CN113528475B - Carbonyl reductase mutant and application thereof in preparation of steroid hormone testosterone - Google Patents

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to a carbonyl reductase mutant and application thereof in preparation of steroid hormone testosterone. The carbonyl reductase mutant PmCRm and the formate dehydrogenase BstFDH_m are co-expressed in escherichia coli, and resting cells of the co-expressed engineering bacteria are used as biocatalysts to catalyze androstane-4-alkene-3, 17-dione to synthesize testosterone. The biocatalyst has higher catalytic activity, regioselectivity and stereoselectivity, can completely convert 28.8 g/L androstane-4-alkene-3, 17-dione into a target product testosterone within 10 h, has no byproducts, has the highest level of testosterone prepared by the domestic biological method in substrate feeding amount, conversion rate and space-time conversion rate, and has the product recovery rate of more than 95 percent after separation and purification, so that the biocatalyst is an efficient catalyst for green synthesis of testosterone.

Description

Carbonyl reductase mutant and application thereof in preparation of steroid hormone testosterone

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to a carbonyl reductase mutant and application thereof in preparation of steroid hormone testosterone.

Background

Testosterone (TS) is an androgenic steroid drug, also known as Testosterone, testosterone or Testosterone, and has the chemical name 17β -hydroxy-androstan-4-en-3-one, and the chemical structure is shown in figure 1. Testosterone can be used in medicine as an androgen medicine for treating male primary or secondary hypogonadism and maintaining secondary sexual characteristics and sexual functions, and can also be used as a key precursor for synthesizing a series of high-efficiency steroid medicines (such as methyltestosterone, nandrolone propionate, testosterone enanthate and the like).

In the conventional process, testosterone is prepared by reacting various starting materials (indanone, indendione, beta-sitosterol, phytosterol, androstane-4-ene-3, 17-dione) in a multi-step chemical reactionAndrostane-1, 4-diene-3, 17-dione, etc.) is converted into a mixture of testosterone and trans-testosterone, and then the mixture is resolved by an enzymatic method or a chemical method to obtain a pure testosterone product. The whole process involves 3-carbonyl protection, 17-carbonyl reduction, 3-deprotection, resolution and the like, has complicated process, severe conditions and serious pollution, and is a testosterone synthesis method with high cost, poor atom economy and low recovery rate. In order to achieve a more environmentally friendly, less costly and easy to handle testosterone synthesis process, scientists have begun to shift their eyes to bioconversion and have made numerous attempts to use androsta-4-ene-3, 17-dione or androsta-1, 4-diene-3, 17-dione as precursors, using various microorganisms (e.g.)Acremonium strictum、Beauveria bassianaAndCephalosporium aphidicolaetc.) to synthesize testosterone. However, the microbial method has low yield and contains byproducts, and is difficult to be used for industrial production of testosterone, so that the chemical method is still adopted for producing testosterone at home at present.

In 2017, CN 2017-10617905 discloses a method for using candida albicansCandida albicansThe enzyme method process for preparing testosterone by 17 beta-hydroxysteroid dehydrogenase from 7103 has substrate load of 3-5 g/L and space-time conversion rate of 0.397 g/L/h, and is the only enzyme method process for preparing testosterone in China. Carbonyl reductase and 17 beta-hydroxysteroid dehydrogenase belong to the family of short-chain dehydrogenases, both of which catalyze the conversion of carbonyl groups to hydroxyl groups. And carbonyl reductase has wide substrate spectrum, is widely applied to the industrialized preparation of various chiral alcohols, and can theoretically reduce androstane-4-alkene-3, 17-dione into testosterone. However, no report on asymmetric reduction of androstane-4-alkene-3, 17-dione by carbonyl reductase is available internationally for synthesizing testosterone.

Disclosure of Invention

In view of the above problems, the present invention aims to provide a carbonyl reductase mutant and its application in preparing steroid hormone testosterone.

In order to achieve the above purpose, the invention adopts the following technical scheme:

a carbonyl reductase mutant PmCRm is obtained by mutating leucine at position 136 of an amino acid sequence shown in SEQ ID No.1 into serine.

