Cytochrome P450 monooxygenase CYP109B2 and application thereof

文档序号：183767 发布日期：2021-11-02 浏览：39次中文

阅读说明：本技术 一种细胞色素p450单加氧酶cyp109b2及其应用 (Cytochrome P450 monooxygenase CYP109B2 and application thereof ) 是由李爱涛郭瑞庭张小栋沈盼盼陈纯琪李倩赵晶邓迪于 2021-05-28 设计创作，主要内容包括：本发明公开了一种细胞色素P450单加氧酶CYP109B2及其应用,所述CYP109B2的氨基酸序列如SEQ ID NO：1所示。本发明首次从索诺拉沙漠芽孢杆菌Bacillus sonorensis中克隆获得了一个新的细胞色素P450单加氧酶CYP109B2蛋白酶基因,并以pRSFDuet-1作为表达载体,大肠杆菌BL21(DE3)作为宿主细胞实现了该蛋白酶的异源表达,发现该蛋白酶能够对甾体化合物的16β位进行高效选择性的羟基化修饰,为甾体化合物的定点羟基化修饰和甾体羟基化产物合成提供了新思路,且催化效率高、经济环保。(The invention discloses a cytochrome P450 monooxygenase CYP109B2 and application thereof, wherein the amino acid sequence of CYP109B2 is shown as SEQ ID NO:1 is shown. The invention clones and obtains a new cytochrome P450 monooxygenase CYP109B2 protease gene from Bacillus sonorensis, pRSFDuet-1 is used as an expression vector, and Escherichia coli BL21(DE3) is used as a host cell to realize heterologous expression of the protease, so that the protease can efficiently and selectively carry out hydroxylation modification on the 16 beta position of a steroid compound, a new thought is provided for site-specific hydroxylation modification of the steroid compound and synthesis of a steroid hydroxylation product, and the invention has high catalytic efficiency, economy and environmental protection.)

1. A cytochrome P450 monooxygenase CYP109B2, wherein the amino acid sequence of CYP109B2 is as shown in SEQ ID NO:1 is shown.

2. The cytochrome P450 monooxygenase CYP109B2 according to claim 1, wherein said CYP109B2 is from Bacillus sonorensis, sonorensis.

3. A gene encoding the cytochrome P450 monooxygenase CYP109B2 of claim 1, having the nucleotide sequence of SEQ ID NO:2, respectively.

4. An amplification primer for amplifying the gene as set forth in claim 3, wherein the nucleotide sequence of the amplification primer is as set forth in SEQ ID NO: 3-4.

5. A vector comprising the gene of claim 3.

6. The vector according to claim 5, further comprising a gene for a redox chaperone protein and/or a gene for a P450 monooxygenase redox domain.

7. A genetically engineered bacterium comprising the vector of claim 5.

8. Use of a cytochrome P450 monooxygenase CYP109B2 according to claim 1, or Bacillus sonorensis according to claim 2, or a vector according to claim 5, or a genetically engineered bacterium according to claim 7 for catalyzing the hydroxylation of steroids.

9. Use according to claim 8, wherein the hydroxylation modification is a 16 β hydroxylation modification of steroids.

10. Use according to claim 8 or 9, characterized in that said steroidal compound comprises: testosterone, nortestosterone, boehmeria, methyldienolone, androstenedione, adrenal ketone, 18-methyldiganone, substance 49, and ethisterone.

Technical Field

The invention belongs to the technical field of biocatalysis enzyme, and particularly relates to cytochrome P450 monooxygenase CYP109B2 and application thereof.

Background

Steroid (hormone) compounds usually have biological active compounds of basic parent nucleus skeleton of cyclopentane-polyhydrophenanthrene, and the steroid hormone is widely involved in metabolism synthesis in organism, and plays a very important regulating role in biological life activity. The steroid drugs mainly comprise adrenocortical hormone and sex hormone, are used as 'life keys' to maintain normal physiological activities, and are widely used for treating cardiovascular diseases, rheumatism, inflammation, endocrine dyscrasia, tumor and other diseases clinically. At present, steroids are second only to antibiotics in the market, with annual production over 100 million tons worldwide and annual market sales of approximately $ 1000 billion.

Chemical modification of the four rings of the steroid can endow the steroid drugs with stronger physiological and pharmacological activities, and chemical modification products of carbon atoms at different positions can also show different pharmacological activities. Compared with steroid substrates, the hydroxylation modification of the non-active carbon on the steroid parent nucleus not only can enhance the physiological and pharmacological activities of the steroid drugs by changing the polarity of the steroid drugs, but also provides a wider modification space for the further derivative modification of steroid drug intermediates. Dexamethasone (dexomethasone) for treating rheumatism, asthma and cerebral edema, hydrocortisone (hydrocortisone) with anti-inflammatory special effect, 11 alpha-hydroxyprogesterone (11 alpha-hydroxylated progetosterone) which is an important intermediate for synthesizing contraceptives, 16 alpha-hydroxylated steroids (16 alpha-hydroxylated steroids) with the function of increasing glucocorticoid activity and the like can be obtained by specific hydroxylation modification. The complexity of the steroid molecular structure determines the diversity of steroid parent nucleus functionalization, and nearly 20 or more hydroxylation sites bring great challenges to site-directed modification of steroids. The chemical synthesis of hydroxylated sterol is very complex, not only harsh synthesis conditions are required, but also the total yield of the product is low, and the processing cost brought by the separation and purification of the product seriously restricts the industrial production of steroid drugs. At present, few effective chemical methods for synthesizing specific hydroxylated sterols exist, and in only a few reports, besides the need of multi-step reactions and harsh reaction conditions, modification such as protection and deprotection of special chemical active groups is often needed, the preparation process is quite complex, the cost is high, and the toxicity of reagents is high.

