by using the extracted mycobacterium LY-1 genome as a template and the primer pcr. The PCR reaction was carried out in a 25. mu.L system under the following conditions: beginning circulation after pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 59 ℃ for 30s, and extension at 72 ℃ for l min for 34 cycles; final extension at 72 ℃ for l0 min. And (3) carrying out agarose gel electrophoresis on the PCR product, and then tapping and recovering.

A one-step cloning kit is selected to construct a plasmid pMA5-17 beta HSDy1 of the recombinant bacillus subtilis.

And (3) converting the successfully constructed plasmid into the Bacillus subtilis WB600 by a chemical conversion method, coating the Bacillus subtilis WB600 on an LB culture medium containing 50mg/L kanamycin for overnight culture, and selecting a positive transformant to obtain the heterologous expression recombinant Bacillus subtilis WB600-pMA5-17 beta HSDy 1.

Example 4: crystal structure simulation of 17 beta-hydroxysteroid dehydrogenase derived from mycobacterium LY-1

The reported type II 3-hydroxyacyl coenzyme A dehydrogenase (PDB code: 1UAY) from Thermus thermophilus HB8 is used as a template (the similarity of the two amino acids is 54.36%), and molecular simulation docking software Autodock is used for simulating docking of a substrate androstene-1, 4-ene-3, 17-dione (ADD) and 17 beta-HSDy 1, and threonine at the 239 th position of the enzyme is selected as an active site of 17 beta-HSDy 1 in the research.

Example 5: effect of 17 beta-hydroxysteroid dehydrogenase active site mutation on enzyme activity expression

A site-directed mutagenesis kit is utilized to design primers T239-F and T239-R as shown in Table 1, constructed pMA5-17 beta-HSDy 1 is used as a template to carry out PCR, and the 239 th tyrosine of 17 beta-hydroxysteroid dehydrogenase is respectively replaced by glutamine, tyrosine, valine, glycine, alanine and leucine. The PCR reaction conditions are as follows: 95 ℃ for 3min, 34 cycles (95 ℃ for 30S, 58 ℃ for 30S, 72 ℃ for 1.5min), 72 ℃ for 10 min. PCR amplification System: 1 μ L of template, 1 μ L of upstream and downstream primers, Prime Star Max (Premix) DNA20 μ L, ddH₂O17. mu.L. And purifying and recovering the PCR product by using a gel recovery kit, and carrying out electrophoresis test on the concentration of the recovered product. This was transformed into competent E.coil JM109, spread on ampicillin LB plates, and positive colonies were picked. After shaking table overnight culture at 37 ℃, extracting plasmid, and then transferring the plasmid into bacillus subtilis WB600 to obtain the recombinant strain with site-directed mutagenesis.

TABLE 1 primer sequences

T239 represents no mutation.

T239Q represents the replacement of the amino acid at position 239 from the parent threonine (Thr, T) to glutamine (Gln, Q),

T239Y represents that the amino acid at the position 239 is replaced by tyrosine (Tyr, Y) from threonine (Thr, T) of a parent, the recombinant expression plasmid is pMA5-17 beta HSDy1-T239Y, and the obtained engineering strain is Bacillus subtilis WB600-pMA5-17 beta HSDy 1-T239Y.

T239V represents the replacement of the amino acid at position 239 from threonine (Thr, T) of the parent to valine (Val, V),

T239G represents the replacement of the amino acid at position 239 from threonine (Thr, T) of the parent to glycine (Gly, G),

T239A represents the replacement of the amino acid at position 239 from threonine (Thr, T) of the parent to alanine (Ala, A),