Wherein the amino acid sequence of the carbonyl reductase mutant PmCRm is shown as SEQ ID No.2。

Gene for coding carbonyl reductase mutant PmCRmpmcr_mThe nucleotide sequence is shown as SEQ ID No. 3.

A recombinant expression vector comprising the gene encoding the carbonyl reductase mutant PmCRm as described abovepmcr_mAnd has a gene encoding formate dehydrogenase BstFDH_m linked downstream thereofbstfdh_m。

Wherein the recombinant expression vector is a recombinant expression vector pET30 a-containing pET30a as a frameworkpmcr_m- bstfdh_m。

Co-expression engineering strain comprising recombinant expression vector pET30a-pmcr_m-bstfdh_m。

Wherein the co-expression engineering strain is recombinant expression vector pET30a-pmcr_m-bstfdh_mCo-expression engineering strain obtained by transforming host microorganism which is Escherichia coliE.coli）BL21（DE3）。

The application of the carbonyl reductase mutant PmCRm in the preparation of steroid hormone testosterone is as follows: recombinant expression vector pET30a-pmcr_m-bstfdh_mConversion to%E.coli) Preparing a co-expression engineering strain in BL21 (DE 3), and preparing wet thalli of resting cells of the co-expression engineering strain; suspending wet thallus in 100 mL phosphate buffer, adjusting the concentration of wet thallus to 100-200 g/L, adding substrate androstane-4-ene-3, 17-dione 2.88 g, tween 80 4 mL, auxiliary substrate sodium formate 1.02 g, NADP ⁺ 15.7 mg,40 ℃, 230 rpm reaction 10 h; and centrifuging to collect precipitate in the reaction liquid, extracting with ethanol or ethyl acetate, concentrating under reduced pressure to obtain crude testosterone product, and washing with water to obtain pure testosterone product.

Compared with the prior art, the invention has the following beneficial effects:

(1) The invention has the advantages of simple and convenient operation, mild catalytic reaction condition, high efficiency (high activity and good selectivity), and coexpression of carbonyl reductase mutant PmCRm and formate dehydrogenase BstFDH_m in the escherichia coliBacteria (fungus)（E.coli）In BL21 (DE 3), resting cells of the coexpression engineering bacteria are used as a biocatalyst, androstane-4-alkene-3, 17-diketone is used as a substrate, the substrate can be completely converted into a target product testosterone in a short time under the condition of higher substrate concentration, no byproducts are generated, and the space-time conversion rate is far higher than the current report level (2.88 vs.0.397 g/L/h).

(2) The invention has high recovery rate (the recovery rate of testosterone is up to 97%), the related organic reagent (ethyl acetate or ethanol) is easy to recover, the cost is low, no pollutant is discharged, the invention is environment-friendly, and the green synthesis of testosterone can be realized.

Drawings

Fig. 1 is a chemical structure of testosterone.

FIG. 2 is a schematic diagram of the construction of co-expression strains.

FIG. 3 is a nucleic acid electrophoretogram; wherein lane M is a DNA marker; lane 1 ispmcr_mThe method comprises the steps of carrying out a first treatment on the surface of the Lane 2 is linearized pET30a; lane 3 is double digested pET30a-pmcr_mThe method comprises the steps of carrying out a first treatment on the surface of the Lane 4 is pET30a-pmcr_mPerforming PCR verification; lane 5 isrbs- bstfdh_mThe method comprises the steps of carrying out a first treatment on the surface of the Lane 6 is pET30a-pmcr_m-rbsA skeleton; lane 7 is linearized pET30a-pmcr_m-bstfdh_mThe method comprises the steps of carrying out a first treatment on the surface of the Lane 8 is pET30a-pmcr_m-bstfdh_mAnd (5) PCR verification.

Fig. 4 is a time curve for whole cell catalyzed synthesis of testosterone.

Fig. 5 is an HPLC detection profile of testosterone pure product.