The biological catalysis method can selectively introduce oxygen atoms between inert C-H bonds to realize one-step synthesis of the hydroxylated sterol, and the utilization of the biological catalysis method to synthesize the hydroxylated steroid shows great advantages of high selectivity, small environmental pollution, mild reaction conditions and the like which are not possessed by the chemical synthesis method. At present, the reaction process of the biological catalysis method for synthesizing the important medical intermediate is relatively simple, the environmental pollution is small, the cost is low, the theoretical yield of the product can reach 100 percent, the method is very in line with the sustainable development concepts of green chemistry and atom economy, and the method increasingly becomes the internationally recognized scheme with the most potential for synthesizing high value-added chemicals. Cytochrome P450 monooxygenase is used as a main body of the reported steroid hydroxylase, provides an important development resource for green synthesis of steroid drugs, can powerfully promote technical innovation of the steroid drugs by site-specific hydroxylation of a steroid substrate, is expected to realize high-efficiency synthesis of a series of hydroxylated sterols, promotes industrial production of the steroid drugs, and makes a new contribution to human health.

Disclosure of Invention

The invention aims to provide a novel cytochrome P450 monooxygenase CYP109B2 and application thereof, the novel cytochrome P450 monooxygenase CYP109B2 protease gene (Sequence ID: WP _029419899.1) is obtained by cloning from Bacillus sonorensis in Sonora desert for the first time, pRSFDuet-1 is used as an expression vector, escherichia coli BL21(DE3) is used as a host cell to realize heterologous expression of the protease, the protease is found to be capable of carrying out efficient and selective hydroxylation modification on 16 beta site of a steroid compound, a novel thought is provided for site-specific hydroxylation modification of the steroid compound and synthesis of steroid hydroxylation products, and the novel cytochrome P109B2 is high in catalytic efficiency, economic and environment-friendly.

One of the purposes of the invention is to provide a cytochrome P450 monooxygenase CYP109B2, wherein the amino acid sequence of CYP109B2 is shown in SEQ ID NO:1 is shown.

Further, the CYP109B2 is derived from Bacillus sonorensis, Bacillus sonorensis.

The second purpose of the invention is to provide a gene for coding the cytochrome P450 monooxygenase CYP109B2, wherein the nucleotide sequence of the gene is shown as SEQ ID NO:2, respectively.

The invention also aims to provide an amplification primer for amplifying the gene, wherein the nucleotide sequence of the amplification primer is shown as SEQ ID NO: 3-4.

The fourth purpose of the present invention is to provide a vector containing the above gene.

Further, the carrier also comprises a gene of a redox chaperone protein and/or a gene of a P450 monooxygenase redox domain.

Further, the redox chaperones are ferredoxin reductase and ferredoxin.

Further, the ferredoxin reductase and ferredoxin are: cytochrome P450 monooxygenase redox chaperones from Synechococcus elongatus PCC7942 or cytochrome P450 monooxygenase redox chaperones from spinach e.c. 1.18.1.2.

Further, the P450 monooxygenase redox domain is: a reductase domain of cytochrome P450 monooxygenase P450-BM3 derived from Bacillus megaterium; or a reductase domain of cytochrome P450 monooxygenase P450-RhF (CYP116B2) derived from Rhodococcus sp.

The fifth purpose of the invention is to provide a genetic engineering bacterium, which comprises the vector.

The invention also aims to provide the application of the cytochrome P450 monooxygenase CYP109B2, or the Bacillus sonorinis, or the carrier, or the genetically engineered bacterium in catalytic steroid hydroxylation modification.

Further, the hydroxylation modification is 16 beta hydroxylation modification of the steroid.

Further, the steroid compound includes: testosterone, nortestosterone, boehmeria, methyldienolone, androstenedione, adrenal ketone, 18-methyldiganone, substance 49, and ethisterone.

