Method for synthesizing 2 α -hydroxylated steroid compound by using cytochrome P450 enzyme
阅读说明：本技术 一种利用细胞色素P450酶合成2α-羟基化类固醇化合物的方法 (Method for synthesizing 2 α -hydroxylated steroid compound by using cytochrome P450 enzyme ) 是由 许莲花 王倩文 马炳炳 于 2019-11-15 设计创作，主要内容包括：本发明公开了一种利用细胞色素P450酶合成2-羟基化类固醇化合物的方法,属于生物技术领域,该方法首先构建了来自阿维链霉菌的CYP154C2基因与还原伴侣RhFRED基因共表达载体,在大肠杆菌中表达；经过培养后,利用静息细胞法喂养底物睾酮、雄烯二酮进行生物转化,获得2α位羟基化产物。此反应具有严格的区域选择性和立体选择性,产生唯一产物,使产物的分离纯化更加快速、效率。(The invention discloses a method for synthesizing a 2-hydroxylated steroid compound by cytochrome P450 enzyme, which belongs to the technical field of biology, and the method comprises the steps of firstly constructing a co-expression vector of CYP154C2 gene from streptomyces avermitilis and a reduction partner RhFRED gene, expressing the co-expression vector in escherichia coli, culturing, and then feeding substrates testosterone and androstenedione by a resting cell method for biotransformation to obtain a 2 α site hydroxylated product.)
1. A method for synthesizing a 2-hydroxylated steroid compound using a cytochrome P450 enzyme, comprising the steps of:
(1) preparation of co-expressed recombinant E.coli: utilizing seamless cloning technology to link pET28b-CYP154C2 and RhFRED gene together to obtain recombinant plasmid pET28b-CYP154C2-RhFRED, transferring the plasmid intoEscherichia coli BL21 (DE)3) And constructing recombinant Escherichia coli. The gene sequence of pET28b-CYP154C2 is shown in SEQ ID NO.1, the gene sequence of pET28b-CYP154C2-RhFRED is shown in SEQ ID NO.2, wherein the gene sequence of CYP154C2 is shown in SEQ ID NO.3, and the gene sequence of RhFRED is shown in SEQ ID NO. 4.
(2) Pre-culture and scale-up culture of recombinant E.coli: and (3) adding the recombinant escherichia coli constructed in the step (1) into an LB culture medium, and culturing for 16 hours at 37 ℃ to obtain a seed solution of the recombinant escherichia coli. Inoculating the seed solution into an M9 culture medium, wherein the volume ratio of the seed solution to the M9 culture medium is 1:100, and performing amplification culture at 37 ℃ until bacterial liquid OD600And (3) when the value reaches 0.8, adding IPTG and 5-aminolevulinic acid salt to induce the bacteria to express protein, reducing the temperature to 20 ℃, continuously culturing for 20-24h, and centrifugally collecting the bacteria. The concentration of IPTG in M9 medium was 0.2mM and the concentration of 5-aminolevulinic acid salt in M9 medium was 0.5 mM.
(3) Biotransformation synthesis of 2-hydroxylated steroid compounds: suspending the collected thallus in sodium phosphate buffer solution, keeping pH at 7.2, adding substrate for biotransformation, oscillating at 30 deg.C for 16h, extracting and separating the fermentation broth to obtain 2-hydroxylated steroid compound. The concentration of the substrate in the sodium phosphate buffer solution is 100 mu M, and the substrate is testosterone or androstenedione.
2. The method of claim 1, wherein the sodium phosphate buffer comprises EDTA at a final concentration of 1mM, dithiothreitol at a final concentration of 2mM, and glycerol in a volume of 10% of the sodium phosphate buffer.
3. The method of claim 1, wherein in step 3 the substrate is testosterone and the 2-hydroxylated steroid compound obtained by biotransformation is 2 α -hydroxytestosterone.
4. The method of claim 1, wherein the substrate in step 3 is androstenedione and the 2-hydroxylated steroid compound obtained by biotransformation is 2 α -hydroxyandrostenedione.
Technical Field
The invention belongs to the technical field of biology, and relates to a method for synthesizing a 2 α -hydroxylated steroid compound by using cytochrome P450 enzyme, in particular to a method for obtaining 2 α hydroxytestosterone and 2 α hydroxyandrostenedione by carrying out biotransformation by using a co-expression recombinant bacterium of novel cytochrome P450 enzyme CYP154C2 and a reduction partner RhFRED.