Detailed Description

In order to make the objects, technical solutions and advantages of the present patent more apparent, the present patent will be described in further detail below with reference to specific embodiments. It will be appreciated by those skilled in the art that the description of the embodiments of the invention is merely exemplary and is not intended to limit the scope of the invention.

EXAMPLE 1 construction of carbonyl reductase mutant (PmCRm) and formate dehydrogenase (BstFDHm) Co-expression engineering Strain

(1) PCR amplification of carbonyl reductase mutant genespmcr_mAnd purifying

The entire construction flow is shown in fig. 2.

The carbonyl reductase mutant PmCRm is obtained by mutating leucine at position 136 of the amino acid sequence (WP_ 060395036.1) shown in SEQ ID No.1 into serine.

Based on the amino acid sequence (SEQ ID No. 2) of the carbonyl reductase mutant PmCRm, the method comprises the following stepsE.coli) Codon optimization for BL21 (DE 3) as target hostpmcr_mGene (SEQ ID No. 3) and was assigned to Shanghai JieR bioengineering Co.Ltd. Two primers were designed according to the sequence shown in SEQ ID No. 3:

F1（SEQ ID No.4）：

5‘-ATCGGATCCATGTCAGCATCTAAAAC-3' (BamHI cleavage site underlined);

R1（SEQ ID No.5）：

5‘-CAGCTCGAGTTACCACGGCAGGGTACG-3' (Xho I cleavage site underlined).

By synthesis ofpmcr_mThe gene is used as a template, and KOD one is utilized to amplify target gene fragments.

The PCR conditions were: 98 ℃ for 2 min; cycling for 30 times at 98 ℃,10 s,55 ℃,10 s,68 ℃,10 s; 68 ℃ for 2 min.

As shown in FIG. 2, the target band was found to be near 705 and bp by 1% wt% agarose gel electrophoresis. The PCR products were purified using a rubber cutting recovery kit (OMEGA, USA) and stored in a-20deg.C refrigerator for use.

(2) Double enzyme digestion, purification and ligation

The step (1) is carried outpmcr_mThe gene fragment and vector pET30a were digested with BamHI-XhoI, and the digested products were purified using DNA purification kit (OMEGA, USA) and confirmed by 1wt% agarose gel electrophoresis, as shown in FIG. 3, and the digested vector was matched to the theoretical molecular weight (5399 bp). The purified gene fragment and the linear vector were ligated with T4 ligase.

(3) Conversion of ligation products

Preparation of competent by calcium chloride method（E.coli）BL21 (DE 3) cells, and the ligation products are transferred to competence by heat shock（E.coli）In BL21 (DE 3), the specific steps are as follows:

removing competence from-80 DEG C（E.coli）BL21 (DE 3) cells were thawed on an ice-water bath for 5 min, then the ligation product obtained in step (2) was transferred into the ice-water bath for 2 min, heat-shocked at 42℃for 90 s and then ice-water-bathed for 2 min, then 900. Mu.L of LB medium was added thereto, incubated at 37℃for 45-60 min at 200 rpm, and finally plated on LB plates containing 50 mg/mL kanamycin, and incubated overnight at 37 ℃.

(4) Positive clone validation

The monoclonal was picked, cultured in LB medium containing 50 mg/mL kanamycin for 8 h, plasmid was extracted using plasmid extraction kit (OMEGA, USA), PCR was performed with T7 universal primer, the plasmid was double digested with BamH I-Xho I, and 1wt% agarose gel electrophoresis was performed to verify that the digested products (705 bp and 5399 bp, respectively) and PCR products (1020 bp) were consistent with theoretical molecular weight, indicating recombinant plasmid pET30a-pmcr_mIs successfully constructed. The sequence of the T7 universal primer is as follows:

T7-F（SEQ ID No.6）：

5‘-TAATACGACTCACTATAGGG--3’；

T7-R（SEQ ID No.7）：

5‘-GCTAGTTATTGCTCAGCGG-3’。

(5) Construction of Co-expression plasmid pET30a by Mega rimer PCRpmcr_m-bstfdh_m