Compared with the prior art, the invention has the beneficial effects that: according to the invention, a steroid substrate testosterone is used as a model substrate, a strain of Bacillus sonorensis capable of selectively metabolizing the substrate testosterone is obtained by screening, a gene of cytochrome P450 monooxygenase CYP109B2 protease of the strain is obtained by cloning from the strain for the first time through a molecular biological means, the full length of the gene is 1218bp, 405 amino acids are encoded, furthermore, pRSFDuet-1 is used as an expression vector, escherichia coli BL21(DE3) is used as a host cell to realize heterologous expression of the protease, and experiments prove that the CYP109B2 protease can carry out efficient and selective hydroxylation modification on 16 beta site of a steroid compound. The invention provides a brand-new cytochrome P450 monooxygenase and verifies the function of the cytochrome P450 monooxygenase on the hydroxylation modification of the steroid, which provides a new thought for the site-specific hydroxylation modification of the steroid and the synthesis of steroid hydroxylation products, has high catalytic efficiency, is economic and environment-friendly, provides more resources for the application of the P450 monooxygenase in the synthetic biology, promotes the industrial production of steroid medicines, and makes a new contribution to the healthy development of human beings.

Drawings

FIG. 1 is a bacterial liquid map of Bacillus sonorensis of Bacillus somnophilus in example 1 of the present invention after culture;

FIG. 2 shows the results of gene detection of CYP109B2 amplified in example 1 of the present invention;

FIG. 3 is a phylogenetic tree of cytochrome P450 monooxygenase CYP109B2 in example 1 of the present invention;

FIG. 4 shows the sequence conservation of cytochrome P450 monooxygenase CYP109B2 in example 1 of the present invention;

FIG. 5 shows the alignment of the homologous sequences of cytochrome P450 monooxygenase CYP109B2 in example 1 of the present invention;

FIG. 6 shows SDS-PAGE of recombinant E.coli (pRSFDuet-1-CYP109B2) heterologously expressed CYP109B2 in example 3 of the present invention;

FIG. 7 shows the results of HPLC analysis of the cytochrome P450 monooxygenase CYP109B2 converting testosterone in example 4 of the present invention;

FIG. 8 shows the HPLC analysis results of the cytochrome P450 monooxygenase CYP109B2 converting steroid substrates testosterone, nortestosterone, androstenedione and adrenal ketone in example 4 of the present invention;

FIG. 9 shows the HPLC analysis and detection results of cytochrome P450 monooxygenase CYP109B2 converting steroid substrates boehmeria, methyldienolone, 18-methyldigolone, 49 and ethisterone in example 4 of the present invention;

FIG. 10 shows the catalytic activity of cytochrome P450 monooxygenase CYP109B2 in example 4 of the present invention for the conversion of various steroids and the results of the measurement of the catalytic products.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 cloning of cytochrome P450 monooxygenase CYP109B2 Gene

1. Culture of Bacillus sonorensis in Sonola desert

Selecting Bacillus Sonorensis (Bacillus sonorensis) bacterial liquid from glycerol tube, streaking and activating on sea water 2216 agar plate culture medium, and inversely culturing at 30 deg.C in constant temperature incubator for 48 h. After single colonies grow on the plate, the single colonies are selected and inoculated in 5mL of non-resistant LB medium, and the shaking culture is carried out at 30 ℃ and 220rpm for 48 h. The whole cultured cells was pale white as shown in FIG. 1. Bacterial body OD₆₀₀About 2.5.

In order to confirm whether the bacteria have the capability of transforming steroid compounds, testosterone is used as a model substrate, and the Bacillus sonorensis is used for carrying out whole-cell reaction detection on the testosterone, which specifically comprises the following steps: after the bacterial culture, the cells were collected by centrifugation at 4000rpm for 10min, resuspended in pH 8.0 potassium phosphate buffer, and OD was adjusted₆₀₀To the bacterial suspension, Glucose (Glucose) was added at a final concentration of 5%, Glycerol (Glycerol) at a final concentration of 5%, Glucose Dehydrogenase (GDH) at a final concentration of 1U, and NADP at a final concentration of 1mM at 20⁺And testosterone dissolved in Dimethylformamide (DMF) at a concentration of 1mM, the biotransformation of testosterone was carried out at 25 ℃ in a shaking table at 200rpm for 9 hours, and samples were taken after the reaction for 9 hours. The reaction solution was sampled and added with an equal volume (500. mu.L) of acetonitrile, centrifuged at 1200rpm for 1min, and subjected to membrane treatment for High Performance Liquid Chromatography (HPLC) analysis and detection. The results of the assay showed that a peak of the liquid phase other than the substrate testosterone was detected in the reacted sample, while no peak of the liquid phase of the substrate testosterone was detected, indicating that an enzyme catalyzing the conversion of testosterone is present in the bacterium and that 1mM testosterone is completely converted within 30 h.

2. Bacillus sonorensis genome information and CYP109B2 gene cloning

The cultured bacterial solution was centrifuged at 12000rpm for 2min to collect the cells, and the genome was extracted according to the instructions of a commercial genome extraction kit. And identifying the extracted genome by running nucleic acid detection gel, and storing at-25 ℃ for later use after the genome is verified to be correct.

Inquiring related genome information in an NCBI (national center for information service) database according to the strain attribute of Bacillus sonorensis, designing a specific primer according to the sequence information of a genome, and cloning from the genome by adopting a molecular cloning technology to obtain a novel P450 monooxygenase gene, which comprises the following specific steps:

taking Bacillus sonorensis genome as a template, and the primers are as follows:

CYP109B 2-F: ATGAACTCGGCAAAACAGCAGAAC (shown in SEQ ID NO: 3)

CYP109B 2-R: TCATGATGAAAGCAGCGCCTCTTTG (shown in SEQ ID NO: 4)

PCR System (50. mu.L): mu.L of genomic template, 1. mu.L each of primers (10. mu.M), 25. mu.L of Prime STAR Max DNA polymerase, sterile distilled water to 50. mu.L.

