CN107312787B - Fusion protein gene, engineering bacterium and application of fusion protein gene and engineering bacterium in preparation of 9 α -hydroxy-androstenedione - Google Patents

Abstract

The invention discloses a fusion protein gene, engineering bacteria and application thereof in preparing 9 α -hydroxy-androstenedione, wherein the fusion protein gene comprises a gene for coding 3-sterone-9 α -hydroxylase and a gene for coding a flavin domain in cytochrome P450BM-3 enzyme, the invention heterologously expresses the 3-sterone-9 α -hydroxylase in escherichia coli, and performs fusion expression on the 3-sterone-9 α -hydroxylase and the flavin domain of the cytochrome P450BM-3 enzyme through a gene recombination technology to obtain a fusion protein KhA-P450, and the recombinant engineering bacteria containing the fusion protein is used for catalyzing a substrate androstenedione to prepare the 9 α -hydroxy-androstenedione, so that the specific activity is improved, and the conversion rate is also improved.

Description

Fusion protein gene, engineering bacterium and application of fusion protein gene and engineering bacterium in preparation of 9 α -hydroxy-androstenedione

Technical Field

The invention relates to the field of biochemical engineering, in particular to a fusion protein gene, engineering bacteria and application thereof in preparation of 9 α -hydroxy-androstenedione.

Background

9 α -OH-AD is an important chemical intermediate, plays an important role in the production of steroid substances, 9 α -OH-AD can be used for synthesizing various steroid raw material medicaments, such as hydrocortisone, dexamethasone, eplerenone, 17 α -OH progesterone, cortisone and the like, the structural formula of 9 α -OH-AD is shown as the formula (1):

at present, 9 α -OH-AD is mainly prepared from Androstenedione (AD) serving as a raw material through multi-step chemical reactions, but the chemical method generally has the problems of long preparation route, poor selectivity, complex reaction, low yield, serious pollution and the like, so the development of an environment-friendly biological preparation method of 9 α -hydroxy-androstenedione is particularly important for overcoming the disadvantages of the chemical synthesis method.

It has now been found that a variety of microorganisms can be used to prepare 9 α -OH-AD, such as Nocardia (Nocardia), Rhodococcus (Rhodococcus) and the like, which can perform 9 α -hydroxylation of Androstenedione (AD) to 9 α -OH-AD, and Mycobacterium (Mycobacterium) and the like, which can perform a series of reactions, such as side chain degradation and 9 α -hydroxylation of phytosterol substrates to 9 α -OH-AD.

In the process of preparing 9 α -OH-AD by a microbial conversion method, a 9 α -hydroxylation reaction carried out on a steroid mother nucleus is a key step, and a key enzyme for catalyzing the reaction is 3-ketosteroid-9 α -hydroxylase (KSH). KSH is a two-component enzyme and consists of 3-ketosteroid-9 α -hydroxylase (KshA) and 3-ketosteroid-9 α -hydroxylase reductase (KshB). wherein the KhA is an active center of the KSH and is responsible for a main reaction of steroid 9 α -hydroxylation, and the KshB is a reductase component of a KSH system and is responsible for transferring free electrons from coenzyme to the KhA to change the KhA from an oxidation state to a reduction state and continuously catalyze the steroid to carry out a 9 α -hydroxylation reaction.

In recent years, the efficiency of synthesizing 9 α -OH-AD by fermentation method using Rhodococcus erythropolis or mycobacteria is continuously improved, but the problems of low thallus activity, low fermentation yield, long reaction period, high cost and the like still exist, so that the preparation of the engineering bacteria with high catalytic activity of 3-ketosteroid-9 α -hydroxylase and the application of the engineering bacteria in the preparation of 9 α -OH-AD are very important for reducing the production cost and shortening the reaction period.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a fusion protein, an engineering bacterium and a fusion protein expressed by the engineering bacterium, wherein the fusion protein catalyzes a substrate androstenedione to react to generate 9 α -hydroxy-androstenedione, and the fusion protein has high specific activity and high product conversion rate.

The invention provides a fusion protein gene, which comprises a gene for coding 3-sterone-9 α -hydroxylase and a gene for coding a flavochrome domain in cytochrome P450BM-3 enzyme.