(1) Amplification ofrbs-bstfdh_mFragments

The amino acid sequence of NADP (H) -dependent formate dehydrogenase BstFDHm as described in SEQ ID No.8 is reported in the literature (H.W. Jiang, Q.Chen, J.Pan, G.W. Zheng and J.H. Xu, appl Biochem Biotechnol, 2020, 192, 530-543.)（E.coli）BL21 (DE 3) is used as target host to carry out codon optimization and determine the gene thereofbstfdh_mAnd was delegated to Shanghai JieRui bioengineering Co.Ltd. To be connected withrbsThe sequence was added upstream of the BstFDHm encoding gene, and two primers were designed according to the sequence shown in SEQ ID No. 9:

F2（SEQ ID No.10）：

5‘-GCACCACAAGAAGGAGATATACCTATGGCAACCGTGCTG TGTGT-3' (homology arm sequences underlined, boldrbsA sequence);

R2（SEQ ID No.11）：

5‘-CGGGCTTTGTTAGCAGCCGGATCTCAGTGTTAGGTCAGGCGATAAGACTG-3' (homology arm sequences underlined).

By synthesis ofbstfdh_mThe gene was used as a template and PCR amplification was performed using KOD one.

The PCR conditions were: 98 ℃ for 2 min; cycling for 30 times at 98 ℃,10 s,55 ℃,10 s,68 ℃,10 s; 68 ℃ for 2 min.

As shown in FIG. 3, the PCR product was confirmed to have a theoretical molecular weight (1214 bp) by 1wt% agarose gel electrophoresisrbs-bstfdh_mFragments.

(2) Amplification of pET30a-pmcr_m-rbsSkeleton frame

Obtaining the linear skeleton fragment pET30a by using PCR amplification technology-pmcr_m-rbs。

F3（SEQ ID No.12）：

5‘-CACTGAGATCCGGCTGCTAACAAAGCCCG-3' (homology arm sequences underlined);

R3（SEQ ID No.13）：

5‘-AGGTATATCTCCTTCTTGTGGTGCCTCGAGTTACCACGGCAGGGTACGAC-3' (homology arm sequences underlined, boldrbsComplementary sequences).

With pET30a-pmcr_mAs a template, PCR amplification was performed using KOD one.

The PCR conditions were: 98 ℃ for 2 min; cycling for 30 times at 98 ℃,10 s,55 ℃,10 s,68 ℃,40 and s; 68 ℃ for 2 min.

As shown in FIG. 2, the PCR product was found to have a theoretical molecular weight (6084 bp) of pET30a by 1wt% agarose gel electrophoresis-pmcr_m-rbsA backbone fragment.

(3) PCR amplification of Co-expression plasmid pET30a-pmcr_m-bstfdh_m

Respectively byrbs-bstfdh_mFragment and pET30a-pmcr_m-rbsFor guidingThe mixture and template were subjected to Mega primer PCR using KOD one.

The PCR conditions were: 98 ℃ for 2 min; cycling for 30 times at 98 ℃,10 s,55 ℃,10 s,68 ℃,50 and s; 68 ℃ for 2 min.

Digestion of the PCR product with DMT (37 ℃,1 h) followed by heat shock (42 ℃,90 s, method same as described in step (3)) converts the PCR product to competent（E.coli）BL21 (DE 3). The single clone culture was selected, the plasmid was extracted, and then subjected to PCR verification with T7 universal primer (sequences as described above) and the extracted plasmid was linearized with BamH I, and 1wt% agarose gel electrophoresis was verified, as shown in FIG. 3, the linearized and PCR products were matched with theoretical molecular weights (7245 bp and 2190 bp, respectively), indicating the co-expression engineering strain（E.coli）BL21 (DE3)-pET30a-pmcr_m-bstfdh_mIs successfully constructed.