The PCR reaction program is: (1) denaturation at 98 deg.C for 3 min; (2) denaturation at 98 ℃ for 10sec, (3) annealing at 55 ℃ for 15sec, (4) extension at 72 ℃ for 20sec, and steps (2) - (4) are performed for 30 cycles in total, and finally extension at 72 ℃ for 5min, and the product is stored at 12 ℃.

And detecting the PCR product obtained by cloning and amplifying by using 0.8% agarose gel nucleic acid electrophoresis, observing a band (shown in figure 2) with the size of 1000-1500 under an ultraviolet nucleic acid imager, wherein the theoretical size of the target band is 1218bp, and the size of the actually obtained target gene is consistent with the theory, thereby indicating that the target gene is obtained by cloning. The obtained PCR product is further delivered to a company for sequencing, and the result shows that the nucleotide sequence and the amino acid sequence of the PCR product obtained by cloning are consistent with those in a database, which indicates that the target gene is accurately cloned, wherein the nucleotide sequence of the gene is shown as SEQ ID NO. 2, and the amino acid sequence is shown as SEQ ID NO. 1.

The NCBI is used for analyzing the homologous sequence of the novel P450 monooxygenase, and the biological sequence analysis software MEGA 6.0 is used for constructing a phylogenetic tree of the novel P450 monooxygenase on the basis of the homologous sequence, the result is shown in figure 3, and the result shows that the homology of the novel P450 monooxygenase and the reported CYP109B1 amino acid sequence of the CYP109 family reaches 61.9 percent, so that the novel P450 monooxygenase is confirmed to be a member of the cytochrome monooxygenase CYP109B subfamily, and the protease is named as CYP109B 2.

Further analyzing the enzymatic structural characteristics of CYP109B2, firstly analyzing the conservation of CYP109B2 by bioinformatics means, and the results are shown in FIG. 4; sequence analysis is carried out on CYP109B2 by combining MEGA 6.0, online Weblog and online ESPript 3.0, differences between CYP109B2 and homologous enzyme gene sequences are explored, and the result of homologous sequence alignment is shown in FIG. 5.

Example 2 acquisition of the Redox chaperone protease Gene or the P450 monooxygenase Redox Domain

Cytochrome P450 monooxygenases require the redox chaperone protease or redox domain for electron transfer in a catalytic process, and several methods for obtaining the redox chaperone protein and the redox domain of the P450 monooxygenase are illustrated in this example.

(1) Cytochrome P450 monooxygenase redox chaperone protease from Synechococcus elongatus PCC7942, specifically: ferredoxin reductase synpc 7942_0978(Fdr _0978) and ferredoxin synpc 7942_1499(Fdx _ 1499).

Respectively taking pET-28a-Fdr _0978 and pET-28a-Fdx _1499 plasmids as templates, and respectively taking primers:

Fdr_0978-F:ATGTTGAATGCGAGTGTGGCTG(SEQ ID NO:5)

Fdr_0978-R:CTAGTAGGTTTCAACATGCCAACGACC(SEQ ID NO:6)；

Fdx_1499-F:ATGGCAACCTACAAGGTTACGCT(SEQ ID NO:7)

Fdx_1499-R:CTAGTAGAGGTCTTCTTCTTTGTGGGTTTCG(SEQ ID NO:8)

PCR System (10. mu.L): 0.1-4 ng of template, 0.5. mu.L (10. mu.M) of each primer, 5. mu.L of Prime STAR Max DNA polymerase, and 10. mu.L of sterilized distilled water were added to perform conventional PCR amplification.

(2) Cytochrome P450 monooxygenase redox partner protease derived from spinach E.C.1.18.1.2, specifically: ferredoxin reductase FNR and ferredoxin Fd I. The gene amplification method is as above, and the amplification primers comprise:

FNR-F:ATGCAGATCGCCTCTGATGTGG

FNR-R:TTAGTAGACTTCAACGTTCCATTGTTCTGCC；

Fd I-F:ATGGCCGCCTACAAGGTGAC

Fd I-R:TTAGGCGGTCAGCTCCTCTTCTT。

(3) the redox domain of cytochrome P450 monooxygenase P450-BM3(CYP102A1) from Bacillus megaterium. The gene amplification method is as above, and the amplification primers comprise:

(CYP109B2)-R_BM3-F:

GCGCTGCTTTCATCACCTTCACCTAGCACTGAACAGTCTG

(CYP109B2)-R_BM3-R:

GCATTATGCGGCCGCTTACCCAGCCCACACGTCTTTTGC。

(4) a redox domain of cytochrome P450 monooxygenase P450-RhF (CYP116B2) derived from Rhodococcus sp. The gene amplification method is as above, and the amplification primers comprise:

(CYP109B2)-R_RhF-F:GCGCTGCTTTCATCAGTGCTGCACCGGCATCAACC

(CYP109B2)-R_RhF-R:GCATTATGCGGCCGCTCAGAGTCGCAGGGCCAGC。

example 3 construction of recombinant plasmid pRSFDuet-1-CYP109B 2-Fdr-0978-Fdx-1499

After the CYP109B2 gene was successfully obtained, it was confirmed by bioinformatics analysis that CYP109B2 belongs to cytochrome P450 monooxygenase of type I. In order to allow CYP109B2 to exert its catalytic activity in Escherichia coli, a suitable redox partner CYP109B2 was selected, wherein CYP109B2 exhibited excellent catalytic activity with the aid of ferredoxin reductase Fdr _0978 and ferredoxin Fdx _1499, thereby constructing a recombinant plasmid.

1. Construction of CYP109B2 recombinant plasmid (pRSFDuet-1-CYP109B2)

(1) Amplification of the Linear vector fragment pRSFDuer-1:

template: plasmid pRSFDuet-1, primers:

pRSFDute-1-F1:GCGGCCGCATAATGCTTAAG(SEQ ID NO:9)

pRSFDute-1-R1:AAGCTTGTCGACCTGCAGGC(SEQ ID NO:10)

PCR System (50. mu.L): 0.5-20 ng of template, 1. mu.L (10. mu.M) of each primer, 25. mu.L of Prime STAR Max DNA polymerase, and 50. mu.L of sterilized distilled water.

The PCR reaction program is: (1) denaturation at 98 deg.C for 3 min; (2) denaturation at 98 ℃ for 10sec, (3) annealing at 55 ℃ for 15sec, (4) extension at 72 ℃ for 40sec, 30 cycles in total of steps (2) - (4), final extension at 72 ℃ for 5min, and storage at 12 ℃.

(2) Amplification of CYP109B2 Gene

Template: amplified CYP109B2 gene fragment

Primer:

(pRSFDuet)-CYP109B2-F:

CAGGTCGACAAGCTTATGAACTCGGCAAAACAGCAGAAC(SEQ ID NO:11)(pRSFDuet)-CYP109B2-R:

GCATTATGCGGCCGCTTACGCCTGGAACGATAAAGATGCCTC(SEQ ID NO:12)

the PCR amplification system and the reaction procedure are the same as those in step (1).

(3) And detecting the PCR product obtained by cloning and amplifying by using 0.8% agarose gel nucleic acid electrophoresis, and observing whether the size of the band is correct or not under an ultraviolet nucleic acid imager. The remaining template in the PCR product was digested with Dpn I after the detection was correct, and the system (50. mu.L) was: mu.L of CutSmart Buffer, 2. mu.L of Dpn I, and 43. mu.L of PCR product. Digestion was carried out at 37 ℃ for 5h, followed by inactivation at 80 ℃ for 15 min. Gel recovery was performed using 1.5% agarose gel nucleic acid electrophoresis and OMEGA recovery kit.

(4) The target gene fragment CYP109B2 and the linear vector fragment pRSFDuer-1 form a 15bp or 20bp sticky end under the action of T5 exonuclease so as to be connected together, and the specific method comprises the following steps: adding the target fragment CYP109B2 and the linearized vector pRSFDuet-1 (controlling the amount of the linearized vector to be 30-50 ng) into a 5-mu-L reaction system according to the molar ratio of 3:1, adding T5 exonuclease and buffer 4.0, and supplementing water to less than 5 mu L. Timing for 5min after adding T5 exonuclease, adding 50 mu L DH5 alpha competent cells immediately after the time is up, transforming according to the basic steps of conventional transformation, adding culture medium for 1h for resuscitation, transferring to LB solid culture medium (peptone 10g/L, yeast powder 5g/L, NaCl 10g/L, 15g/L agar powder) containing corresponding resistant Kan (50 mu g/mL) for overnight culture, and taking the corresponding transformant to a sequencing company for DNA sequencing to finally obtain the correct recombinant pRSFDuet-1-CYP109B 2.

(5) The recombinant pRSFDuet-1-CYP109B2 was transformed into E.coli BL21(DE3) and cultured in LB solid medium containing resistant Kan (50. mu.g/mL) to obtain recombinant E.coli (pRSFDuet-1-CYP109B2) which alone expressed cytochrome CYP109B2 monooxygenase, and the recombinant E.coli (pRSFDuet-1-CYP109B2) was allowed to heterologously express CYP109B2, as shown in FIG. 6 by SDS-PAGE. The results show that there is a relatively bright protein band around 45kd, which is consistent with the actual protein size of 44.5kd for CYP109B2, indicating that CYP109B2 is expressed heterologously in E.coli.

2. Construction of recombinant plasmid for redox chaperone (pETDuet-1-Fdr-0978-Fdx-1499)

(1) Amplification of the linear vector fragment pETDuet-1:

template: plasmid pETDuet-1, primers:

pETDuet-F:GCCTGCAGGTCGACAAGCTT(SEQ ID NO:13)

pETDuet-R:GCGCCGAGCTCGAATTCG(SEQ ID NO:14)

the PCR amplification system and reaction procedure were as above.