P450BM-3 is a soluble fusion protein, which is composed of a catalytic domain (hemoglobin domain) and a reducing domain (flavin domain), which is divided into FMN sub-domain and FAD sub-domain, and has a structure similar to that of KshB. The catalytic mechanism of P450BM-3 is as follows: the substrate and oxygen molecule combine with heme on the heme domain and simultaneously obtain an electron from NADPH, the electron is transmitted to heme through FAD and FMN of the heme domain, the oxygen molecule is reduced and decomposed, one oxygen is added into the substrate, and the other oxygen generates water, and the synergetic catalytic mechanism of KshA and KshB is very similar to that of KshA and KshB.

Therefore, the flavin domain of the P450BM-3 enzyme is selected as the electron transport group to provide electrons for KshA to enhance the catalytic ability of the KshA enzyme. In molecular operation, the part of P450bm-3 gene encoding the catalytic domain is excised and replaced with kshA gene to complete the fusion expression of kshA and P450BM-3 flavochrome domain.

We carried out expression studies on 3-sterone-9 α -hydroxylase (the base sequence of gene coding in GenBank database is accession No. Rv3526) derived from Mycobacterium tuberculosis H37Rv and 3-sterone-9 α -hydroxylase of Rhodococcus erythropolis (Rhodococcus erythropolis) SQ1 (the base sequence of which is accession No. AY083508.1 in GenBank), respectively.

Preferably, the 3-ketosteroid-9 α -hydroxylase is derived from Rhodococcus erythropolis (Rhodococcus erythropolis) SQ1, the base sequence of which is deposited under GenBank under accession number AY083508.1, and the cytochrome P450BM-3 enzyme is derived from Bacillus megaterium (Bacillus megaterium NBRC 15308), which is also designated as (Bacillus megaterium ATCC 14581), i.e., the strain is deposited under NBRC strain bank under accession number 15308, and is deposited under ATCC strain bank under accession number 14581, the base sequence of which is deposited under GenBank under accession number CP 009920.1.

Further, the base sequence of the fusion protein gene is shown as SEQ ID NO. 1.

The 3-sterone-9 α -hydroxylase gene and the cytochrome P450BM-3 enzyme gene can be obtained by cloning or chemical synthesis.

The invention provides an expression vector or a recombinant plasmid containing the fusion protein gene.

The invention also provides a fusion protein encoded by the fusion protein gene.

The fusion protein can be applied to the reaction of catalyzing androstenedione reaction to generate 9 α -hydroxy-androstenedione.

Further, the base sequence of the recombinant gene encoding the fusion protein is shown in SEQ ID No.1, and the schematic diagram of the plasmid structure is shown in FIG. 5. The amino acid sequence of the fusion protein is shown as SEQ ID NO. 2.

In the invention, a protein sequence of KshA from Rhodococcus erythropolis (Rhodococcus erythropolis) SQ1 is obtained by utilizing a GenBank (accession number AY083508.1) database, the protein sequence is converted into a nucleic acid sequence, codons on the nucleic acid sequence are replaced by codons with high use frequency in Escherichia coli, a gene sequence capable of efficiently expressing kshA-reyh in the Escherichia coli is obtained, then whole-gene synthesis is carried out on the optimized sequence, Nhe I and Xho I enzyme cutting sites are added to two ends of the optimized sequence, the synthesized gene is connected into a pET28a (+) vector, a recombinant expression vector pET28a-kshA-reyh is obtained, and finally, the pET28a-kshA-reyh expression vector is converted into an Escherichia coli BL21(DE3) cell, and the 3-sterone-9 α -hydroxylation enzyme engineering bacterium BL21(DE3) -pET 28-28 a-kshA-reyh is obtained.

The invention discloses a method for preparing 3-sterone-9 α -hydroxylase from KshA of Rhodococcus erythropolis SQ1 by heterologous expression in Escherichia coli, and by means of gene recombination technology, the KshA and a flavin domain of P450BM-3 are subjected to fusion expression to finally obtain a fusion protein KshA-P450BM-3, and 9 α -hydroxy-androstenedione (9 α -OH-AD) is prepared by using a recombinant engineering bacterium catalytic substrate Androstenedione (AD).