EXAMPLE 2 preparation of Co-expression engineering Strain resting cells

Picking up（E.coli）BL21 (DE3)-pET30a-pmcr_m-bstfdh_mSingle colonies of the engineering strain were cultured in LB liquid medium containing 50 mg/L kanamycin at 37℃and 200 rpm for 8-12 h as seed solution to 20 mL. Transfer to 1L shake flask containing 300 mL LB medium plus 50 mg/L kanamycin at 2vol% inoculum, continue culturing until OD ₆₀₀ Lactose at a final concentration of 8% (m/v) was then added at 0.6-0.7 and placed at 25℃and continued to oscillate 12 h to induce expression of the gene of interest. Then the culture at 7000 rpm,16 ℃ in the centrifugal for 3 minutes, the supernatant, collecting wet bacterial cells, weighing and at-20 ℃ for storage.

EXAMPLE 3 Co-expression method for converting androstane-4-ene-3, 17-dione into testosterone by resting cells of engineering bacteria

Resting cells of the co-expressed engineering strain were prepared as described in example 2 and used as biocatalysts.

The product was measured by high performance liquid chromatography: eli ultra-high performance liquid chromatograph, chromatographic column: chiralpak AD-H Column (4.6 mm X250 mm, 5 μm), mobile phase: n-hexane-isopropanol (v/v=90/10), detection wavelength: 254 nm, column temperature: 30 ℃, flow rate: 1.4 mL/min. Under the detection conditions, the peak-off times of androstane-4-ene-3, 17-dione and testosterone were respectively: 10.39 min and 11.47 min.

(1) The wet cell was resuspended in 100 mL phosphate buffer (100 mM, pH=7.5) to a wet cell concentration of 200 g/L, and the substrate androstane-4-ene-3, 17-dione 2.88 g, tween 80 4 mL, co-substrate sodium formate 1.02 g, NADP was added ⁺ 15.7 After reaction at 40℃and 230 rpm for 4. 4 h mg, 200. Mu.L of the reaction mixture was extracted with ethyl acetate 1. 1 mL, the solvent was removed under reduced pressure, dissolved in isopropanol and diluted appropriately, and the mixture was dried over anhydrous sodium sulfate and subjected to HPLC detection, with a conversion of 82.07% and a space-time yield of 5.745 g/L/h.

(2) The wet cell was resuspended in 100 mL phosphate buffer (100 mM, pH=7.5) to a wet cell concentration of 100 g/L, and the substrate androstane-4-ene-3, 17-dione 2.88 g, tween 80 4 mL, co-substrate sodium formate 1.02 g, NADP was added ⁺ 15.7 mg,40 ℃, 230 rpm for reaction 10 h, 200 mu L of reaction solution is extracted with 1 mL ethyl acetate, the solvent is removed under reduced pressure, then the solution is dissolved and properly diluted with isopropanol, and after drying with anhydrous sodium sulfate, HPLC detection is carried out, the conversion rate is 96.85%, and the space-time yield is 2.712 g/L/h.

(3) The wet cell was resuspended in 100 mL phosphate buffer (100 mM, pH=7.5) to a wet cell concentration of 150 g/L, and the substrate androstane-4-ene-3, 17-dione 2.88 g, tween 80 4 mL, co-substrate sodium formate 1.02 g, NADP was added ⁺ 15.7 mg,40 ℃, 230 rpm for reaction 10 h, 200 μl of reaction solution was extracted with ethyl acetate 1 mL, the solvent was removed under reduced pressure, dissolved with isopropanol and diluted appropriately, dried over anhydrous sodium sulfate and subjected to HPLC detection, the conversion was 97.95%, and the space-time yield was 2.743 g/L/h.

(4) The wet cell was resuspended in 100 mL phosphate buffer (100 mM, pH=7.5) to a wet cell concentration of 200 g/L, and the substrate androstane-4-ene-3, 17-dione 2.88 g, tween 80 4 mL, co-substrate sodium formate 1.02 g, NADP was added ⁺ 15.7 mg,40 ℃, 230 rpm reaction 10 h, 200 mu L reaction solution is extracted with 1 mL ethyl acetate, after the solvent is removed under reduced pressure, the solution is dissolved and diluted properly with isopropanol, and dried with anhydrous sodium sulfateThe conversion was 100% by HPLC and the space-time yield was 2.88 g/L/h.