(2) Fdr _0978 and Fdx _1499 Gene amplification

Template: fdr _0978 target gene fragment or Fdx _1499 target gene fragment; primer:

(pETDuet)-Fdr_0978-F:CGAATTCGAGCTCGGCGCATGTTGAATGCGAGTGTGGCTG(SEQ ID NO:15)

Fdr_0978-(rbs)-R:GATATATCTCCTTAGGTACCCTAGTAGGTTTCAACATGCCAACGACC(SEQ ID NO:16)；

Fdx_1499-(rbs)-F:GGTACCTAAGGAGATATATCATGGCAACCTACAAGGTTACGCT(SEQ ID NO:17)

(pETDuet)-Fdx_1499-R:GCTTGTCGACCTGCAGGCGTAGAGGTCTTCTTCTTTGTGGGTTTCG(SEQ ID NO:18)

the PCR amplification system and reaction procedure were as above.

(3) The linearized vector and the target gene fragment Fdr _0978-rbs-Fdx _1499 and the linearized vector fragment pETDuet-1 are linked together by forming a 15bp or 20bp cohesive end under the action of T5 exonuclease. The specific method is the same as the construction of a recombinant pRSFDuet-1-CYP109B2, wherein the molar ratio of target fragments Fdr _0978, Fdx _1499 to a linearization vector pETDuet-1 is 3:3: 1. Finally, the correct recombinant pETDuet-1-Fdr _0978-Fdx _1499 is obtained.

3. Construction of recombinant plasmid pRSFDuet-1-CYP109B2-Fdr _0978-Fdx _1499

(1) Template: plasmid pRSFDuet-1-CYP109B 2; primer:

pRSFDute-1-F2:GGCCGGCCACGCGATCGCT(SEQ ID NO:19)

pRSFDute-1-R2:GATATCCAATTGAGATCTGCCATATGTATATCTCCT(SEQ ID NO:20)

the linearized vector pRSFDuet-1-CYP109B2 was obtained by conventional PCR amplification.

(2) Fdr _0978-rbs-Fdx _1499 Gene amplification

Template: plasmid pETDuet-1-Fdr _0978-Fdx _1499

Primer:

(pRSFDuet)-Fdr_0978-F1:TCTCAATTGGATATCATGTTGAATGCGAGTGTGGCTG(SEQ ID NO:21)

(pRSFDuet)-Fdx_1499-R1:ATCGCGTGGCCGGCCCTAGTAGAGGTCTTCTTCTTTGTGGGTTTCG(SEQ ID NO:22)

obtaining the target fragment Fdr _0978-rbs-Fdx _1499 by conventional PCR amplification

(3) The linearized vector and the gene fragment amplified by PCR are connected together by forming a 15bp or 20bp cohesive end under the action of T5 exonuclease. The specific method is the same as the construction of the recombinant pRSFDuet-1-CYP109B2, wherein the molar ratio of the target fragment Fdr-0978-rbs-Fdx-1499 to the linearized vector pRSFDuet-1-CYP109B2 is 3: 1. Obtaining correct recombinant plasmid after sequencing: pFSFDuet-1-CYP109B2-Fdr _0978-Fdx _ 1499.

4. Protein expression recombinant cell construction

Two recombinant plasmids pRSFDuet-1-CYP109B2 and pETdet-1-Fdr _0978-Fdx _1499 can be simultaneously transformed into BL21(DE3) and cultured to obtain recombinant cells E.coli (pRSFDuet-1-CYP109B 2/pETdet-1-Fdr _0978-Fdx _1499) which can simultaneously express cytochrome monooxygenase CYP109B2, ferredoxin reductase Fdr _0978 and ferredoxin Fdx _ 1499.

The recombinant plasmid pFSFDDuet-1-CYP 109B2-Fdr _0978-Fdx _1499 can also be transformed into BL21(DE3) and cultured in LB solid medium containing resistant Kan (50. mu.g/mL) to obtain recombinant cell E.coli (pFSSFDuet-1-CYP 109B2-Fdr _0978-Fdx _1499) capable of simultaneously expressing cytochrome monooxygenase CYP109B2, ferredoxin reductase Fdr _0978 and ferredoxin Fdx _ 1499.

Example 4 protein expression and functional verification

Coli (pFSFDuet-1-CYP109B 2-Fdr-0978-Fdx-1499) was used in the subsequent biotransformation studies, as the recombinant cell constructed in example 3.

Coli (pRSFDuet-1-CYP109B2-Fdr _0978_ Fdx _1499) in 3mL of LB liquid medium containing 50. mu.g/mL kanamycin at 37 ℃ for about 6 to 8 hours, transferred at 2% inoculum size to 50mL of TB medium containing 50. mu.g/mL kanamycin at 37 ℃ for about 2 to 3 hours at 220rpm until OD reached₆₀₀Adding isopropyl-beta-D-thiogalactopyranoside (IPTG) with the final concentration of 0.2mM into the bacterial liquid in an ultra-clean bench, and carrying out induction culture for 14-16h at the temperature of 25 ℃ and the rpm of 200. Centrifuging the induced thallus at 15 deg.C and 4000rpm for 10min to collect thallus, washing thallus twice with 100mM potassium phosphate buffer solution with pH 8.0, and re-suspending and adjusting OD₆₀₀20. And (3) quickly freezing the cells by using liquid nitrogen (enhancing the permeability of cell membranes and promoting substrates to enter the cells), and freezing and thawing at room temperature for sterol conversion.