T239L represents the replacement of the amino acid at position 239 from threonine (Thr, T) to leucine (Leu, L) of the parent,

carrying out ultrasonic cell disruption on different mutant recombinant bacteria B.Subtilis-pMA5-17 beta-HSDy 1 with good fermentation growth for 20min, centrifuging at 8000rpm for 20min, and taking the supernatant to obtain a crude enzyme solution. And (3) using the supernatant crude enzyme solution for enzyme activity determination. The enzyme activities of different mutants are shown in figure 1, wherein the enzyme activity of the mutant 2 is the highest, the 239 th threonine of the 17 beta-hydroxysteroid dehydrogenase is mutated into tyrosine, the enzyme activity is improved by 19.2 percent and reaches 9571.77 U.mg^-1。

Example 6: determination of recombinant bacillus subtilis WB600-pMA5-17 beta HSDy1-T239Y enzyme activity-growth curve

The recombinant strain was inoculated into LB liquid seed medium (10.0 g. L)^-1Peptone, 5 g. L^-1Yeast powder, 10.0 g.L^-1NaCl) at 37 ℃ and at a rotation speed of 220 rpm/min. Sampling every 8h from 12h of fermentation, determining the OD of the thalli, and performing corresponding enzyme activity determination of sampling at each time. Drawing an enzyme activity-growth curve, as shown in figure 2, when fermenting for 28h, the specific enzyme activity of the recombinant bacillus subtilis WB600-pMA5-17 beta HSDy1-T239Y is 9571.77U mg^-1At this time, the OD600 of the recombinant bacterium was 13.29. With the extension of the fermentation time of the recombinant bacteria, the growth and the enzyme production capacity of the bacterial strain show a decline trend.

Example 7: investigation of relevant properties of crude enzyme solution of 17 beta-hydroxysteroid dehydrogenase mutant

(1) Optimum reaction temperature study of crude enzyme

Will contain 0.3 mmol. multidot.L^-12% methanol solution of substrate ADD and 1mL solution containing 1.5 mmol. multidot.L^-150 mmol. L of NADPH^-1Potassium phosphate buffer (pH 6.0) was mixed and incubated at 20, 25, 30, 35, 40 ℃ in a metal bath, and then 500. mu.L of the crude enzyme solution of the disrupted supernatant was added and incubated at the corresponding temperature in a metal bath for 5min, and the change in OD340 was recorded in triplicate for each group.

The results of the optimal reaction temperature of the crude enzyme solution are shown in FIG. 3, and the optimal reaction temperature of the crude enzyme solution is 30 ℃.

(2) Exploration of optimum reaction pH of enzyme

Will contain 0.3 mmol. multidot.L^-12% methanol solution of substrate ADD and 1mL solution containing 1.5 mmol. multidot.L^-150 mmol. L of NADPH^-1Potassium phosphate buffers (pH 5.0, 6.0, 7.0, 8.0, 9.0) of different pH were mixed and placed in a metal bath at 30 ℃ for incubation, and then 500. mu.L of crude enzyme solution of the supernatant after disruption was added and placed in the corresponding metal bath for reaction for 5min, with triplicate sets, and the change in OD340 was recorded.

The results of the optimum reaction pH of the crude enzyme solution are shown in FIG. 4, and the optimum reaction pH of the crude enzyme solution was 6.

(3) Substrate profiling of crude enzymes

According to the action substrate of 17 beta-HSDy 1 in mycobacteria LY-1, the relative substrate specificity analysis is carried out on 17 beta-HSDy 1 by using the crushed supernatant of the heterologous expression strain of recombinant bacillus subtilis WB600-pMA5-17 beta HSDy 1-T239Y. The relative activity of the enzyme on ADD and 9 alpha-OH-AD was characterized by taking AD as a reference substrate and setting the enzyme activity to 100%. The construction method of the recombinant Bacillus subtilis WB600-pMA5-17 beta HSDy1 is as in example 3.

The recombinant bacillus subtilis WB600-pMA5-17 beta HSDy1-T239Y has characteristic substrate of ADD, and AD and 9 alpha-OH-AD, and compared with a heterologous expression strain before mutation, the substrate preference is unchanged.