EXAMPLE 4 time curve for transformation of resting cells of Co-expressed engineering bacteria into androsta-4-ene-3, 17-dione to testosterone

The wet cell was resuspended in 100 mL phosphate buffer (100 mM, pH=7.5) to a wet cell concentration of 200 g/L, and the substrate androstane-4-ene-3, 17-dione 2.88 g, tween 80 4 mL, co-substrate sodium formate 1.02 g, NADP was added ⁺ 15.7 mg,40℃and 230 rpm. At different times, 200 μl of reaction solution was extracted with 1 mL ethyl acetate, the solvent was removed under reduced pressure, dissolved and properly diluted with isopropanol, dried over anhydrous sodium sulfate, and subjected to HPLC detection, and the time profile of the reaction was plotted according to the conversion at each time, as shown in fig. 4, the conversion increased rapidly to 91.8% within 4 hours, the reaction continued, the conversion increased slowly, and finally the substrate was converted to testosterone entirely at 10 h.

EXAMPLE 5 purification of testosterone

(1) The 100 mL reaction was centrifuged (theoretical testosterone content 2.90 g) and the precipitate was collected. The precipitate was extracted several times with ethyl acetate (58 mL) of 20 times the mass of testosterone, and the organic solution was collected and concentrated under reduced pressure to crystallize. The crystals were rinsed with 20 times the mass of testosterone (58 mL), collected and dried to give 2.76 g crystals in 95.1% yield.

(2) The 100 mL reaction was centrifuged (theoretical testosterone content 2.90 g) and the precipitate was collected. The precipitate was extracted with 20 times the mass of testosterone in ethanol (58, mL) and the organic solution was collected and concentrated under reduced pressure to crystallize. The crystals were rinsed with 20 times the mass of testosterone (58 mL), collected and dried to give 2.82 g crystals in 97.2% yield.

EXAMPLE 6 quality testing of testosterone finished products

Detecting the quality of the obtained crystal, and measuring the melting point by using a WRS-1C melting point instrument; the specific optical rotation is measured by using a full-automatic polarimeter P850, and the concentration of a sample is 1% (m/m and dissolved in ethanol); the water content was measured by an infrared ray moisture measurement balance MA 35. The results were as follows:

melting point is 151-156 deg.C, purity is 99.9% (HPLC method, shown in figure 5), specific rotation measured under D line of sodium light at 25deg.C is 107 deg.C, and water content is < 0.1%.

The product was tested to be testosterone. No by-product is produced, and the recovery rate of testosterone reaches more than 95%.

According to the public knowledge in the art, any gene can change its sequence by means of DNA mutation technology, and various mutants are produced, and the proteins expressed by these mutants often have the same functions. The genes and products thereof related to the invention also have the same characteristics. Thus, any enzyme which is mutated by an allele or has one or more amino acid insertions, deletions or substitutions and has the function of catalyzing androsta-4-ene-3, 17-dione to testosterone is within the scope of the invention.

SEQUENCE LISTING

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atggcaaccg tgctgtgtgt gctgtatcca gacccggtgg atggctatcc gccgcattat 60