Specifically, the invention replaces the hemoglobin domain of the p450bm-3 gene with kshA-reyh gene, connects the kshA-reyh gene with the gene sequence of p450bm-3 coding the flavonol domain, constructs a recombinant plasmid pET-28a-kshA-p450, and transforms the recombinant plasmid into an escherichia coli expression host.

Furthermore, the invention uses site-directed mutagenesis to carry out vector transformation on pET28a-p450bm-3 recombinant plasmid, carries out silent mutagenesis between gene sequences encoding a hemoglobin domain and a biotin domain, inserts an Xho I enzyme cutting site, and then uses silent mutagenesis to remove the Xho I enzyme cutting site at the tail end of the vector; then, using Nhe I and Xho I restriction enzyme cutting sites, linking the kshA-reyh gene into the modified pET28a-p450bm-3 plasmid vector to obtain a recombinant expression vector pET28a-kshA-p 450; finally, the pET28a-kshA-p450 expression vector was transformed into E.coli BL21(DE3) cells to obtain the fusion protein expression engineering bacterium BL21(DE3) -pET28a-kshA-p 450.

Therefore, the invention also provides an engineering bacterium, which comprises a host cell and a fusion protein gene transferred into the host cell, wherein the fusion protein gene is described above.

Wherein, the host cell can be Escherichia coli (E.coli) BL21, Escherichia coli (E.coli) BLR, Escherichia coli (E.coli) Origami, Escherichia coli (E.coli) NovaBlue or Escherichia coli (E.coli) Rosetta.

The recombinant gene is connected to a recombinant expression vector and then transferred into a host cell; wherein, the recombinant expression vector can be pET-28a (+), pET-21a (+), and pET-Duet.

The invention also provides application of the engineering bacteria in catalyzing androstenedione reaction to generate 9 α -hydroxy-androstenedione.

Specifically, the method for preparing 9 α -hydroxy-androstenedione by using the engineering bacteria comprises the following steps:

(1) culturing engineering bacteria and inducing enzyme expression, centrifuging to take cells, and resuspending with buffer solution to obtain resting cell suspension;

(2) adding a substrate androstenedione into the resting cell suspension for reaction, and after the reaction is finished, separating and purifying the reaction solution to obtain 9 α -hydroxy-androstenedione.

Preferably, in step (1), the method for inducing enzyme expression is: to OD of the culture broth₆₀₀When the temperature reaches 0.5-1.0, adding 0.01-0.5 mM IPTG, and inducing for 6-12 h at the temperature of 25-30 ℃; in the step (2), the reaction temperature is 25-30 ℃ and the reaction time is 1-24 h.

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

the 3-sterone-9 α -hydroxylase is heterologously expressed in escherichia coli, the 3-sterone-9 α -hydroxylase and a flavin domain of cytochrome P450BM-3 enzyme are subjected to fusion expression through a gene recombination technology to obtain a fusion protein KshA-P450, and the recombinant engineering bacteria containing the fusion protein are used for catalyzing a substrate androstenedione to prepare 9 α -hydroxy-androstenedione, so that the specific activity is improved, and the conversion rate is also improved.

Drawings

FIG. 1 is a structural diagram of the KshA-P450BM-3 fusion protein of the invention.

FIG. 2 shows the nucleic acid electrophoresis of recombinant vectors pET28a-kshA-reyh and pET28a-kshA-mtyh after double digestion with Nhe I and Xho I.

FIG. 3 is a plasmid construction map of plasmid pET28 a-kshA-reyh.

FIG. 4 shows the results of the double restriction enzyme digestion of the recombinant plasmid pET28a-kshA-p 450;

wherein, lanes 1-5: performing double enzyme digestion on Nhe I and Xho I; lanes 6-10: xho I and EcoR I double digestion.

FIG. 5 is a plasmid construction map of recombinant plasmid pET28a-kshA-p 450.

FIG. 6 shows the SDS-PAGE protein electrophoresis of KshA and the fusion protein.

Detailed Description

The present invention will be further described with reference to the following specific examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.