Example 7: WB600-pMA5-17 beta HSDy1-T239Y converts ADD to generate boldenone

As ADD is a fat-soluble substance, in the process of converting the substrate ADD by utilizing the recombinant bacillus subtilis WB600-pMA5-17 beta HSD y1-T239Y, the cosolvent is added, so that the final concentration of the ADD is 3.0 g/L. Finally, the recombinant bacteria WB600-pMA5-17 beta HSDy1-T239Y are utilized to successfully convert ADD to generate the boldenone, and the result is shown in Table 3, compared with the original Bacillus subtilis, the recombinant Bacillus subtilis after heterologously expressing 17 beta HSDy1 (the construction method of the recombinant Bacillus subtilis WB600-pMA5-17 beta HSDy1 is shown in example 3) realizes the conversion of ADD to generate the boldenone, which shows that the 17 beta HSDy1 realizes the successful expression in the Bacillus subtilis; the mutated 17 beta HSDy1-T239Y also realizes successful expression in the bacillus subtilis, and the conversion rate of the ADD to generate the pagodanone is greatly improved and is 150 percent of the un-mutated 17 beta HSDy1 recombinant engineering strain.

TABLE 3 conversion of ADD to Plumbum Preparatium Ketone

The above description is only of the preferred embodiments of the present invention, and it should be noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the invention and these are intended to be within the scope of the invention.

Sequence listing

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Claims

1. A 17 β -hydroxysteroid dehydrogenase mutant, characterized by: the amino acid sequence is shown as SEQ ID NO. 4.

2. The 17 β -hydroxysteroid dehydrogenase mutant as set forth in claim 1, wherein: the 17 beta-hydroxysteroid dehydrogenase mutant is obtained by mutating threonine at the 239 th site of 17 beta-hydroxysteroid dehydrogenase 17 beta HSDy1 of parent Mycobacterium LY-1 with an original amino acid sequence shown as SEQ ID NO.2 into tyrosine, wherein the gene of the 17 beta-hydroxysteroid dehydrogenase is derived from Mycobacterium sp LY-1 CGMCC No. 13031.

3. A gene of a 17 β -hydroxysteroid dehydrogenase mutant, characterized in that: the nucleotide sequence is any one of the following:

a) a base sequence shown as SEQ ID N0: 3;

4. A recombinant expression plasmid for encoding a 17 β -hydroxysteroid dehydrogenase mutant of mycobacterium (Mcobacterium sp.) LY-1, wherein: containing the nucleotide sequence of claim 3, wherein the recombinant expression plasmid is pMA5-17 β HSDy 1-T239Y.

5. An engineered strain which expresses 17 β -hydroxysteroid dehydrogenase at a high efficiency, wherein the engineered strain incorporates the gene of the 17 β -hydroxysteroid dehydrogenase mutant according to claim 3, or comprises the recombinant expression plasmid according to claim 4.

6. The engineering strain of claim 5, wherein the host microorganism of the engineering strain is Bacillus subtilis WB600, and the obtained engineering strain is Bacillus subtilis WB600-pMA5-17 β HSDy 1-T239Y.

7. The engineering strain or/and the crude enzyme solution secreted and expressed by the engineering strain according to claim 5, and application thereof in the preparation of the pagodanone, wherein the preparation of the pagodanone takes androstene-1, 4-ene-3, 17-dione as a substrate to generate the pagodanone.

8. The use of the engineered strain of claim 7, wherein the reaction conditions of the crude enzyme solution are as follows: the temperature is 30 ℃, the pH is 6, and the fermentation time is 30 h.

9. The use of the engineering strain according to claim 7, wherein the engineering strain or/and the crude enzyme solution secreted and expressed by the engineering strain is transferred into a culture medium containing 3g/L androstene-1, 4-ene-3, 17-dione at 30-37 ℃ and 220 r-min^-1And (5) converting for 24-30h to obtain the boldenone.

10. The pagodanone prepared by the use of the engineered strain according to any of claims 7-9.