4mL of the suspension was placed in a 50mL reaction flask, and Glucose (Glucose) at a final concentration of 5%, Glycerol (Glycerol) at a final concentration of 5%, Glucose Dehydrogenase (GDH) at a final concentration of 1U, and NADP at a final concentration of 1mM were added⁺And 1mM testosterone, a sterol dissolved in Dimethylformamide (DMF), were subjected to biotransformation of testosterone in a shaking shaker at 25 ℃ and 200rpm for 9h, and sampled for detection at reaction times of 1h, 5h and 9h, respectively. Adding equal volume (500 μ L) of ethyl acetate into the sampled reaction solution for extraction, centrifuging at 1200rpm for 1min, taking the upper layer of organic phase, adding 500 μ L of acetonitrile after the ethyl acetate is volatilized, carrying out membrane treatment, and carrying out High Performance Liquid Chromatography (HPLC) analysis and detection, wherein the detection result is shown in FIG. 7.

The result shows that the reaction is carried out for 9h, the testosterone substrate is completely reacted, the HPLC chromatogram has no detected substrate peak pattern, and four peaks different from the substrate appear, which indicates that CYP109B2 can efficiently catalyze the conversion of the testosterone substrate.

And (3) separation, purification and identification of reaction products: large scale preparative reactions were carried out according to the above system, samples were taken after biotransformation was complete, the product was extracted with an equal volume (50mL) of ethyl acetate, and the organic phase was dried overnight with anhydrous sodium sulfate. The ethyl acetate was removed by evaporation using a rotary evaporator at 40 deg.C, 100rpm and-0.1 Kpa and the reaction product was concentrated. The substrate standard and the reaction product were analyzed simultaneously with a 25X 75mm TLC silica gel plate under methanol and methylene chloride as developing agents, and the conditions for separating the substrate and the product were investigated. The separating developer conditions determined were methanol: 1-dichloromethane: 15. before filling the purified silica gel column (the pre-packed column is ensured to be clean and dry), firstly, a developing agent is used for rinsing the pre-packed column, then, silica gel which is uniformly mixed by the developing agent is added into the pre-packed column, and the outer wall of the column is knocked by rubber while adding, so that the silica gel column is ensured to be uniformly filled, compact and free of bubbles. And stopping adding the silica gel when the silica gel plane reaches 4/5 of the pre-packed column, adding a developing agent with the volume being 3 times that of the column into the silica gel column, further compacting the silica gel column, finally discharging the redundant developing agent in the silica gel column until the liquid level of the developing agent is tangent to the silica gel plane, and closing a silica gel column piston. And (3) loading the silica gel column on a wet column, carefully and slowly adding the crude product along the wall of a pre-loaded column tube, and adding proper anhydrous sodium sulfate to the upper-layer product for covering so as to ensure that the silica gel column is in an anhydrous condition. And then slowly adding a developing solvent along the wall of the silica gel column, opening a piston at the lower end of the silica gel column, collecting effluent liquid by using a small test tube, sampling in time, and detecting the product distribution condition of each test tube by using a TLC silica gel plate. And collecting effluent containing only the target product, and performing concentration crystallization by using a rotary evaporator to prepare the product. And (3) separating the obtained product by nuclear magnetic analysis, and determining the molecular structure of the reaction product by a nuclear magnetic carbon spectrum, a nuclear magnetic hydrogen spectrum and a substrate structure.

Further, kinetic parameters of the reaction of CYP109B2 with testosterone were determined, specifically: respectively constructing plasmids containing CYP109B2, Fdr _0978 and Fdx _1499, expressing, purifying and determining protein concentration, preparing a protein mixed solution from the three proteins according to the concentration ratio of 1:4:20, and adding MgCl with the final concentration of 1mM into the protein mixed solution₂Glucose and glycerol with the final concentration of 5 percent and Glucose Dehydrogenase (GDH) with the final concentration of 1U are fully and uniformly mixed and then are respectively filled into 30 EP tubes with the concentration of 1.5mL, testosterone substrates diluted in dimethyl sulfoxide (DMSO) with the concentration gradient of 25-1000 mu M are respectively added into the 30 EP tubes, three parallel samples are arranged in each gradient, and the glucose and glycerol with the final concentration of 5 percent and the testosterone substrate with the concentration gradient of 700rpm are fully and uniformly mixed on a vibration metal bath at the temperature of 30 ℃ and 700rpmHomogenizing for 2 min. Add NADP to each EP tube to a final concentration of 1mM⁺The reaction was initiated by controlling the total reaction system to 200. mu.L (made up with potassium phosphate buffer), mixing well for 5min at 700rpm with shaking at 30 ℃ on a metal bath, and then rapidly adding an equal volume (200. mu.L) of acetonitrile to each EP tube to terminate the reaction. Centrifuging at 1200rpm for 2min, filtering with 0.22 μm filter membrane, analyzing by HPLC, detecting substrate reaction, and determining kinetic parameters of CYP109B2 according to Mie's equation by calculating the relationship between protease reaction rate and substrate concentration, wherein K is_mThe value is 0.21. + -. 0.02mM, k_catThe value was 3.93. + -. 0.15min^-1Through k_cat/k_mThe catalytic activity is measured to be 1.9 multiplied by 10⁴M^-1min^-1。