Example 13 construction of sterone-9 α -Hydroxylase engineering bacteria

Since Escherichia coli has a certain difference in codon preference between the KshA-derived bacteria Mycobacterium (Mycobacterium tuberculosis H37Rv) and Rhodococcus erythropolis (Rhodococcus erythropolis) SQ1, to achieve high expression of KshA in Escherichia coli, codon preference is first adjusted by synonymous conversion, and codons frequently used in Escherichia coli are substituted for the codons of the kshA gene. The optimization software is Jcat: (http:// www.jcat.de).

The similarity of the sequence after kshA-re transformation (kshA-reyh) and the original sequence is 81.24 percent, and 224 bases are changed in total; meanwhile, Nhe I and Xho I enzyme cutting sites are added at the front end and the rear end of the optimized sequence respectively.

The similarity of the sequence after kshA-mt modification (kshA-mtyh) and the original sequence is 80.92 percent, and 221 basic groups are changed; meanwhile, Nhe I and Xho I enzyme cutting sites are added at the front end and the rear end of the optimized sequence respectively.

The optimized gene sequence is synthesized by Shanghai Czeri company, the target gene and the vector are subjected to double enzyme digestion by using Nhe I and Xho I, the enzyme digestion product is subjected to agarose gel electrophoresis, and the target gene and the vector fragment are recovered. The recovered target gene and vector fragment are treated with T₄DNA ligase is used for ligation.

The above-mentioned ligation products were transformed into E.coli DH5 α competent cells by heat shock method, and the transformation products were spread on LB solid medium containing 50. mu.g/mL kanamycin and cultured at 37 ℃ for 12 hours.

The grown escherichia coli colony is picked up and cultured for 12-15h under the condition of oscillation at the temperature of 37 ℃ and the speed of 200rpm, plasmids are extracted by a plasmid extraction kit, the extracted plasmids are verified by double enzyme digestion by Nhe I and XhoI, the electrophoresis result is shown in the attached figure 2, a transformant with the correct enzyme digestion result is sent to a company Limited in Biotechnology engineering (Shanghai) to verify, recombinant plasmids with the correct sequence (shown in figure 3) are transformed into escherichia coli BL21(DE3) competent cells, and 3-sterone-9 α hydroxylase engineering bacteria BL21(DE3) -pET28a-kshA-reyh (kshA-reyh is from rhodococcus erythropolis) and BL21(DE3) -pET28a-kshA-mtyh (kshA-mtyh is from mycobacteria) are obtained.

Example 2 Activity verification of two recombinant strains BL21(DE3) -pET28a-kshA-reyh and BL21(DE3) -pET28a-kshA-mtyh

The engineered bacteria BL21(DE3) -pET28a-kshA-mtyh and BL21(DE3) -pET28a-kshA-reyh were activated and inoculated into TB medium at an inoculum size of 2%. Culturing at 37 deg.C until OD600 is 0.5-1.0, adding IPTG to final concentration of 0.5 mmol.L^-1Expression was induced while substrate AD dissolved in dimethyl sulfoxide (DMSO) was added to a final concentration of 300. mu. mol. multidot.L^-1. Reacting in a shaking table at 30 ℃ for oscillation reaction, sampling 24h after adding substrate AD, centrifuging at 12000rpm for 2min, taking supernatant, filtering with an aqueous phase filter membrane, and detecting by HPLC.

The detection conditions are as follows: the liquid phase column was Hypersil ODS2C18(4.6 mm. times.250 mm), and the 5 μm mobile phase was methanol: water 5: 5; the flow rate is 1 mL/min; the column temperature is 40 ℃; ultraviolet detection wavelength 254 nm; the injection volume was 20. mu.L.

The detection shows that the yield of 9 α -OH-AD generated by transforming the recombinant strain BL21(DE3) -pET28a-kshA-mtyh into AD is very low, the yield of 9 α -OH-AD is only 2.7% through 24-hour reaction in a TB culture medium, while the yield of 9 21(DE3) -pET28a-kshA-reyh can better transform AD into 9 α -OH-AD, and the occurrence of 9 α -OH-AD absorption peak, the reduction of substrate AD absorption peak and the yield of 9 α -OH-AD of 63.86% are 23.57 times of the yield of the strain BL21(DE3) -pET28a-kshA-mtyh obviously in a chromatogram.