gttcgcgata ccattccggt tattacccgc tatgcagatg gccagaccgc accgaccccg 120

gcaggcccgc cgggctttcg ccctggtgaa ctggtgggtt cagtgagcgg cgcactgggc 180

ctgcgcggct atctggaagc acatggtcat accctgattg ttaccagtga taaagatggt 240

ccggatagcg aatttgaacg tcgtctgccg gatgcagatg ttgtgatttc tcagccgttt 300

tggccggcat atctgaccgc agaacgtatt gcacgcgcac cgaaactgcg tctggcactg 360

accgcaggca ttggtagcga tcatgtggat ctggatgcag cagcacgtgc acatattacc 420

gttgcagaag ttaccatgag taatagtatt agcgttgcag aacatgttgt tatgaccacc 480

ctggcactgg tgcgtaatta tctgccgagc catgcaattg cacagcaggg cggttggaat 540

attgcagatt gtgtgtctcg tagctatgat gtggaaggta tgcattttgg tactgtgggc 600

gcaggtcgta ttggtctggc agtgctgcgt cgcctgaaac cgtttggcct gcatctgcat 660

tatacccagc gtcatcgcct ggatgcagca attgaacagg aactgggtct gacctatcat 720

gcagatccgg caagcctggc agcagcagtt gatattgtta atcttcaaat tccgctgtat 780

ccgtcaaccg aacatctgtt tgatgcagca atgattgcac gtatgaaacg cggcgcatat 840

ctgattaata ccgcacgcgg taaactggtt gatcgcgatg cagttgttcg cgcagtgacc 900

tcaggccatc tggcaggcta tggcggcgat gtttggtttc cgcagccggc accggcagat 960

catccgtggc gcgcaatgcc gtttaatggt atgaccccgc atatttcagg gacatcactg 1020

tcagcacagg cacgctatgc agcagggacg ctggaaattc tacagtgttg gtttgatggt 1080

cgtccgattc gtaatgaata tctgattgtg gatggcggca ccctggcagg tacgggcgca 1140

cagtcttatc gcctgaccta a 1161

<210> 10

<211> 44

<212> DNA

<213> artificial sequence

<400> 10

gcaccacaag aaggagatat acctatggca accgtgctgt gtgt 44

<210> 11

<211> 50

<212> DNA

<213> artificial sequence

<400> 11

cgggctttgt tagcagccgg atctcagtgt taggtcaggc gataagactg 50

<210> 12

<211> 29

<212> DNA

<213> artificial sequence

<400> 12

cactgagatc cggctgctaa caaagcccg 29

<210> 13

<211> 50

<212> DNA

<213> artificial sequence

<400> 13

aggtatatct ccttcttgtg gtgcctcgag ttaccacggc agggtacgac 50

Claims (8)

1. A carbonyl reductase mutant PmCRm, characterized in that: the mutant is obtained by mutating leucine at 136 th position of an amino acid sequence shown in SEQ ID No.1 into serine.

2. Encoding carbonyl reductase mutant PmCRmGenepmcr_mThe method is characterized in that: the nucleotide sequence of the gene is shown as SEQ ID No. 3.

3. A recombinant expression vector, characterized in that: the recombinant expression vector comprising the gene encoding the carbonyl reductase mutant PmCRm as claimed in claim 2pmcr_mAnd has a downstream encoding formate dehydrogenase BstFDH_m genebstfdh_mThe method comprises the steps of carrying out a first treatment on the surface of the The formate dehydrogenase BstFDH_The amino acid sequence of m is shown as SEQ ID No.8, and the genebstfdh_mThe nucleotide sequence of (2) is shown as SEQ ID No. 9.

4. A recombinant expression vector according to claim 3, wherein: the recombinant expression vector is a recombinant expression vector pET30a-pmcr_m-bstfdh_m taking pET30a as a framework.

5. A co-expression engineering strain, characterized in that: the coexpression engineering strain comprises the recombinant expression vector as claimed in claim 4.

6. The co-expression engineering strain according to claim 5, wherein: it is to use recombinant expression vector pET30a-pmcr_m-bstfdh_m into host microorganism; the host microorganism is Escherichia coliE.coli BL21（DE3）。

7. Use of the carbonyl reductase mutant PmCRm according to claim 1 for the preparation of the steroid hormone testosterone.

8. The use according to claim 7, characterized in that: transformation of the recombinant expression vector pET30a-pmcr_m-bstfdh_m as defined in claim 4 intoE.coli Preparing a co-expression engineering strain in BL21 (DE 3), and preparing wet thalli of resting cells of the co-expression engineering strain; suspending wet thallus in 100 mL phosphate buffer, adjusting the concentration of wet thallus to 100-200 g/L, adding substrate androstane-4-ene-3, 17-dione 2.88 g, and tween 80 4mL, co-substrate sodium formate 1.02 g, NADP ⁺ 15.7 mg,40 ℃, 230 rpm reaction 10 h; and centrifuging to collect precipitate in the reaction liquid, extracting with ethanol or ethyl acetate, concentrating under reduced pressure to obtain crude testosterone product, and washing with water to obtain pure testosterone product.

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