Then, the catalytic ability of CYP109B2 on other steroids is verified, 14 steroids such as testosterone, nortestosterone, boehmeria, progesterone, methyldienolone, androstenedione, adrenal ketone, 18-methyldigenone, 49 substance, ethisterone, canrenone, prednisolone, pregnenolone, estradiol and the like are used as substrates, the biotransformation of CYP109B2 on other steroids is verified according to the above method, the catalytic selectivity is determined by high performance liquid chromatography, the system is amplified, the hydroxylation product of the steroid substrate is separated and purified by silica gel column chromatography, the product preparation yield is calculated, the molecular structure is further verified by mass spectrometry and nuclear magnetism, and the result is shown in fig. 8-10, and the asterisk in fig. 8 and fig. 9 is the peak position corresponding to the steroid substrate. The results show that CYP109B2 has a high efficiency 16 β hydroxylation on partial steroids including testosterone, nortestosterone, boehmeria, and methyldialkenolone, with selectivity for 16 β hydroxylation of testosterone of 83%, conversion > 99%, yield 63%; the selectivity of nortestosterone 16 beta hydroxylation is 92%, the conversion rate is about 75%, and the yield is 36%; the selectivity to the 16 beta hydroxylation of the earthworm is 86%, the conversion rate is more than 99%, and the yield is 79%; the selectivity to the 16 β hydroxylation of methyl diketene was 71%, the conversion was > 99%, and the yield was 62%. And CYP109B2 also has relatively high catalytic action on androstenedione, adrenal ketone, 18-methyl diketone, 49 substances and ethisterone steroid substrates, wherein the conversion rate on androstenedione, 18-methyl diketone and 49 substances is more than 99%, and the conversion rate on adrenal ketone and ethisterone is more than 50%. However, CYP109B2 did not exhibit catalytic activity on some steroid substrates with relatively large side chains, such as progesterone, canrenone, prednisolone, pregnenolone, and estradiol, indicating that the steroid substrates with large side chains are difficult to enter the active pocket of CYP109B2 protease.

Example 5 Experimental procedures

1. Purification and concentration determination of CYP109B2, Fdr _0978 and Fdx _1499 proteins

(1) Purification of CYP109B2, Fdr _0978, and Fdx _1499 proteins can be obtained by methods conventional in the art. The prepared purified target protein pure enzyme solution is quickly frozen by liquid nitrogen and then is stored at-80 ℃ for later use. After thawing the pure enzyme solution taken at-80 ℃ on ice, the protease concentration of CYP109B2 was determined by CO differential spectroscopy. Determination of the concentration of Fdr _0978 and Fdx _1499 proteins can be determined using a Bradford protein concentration assay kit according to methods routine in the art.

(2) 50 μ L of CYP109B2 purified enzyme solution was diluted to 2mL with 100mM potassium phosphate buffer pH 8.0, CO was bubbled at 1 bubble per second for one minute, and 1mL was dispensed into another cuvette using a pipette. And adding 20mg of sodium hydrosulfite into one cuvette, turning upside down and uniformly mixing, and immediately scanning the wavelength by using a spectrophotometer, wherein the wavelength range is set to be 400-500 nm and 2 nm/point. The group without sodium dithionite added was used as a blank and the other groups were used as experimental groups for determining the actual P450 content, and the changes in absorbance at a wavelength of 450nm and at a wavelength of 490nm were recorded as compared with the blank. Molar extinction coefficient value of 0.091M^-1·cm^-1The concentration of P450 protein was calculated according to the following formula:

p450 protein concentration (μmol) ═ a₄₅₀-ΔA₄₉₀) Dilution factor/0.091M^-1·cm^-1

2. HPLC analysis

To detect the reaction of steroids, the samples were analyzed using a SHIMADZU LC2030C system equipped with a FAD detector and an Agilent Zorbax eclipse XDB-C18 column (4.6X 250mm,5 μm; Agilent Technologies, Santa Clara, Calif., USA) chromatography column. The flow rate is 1.5mL/min, the column temperature is 40 ℃, and the sample injection amount is 10 mu L. The analytical procedure used gradient elution, the elution procedure is shown in the following table:

TABLE 1 steroid substrates liquid phase detection gradient elution procedure

Note: the mobile phase A is ultrapure water, the mobile phase B is chromatographic grade methanol, and the mobile phase C is chromatographic grade acetonitrile.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention.

Sequence listing

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