Background
Cytochrome P450 enzymes are a class of thiol-heme-containing terminal monooxygenases that are widely found in animals, plants, and microorganisms. The P450 enzyme can play a role in efficiently catalyzing compounds with complex structures under mild conditions, and researches show that the substrate structure types recognized by the P450 enzyme comprise fatty acids, terpenes, polypeptides, macrolides, alkaloids, steroids, aromatic compounds and the like, and the catalytic reaction types comprise hydroxylation, epoxidation, dealkylation, deamination, desulfurization and the like. In view of the high catalytic activity of P450 enzyme and the diversity of substrate selection, more and more P450 enzymes are applied to the pharmaceutical field as biocatalysts, the application of synthetic drugs and drug intermediates is an important direction of cytochrome P450 enzyme industrialization, and the industrial success cases of drug intermediates such as artemisinin, pravastatin and the like and drugs synthesized by P450 enzyme catalysis have been existed.
The steroid compound takes cyclopentanoperhydrophenanthrene as a mother nucleus, consists of three six-membered rings and one five-membered ring, which are respectively called A, B, C and D rings, has unique physiological functions in the aspects of biological metabolism, reproductive development, immunoregulation, cancer treatment and the like, and plays an important role in the field of medicine.
Disclosure of Invention
The invention aims to disclose a method for synthesizing a 2 α -hydroxylated steroid compound by using cytochrome P450 enzyme, and the aim of the invention is realized by the following technical scheme that 1, the method for synthesizing the 2-hydroxylated steroid compound by using the cytochrome P450 enzyme comprises the following steps:
(1) preparation of co-expressed recombinant E.coli: the pET28b-CYP154C2 and the RhFRED gene are connected together by utilizing a seamless cloning technology to obtain a recombinant plasmid pET28b-CYP154C2-RhFRED, and the plasmid is transferred into escherichia coli BL21 (DE)3) And constructing recombinant Escherichia coli. The gene sequence of pET28b-CYP154C2 is shown in SEQ ID NO.1, the gene sequence of pET28b-CYP154C2-RhFRED is shown in SEQ ID NO.2, wherein the gene sequence of CYP154C2 is shown in SEQ ID NO.3, and the gene sequence of RhFRED is shown in SEQ ID NO. 4.
(2) Recombinant large intestinePre-and scale-up of bacilli: and (3) adding the recombinant escherichia coli constructed in the step (1) into an LB culture medium, and culturing for 16 hours at 37 ℃ to obtain a seed solution of the recombinant escherichia coli. Inoculating the seed solution into an M9 culture medium, wherein the volume ratio of the seed solution to the M9 culture medium is 1:100, and performing amplification culture at 37 ℃ until bacterial liquid OD600And (3) when the value reaches 0.8, adding IPTG and 5-aminolevulinic acid salt to induce the bacteria to express protein, reducing the temperature to 20 ℃, continuously culturing for 20-24h, and centrifugally collecting the bacteria. The concentration of IPTG in M9 medium was 0.2mM and the concentration of 5-aminolevulinic acid salt in M9 medium was 0.5 mM.
(3) Biotransformation synthesis of 2-hydroxylated steroid compounds: suspending the collected thallus in sodium phosphate buffer solution, keeping pH at 7.2, adding substrate for biotransformation, oscillating at 30 deg.C for 16h, extracting and separating the fermentation broth to obtain 2-hydroxylated steroid compound. The concentration of the substrate in the sodium phosphate buffer solution is 100 mu M, and the substrate is testosterone or androstenedione.
Further, the sodium phosphate buffer solution consists of EDTA with a final concentration of 1mM, dithiothreitol with a final concentration of 2mM and glycerol, and the volume of the glycerol is 10% of that of the sodium phosphate buffer solution.
Further, in
Further, in the
① CYP154C2 is a new enzyme not disclosed, its recombinant bacteria can convert testosterone and androstenedione to produce 2 α -position hydroxylation product, which can only be obtained from chemical synthesis method at present, ② CYP154C2 enzyme has strict regioselectivity and stereoselectivity to steroid compounds, and can produce the only product, i.e. 2 α -position hydroxytestosterone or 2 α -position hydroxyandrostenedione, which can improve the efficiency of separation and extraction, ③ the catalytic system constructed by the invention can selectively carry out oxidation reaction under mild conditions, compared with the complex process flow of traditional chemical reaction, it has great application value in the field of biological pharmacy, ④ can greatly reduce side reaction products by utilizing biotransformation of recombinant bacteria, and is easy to separate out the desired product, thus achieving high efficiency.
Drawings
FIG. 1 is a scheme showing the biotransformation of CYP154C2 with a substrate;
FIG. 2 is an HPLC profile of experimental samples, wherein FIG. 2A is testosterone and FIG. 2B is androstenedione;
FIG. 3 is a mass spectrum corresponding to the substrate testosterone and its product after 16 hours of reaction: FIG. 3a is testosterone and FIG. 3b is the product thereof after 16 hours of reaction;
FIG. 4 is a mass spectrum corresponding to the substrate androstenedione and its product after 16 hours of reaction: FIG. 4a is androstenedione and FIG. 4b is the product after 16 hours of reaction.
Detailed Description
The present invention will be further described in detail with reference to examples. The embodiments are provided to facilitate a better understanding of the invention and are not intended to limit the invention.