From this experiment, we can conclude that KshA derived from Rhodococcus erythropolis SQ1 can produce 9 α -OH-AD with AD as a rational substrate, while KshA derived from Mycobacterium tuberculosis H37Rv is not an ideal substrate for AD.

Example 3 construction of engineering bacteria for expressing the fusion protein KshA-P450BM-3

First, the pET28a-p450bm-3 recombinant plasmid was vector-engineered using site-directed mutagenesis:

(1) a silent mutation is carried out between the gene sequences of the heme domain coding for p450bm-3 and the heme domain coding for p450bm-3, and an Xho I enzyme cutting site is inserted;

(2) the Xho I cleavage site at the end of the multiple cloning site of pET28a-p450bm-3 vector was removed by using a silent mutation.

Performing double enzyme digestion on the target gene and the modified vector by using Nhe I and Xho I, performing agarose gel electrophoresis on the enzyme digestion product, and recovering the target gene and the vector fragment. The recovered target gene and the vector fragment are ligated with T4DNA ligase.

The above-mentioned ligation products were transformed into E.coli DH5 α competent cells by heat shock method, and the transformation products were spread on LB solid medium containing 50. mu.g/mL kanamycin and cultured at 37 ℃ for 12 hours.

And (3) selecting the grown escherichia coli colony, carrying out shake culture at 37 ℃ and 200rpm for 12-15h, extracting plasmids by using a plasmid extraction kit, and carrying out two-group double enzyme digestion verification on the extracted plasmids by using Nhe I, XhoI and EcoR I. The electrophoresis results are shown in FIG. 4.

The transformant with correct enzyme digestion result is sent to the company Limited in Biotechnology engineering (Shanghai) for verification. The recombinant plasmid with the correct sequence (shown in figure 5) is transformed into competent cells of Escherichia coli BL21(DE3) to obtain the fusion protein expression engineering bacterium BL21(DE3) -pET28a-kshA-p 450. The gene sequence of the fusion protein is shown in a sequence table SEQ ID NO. 1.

Example 43 inducible expression of sterone-9 α -hydroxylase (KshA) and fusion protein KshA-P450BM-3

Picking single colonies of BL21(DE3) -pET28a-kshA-reyh and BL21(DE3) -pET28a-kshA-p450, and carrying out shake culture at 37 ℃ and 200rpm for 12-15 h; then inoculating the strain into a 250mL triangular flask containing 50mL LB medium containing 50 ug/mL kanamycin in an inoculation amount of 2%, and performing shaking culture at 37 ℃ and 200 rpm; after the bacterial liquid grows to OD₆₀₀When the concentration is 0.6-0.8, adding IPTG with the final concentration of 0.5 mM; inducing expression at 28 ℃ and 150 rpm; after the induction expression for 6h, centrifugation is carried out, supernatant is discarded, thalli sediment is collected, the thalli is washed by phosphate buffer (0.2mM, pH 7.5),adding 5mL of cell-breaking buffer solution to resuspend the thalli, breaking cells by using an ultrasonic cell-breaking instrument (power is 300W, 90 cycles: ultrasonic for 3s, and interval for 6s), centrifuging for 30min at 4 ℃ and 13000rpm, and collecting supernatant to obtain a crude enzyme solution.

And (3) adding 10 mu L of the two crude enzyme solutions into 10 mu L of 6 Xprotein electrophoresis buffer solution, adding 40 mu L of ultrapure water, uniformly mixing, heating the sample in a water bath kettle at 100 ℃ for 5min, and taking out the sample after the reaction is finished, and immediately placing the sample on ice to prevent protein renaturation. The protein expression results were analyzed by SDS-PAGE using 5% concentrated gel and 10% separation gel.

Coli BL21(DE3) -pET28a-p450bm-3 was used as a negative control, and other treatment conditions were the same as described above.

The electrophoresis result is shown in figure 6, and the recombinant strain BL21(DE3) -pET-28a-kshA-reyh is between the 40kD and 50kD bands, the sample group has more bands compared with the control group, and the bands are obvious, and the molecular weight is identical with the KshA of 44.5 kD; the recombinant strain BL21(DE3) -pET28a-kshA-reyh-p450bm-3 has a band between 100kD and 120kD, a sample group has more bands compared with a control group, and the bands are obvious, and the molecular weight of the band is matched with that of a fusion protein of 111.68 kD.

Example 5

The glycerol bacteria stored in a refrigerator are taken, 10 mu L of glycerol bacteria are inoculated into 5mL of LB culture medium containing 50 mu g/mL of kanamycin, and the mixture is shaken at 37 ℃ and 200rpm for overnight; the fresh bacterial solution from the previous step was inoculated into 50mL TB medium (tryptone 12g/L, yeast extract 24g/L, glycerol 4mL/L, KH) containing 50. mu.g/mL kanamycin₂PO₄17mmol/L、K₂HPO₄72mmol/L), the inoculum size is 2 percent, and the shaking culture is carried out at 37 ℃ and 200 rpm; after the bacterial liquid grows to OD₆₀₀When the concentration is 0.6-0.8, IPTG with a final concentration of 0.5mM is added, and then induction expression is carried out at 28 ℃ and 150 rpm.

After 11h of induction expression, the cells were sampled and OD of the cells was measured at a wavelength of 600nm using the sterile fermentation broth as a blank₆₀₀The biomass was measured as a value of bacterial biomass. The cells after induction expression were collected, washed once with PBS buffer, and then again washed with PBS bufferThe cells were resuspended, substrate AD (added substrate dissolved in ethanol) was added to a final concentration of 200. mu.M, and the reaction was carried out at 30 ℃ and 800rpm using a constant temperature metal shaker.

Samples were taken at various time points of the reaction and the product content of the system was determined. Centrifuging the sample at 12000rpm for 1min, collecting supernatant, filtering with water phase filter membrane, and analyzing the content of the product with HPLC. Liquid chromatography conditions: using Hypersil ODS2C18(4.6 mm. times.250 mm), 5 μm mobile phase was methanol: water 5: 5; the flow rate is 1 mL/min; the column temperature is 40 ℃; ultraviolet detection wavelength 254 nm; the injection volume was 20. mu.L. And correlating the peak area of the product with a standard curve to obtain the concentration of the product.

All experimental procedures were consistent with the experimental group using the KshA-reyh expressing strain alone as the experimental control group.

The experimental results show that: after two hours of reaction, the conversion rate of the experimental group is 43.49%, and the specific activity of the thallus is 264.96nmol/(min g) which is 2.3 times of that of the control group. After the reaction for twelve hours, the conversion rate of the reaction group reached 71.69%, the specific activity of the cells was 72.79nmol/(min g), and the specific activity was 3.26 times that of the control group.

Example 6

The induction time in example 5 was changed to 6 hours, the other conditions were kept unchanged, and when the reaction time was 4 hours, the specific activity of the cells was 48.30nmol/(min g), which was 3.14 times that of the control group.

SEQUENCE LISTING

<110> Ningbo theory of Zhejiang university college

<120> fusion protein, engineering bacteria and application thereof in preparation of 9 α -hydroxy-androstenedione

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115 120 125

Ala Trp Thr Thr Leu Glu Lys Asn Gly Gln Leu Phe Val Trp His Asp

130 135 140

Pro Gln Gly Asn Pro Pro Pro Ala Glu Val Thr Ile Pro Asp Ile Glu

145 150 155 160

Gly Phe Gly Ser Asp Glu Trp Ser Asp Trp Ser Trp Asn Thr Leu Thr

165 170 175

Ile Glu Gly Ser His Cys Arg Glu Ile Val Asp Asn Val Val Asp Met

180 185 190

Ala His Phe Phe Tyr Val His Tyr Ser Phe Pro Lys Tyr Phe Lys Asn

195 200 205

Ile Phe Glu Gly His Val Ala Ser Gln Tyr Met Glu Ser Val Gly Arg

210 215 220

Glu Asp Ile Ile Ser Gly Thr Asn Tyr Gly Asp Pro Asn Ala Val Leu

225 230 235 240

Arg Ser Asp Ala Ser Tyr Phe Gly Pro Ser Tyr Met Ile Asp Trp Ile

245 250 255

Lys Ser Glu Ala Asn Gly Gln Ile Ile Glu Thr Val Leu Ile Asn Cys

260 265 270

His Tyr Pro Ile Ser Asn Asn Ala Phe Val Leu Gln Tyr Gly Ala Met

275 280 285

Val Lys Lys Leu Pro Gly Met Asp Asp Glu Ser Thr Ala Ala Met Ala

290 295 300

Ala Gln Phe Thr Glu Gly Val Glu Met Gly Phe Leu Gln Asp Val Glu

305 310 315 320

Ile Trp Lys Asn Lys Ala Pro Ile Asp Asn Pro Leu Leu Ser Glu Glu

325 330 335

Asp Gly Pro Val Tyr Gln Leu Arg Arg Trp Tyr Asn Gln Phe Tyr Thr

340 345 350

Asp ValGlu Asn Val Thr Glu Asp Met Thr Lys Arg Phe Glu Phe Glu

355 360 365

Ile Asp Thr Thr Arg Ala Val Glu Ser Trp Lys Arg Glu Val Ala Glu

370 375 380

Asn Val Ala Ala Arg Asp Ala Gln Ala Leu Glu Thr Thr Ser Leu Glu

385 390 395 400

Gly Phe Val Val Lys Ala Lys Ser Lys Lys Ile Pro Leu Gly Gly Ile

405 410 415

Pro Ser Pro Ser Thr Glu Gln Ser Ala Lys Lys Val Cys Lys Lys Ala

420 425 430

Glu Asn Ala His Asn Thr Pro Leu Leu Val Leu Tyr Gly Ser Asn Met

435 440 445

Gly Thr Ala Glu Gly Thr Ala Arg Asp Leu Ala Asp Ile Ala Met Ser

450 455 460

Lys Gly Phe Ala Pro Gln Val Ala Thr Leu Asp Ser His Ala Gly Asn

465 470 475 480

Leu Pro Arg Glu Gly Ala Val Leu Ile Val Thr Ala Ser Tyr Asn Gly

485 490 495

His Pro Pro Asp Asn Ala Lys Gln Phe Val Asp Trp Leu Asp Gln Ala

500 505 510

Ser Ala Asp GluVal Lys Gly Val Arg Tyr Ser Val Phe Gly Cys Gly

515 520 525

Asp Lys Asn Trp Ala Thr Thr Tyr Gln Lys Val Pro Ala Phe Ile Asp

530 535 540

Glu Thr Leu Ala Ala Lys Gly Ala Glu Asn Ile Ala Asp Arg Gly Glu

545 550 555 560

Ala Asp Ala Ser Asp Asp Phe Glu Gly Thr Tyr Glu Glu Trp Arg Glu

565 570 575

His Met Trp Ser Asp Val Ala Ala Tyr Phe Asn Leu Asp Ile Glu Asn

580 585 590

Ser Glu Asp Asn Lys Ser Thr Leu Ser Leu Gln Phe Val Asp Ser Ala

595 600 605

Ala Asp Met Pro Leu Ala Lys Met His Gly Ala Phe Ser Thr Asn Val

610 615 620

Val Ala Ser Lys Glu Leu Gln Gln Pro Gly Ser Ala Arg Ser Thr Arg

625 630 635 640

His Leu Glu Ile Glu Leu Pro Lys Glu Ala Ser Tyr Gln Glu Gly Asp

645 650 655

His Leu Gly Val Ile Pro Arg Asn Tyr Glu Gly Ile Val Asn Arg Val

660 665 670

Thr Ala Arg Phe Gly LeuAsp Ala Ser Gln Gln Ile Arg Leu Glu Ala

675 680 685

Glu Glu Glu Lys Leu Ala His Leu Pro Leu Ala Lys Thr Val Ser Val

690 695 700

Glu Glu Leu Leu Gln Tyr Val Glu Leu Gln Asp Pro Val Thr Arg Thr

705 710 715 720

Gln Leu Arg Ala Met Ala Ala Lys Thr Val Cys Pro Pro His Lys Val

725 730 735

Glu Leu Glu Ala Leu Leu Glu Lys Gln Ala Tyr Lys Glu Gln Val Leu

740 745 750

Ala Lys Arg Leu Thr Met Leu Glu Leu Leu Glu Lys Tyr Pro Ala Cys

755 760 765

Glu Met Lys Phe Ser Glu Phe Ile Ala Leu Leu Pro Ser Ile Arg Pro

770 775 780

Arg Tyr Tyr Ser Ile Ser Ser Ser Pro Arg Val Asp Glu Lys Gln Ala

785 790 795 800

Ser Ile Thr Val Ser Val Val Ser Gly Glu Ala Trp Ser Gly Tyr Gly

805 810 815

Glu Tyr Lys Gly Ile Ala Ser Asn Tyr Leu Ala Glu Leu Gln Glu Gly

820 825 830

Asp Thr Ile Thr Cys Phe Ile SerThr Pro Gln Ser Glu Phe Thr Leu

835 840 845

Pro Lys Asp Pro Glu Thr Pro Leu Ile Met Val Gly Pro Gly Thr Gly

850 855 860

Val Ala Pro Phe Arg Gly Phe Val Gln Ala Arg Lys Gln Leu Lys Glu

865 870 875 880

Gln Gly Gln Ser Leu Gly Glu Ala His Leu Tyr Phe Gly Cys Arg Ser

885 890 895

Pro His Glu Asp Tyr Leu Tyr Gln Glu Glu Leu Glu Asn Ala Gln Ser

900 905 910

Glu Gly Ile Ile Thr Leu His Thr Ala Phe Ser Arg Met Pro Asn Gln

915 920 925

Pro Lys Thr Tyr Val Gln His Val Met Glu Gln Asp Gly Lys Lys Leu

930 935 940

Ile Glu Leu Leu Asp Gln Gly Ala His Phe Tyr Ile Cys Gly Asp Gly

945 950 955 960

Ser Gln Met Ala Pro Ala Val Glu Ala Thr Leu Met Lys Ser Tyr Ala

965 970 975

Asp Val His Gln Val Ser Glu Ala Asp Ala Arg Leu Trp Leu Gln Gln

980 985 990

Leu Glu Glu Lys Gly Arg Tyr Ala Lys AspVal Trp Ala Gly

995 1000 1005

Claims (8)

1. A fusion protein gene comprising a gene encoding 3-sterone-9 α -hydroxylase and a gene encoding the flavin domain of cytochrome P450BM-3 enzyme,

the 3-sterone-9 α -hydroxylase is derived from Rhodococcus erythropolis (R)Rhodococcus erythropolis) SQ1, accession No. AY083508.1 of GenBank of its base sequence; the cytochrome P450BM-3 enzyme is derived from Bacillus megaterium (C.) (Bacillus megaterium) NBRC 15308 having a nucleotide sequence of GenBank accession number CP009920.1,

the base sequence of the fusion protein gene is shown as SEQ ID NO. 1.

2. An expression vector or recombinant plasmid comprising the fusion protein gene of claim 1.

3. A fusion protein encoded by the fusion protein gene of claim 1.

4. Use of the fusion protein of claim 3 to catalyze the androstenedione reaction to 9 α -hydroxy-androstenedione.

5. An engineered bacterium comprising a host cell and a fusion protein gene transferred into the host cell, wherein the fusion protein gene is according to claim 1.

6. The use of the engineered bacteria of claim 5 to catalyze androstenedione reaction to produce 9 α -hydroxy-androstenedione.

7. A method for preparing 9 α -hydroxy-androstenedione by using the engineering bacteria of claim 5, comprising the following steps:

(1) culturing engineering bacteria and inducing enzyme expression, centrifuging to take cells, and resuspending with buffer solution to obtain resting cell suspension;

(2) adding a substrate androstenedione into the resting cell suspension for reaction, and after the reaction is finished, separating and purifying the reaction solution to obtain 9 α -hydroxy-androstenedione.

8. The method of claim 7, wherein in step (1), the method for inducing expression of enzymes comprises: to OD of the culture broth₆₀₀When the temperature reaches 0.5-1.0, adding 0.01-0.5 mM IPTG, and inducing for 6-12 h at the temperature of 25-30 ℃; in the step (2), the reaction temperature is 25-30 ℃ and the reaction time is 1-24 h.

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