3-甾酮-9α-羟基化酶基因在分枝杆菌中强化表达
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摘要
9α-羟基雄烯二酮(9α-OH-AD)是制备倍他米松及地塞米松等甾体类药物的重要中间产物之一,在工业应用中,常使用谷甾醇、豆甾醇和菜油甾醇等植物甾醇作为底物,通过分枝杆菌等菌种生物转化获得9α-OHAD。在此过程中,3-甾酮-9α-羟基化酶(KSH)是生物转化植物甾醇为9α-OH-AD的关键酶,该酶由3-甾酮-9α-羟基化酶(Ksh A)和3-甾酮-9α-羟基化酶还原酶(Ksh B)两个亚基构成。因此,提高3-甾酮-9α-羟基化酶的活性对促进9α-OH-AD的合成具有极其重要的作用。为了获得高效积累9α-OH-AD产物的重组菌株,根据NCBI提供的基因序列为主,相关设计引物软件Prime 5和DNAMAN等辅助工具设计引物Ksh A和Ksh B,PCR扩增,经过EcoR I、Nde I和EcoR I、Hind III分别双酶切Ksh A和Ksh B,同时与载体p NIT进行连接,获得了含有Ksh A基因和Ksh B基因的重组质粒p NIT-Ksh A-Ksh B,最后再将构建好的质粒电转化入分枝杆菌中,根据最小抑菌浓度卡那霉素抗性平板上筛选得到1株阳性转化子M-ks H。发酵催化积累9α-OH-AD的实验结果表明,当底物植物甾醇投料为10 g/L时,M-ks H积累9α-OH-AD产量相比较出发菌株提高了0.486 g/L,重组菌的转化率提高了50.78%。研究结果可为提高甾体类药物9-OH-AD生产效率提供一定的基础。
9α-Hydroxy-4-androstene-3,17-dione(9α-OH-AD) is one of the significant intermediates for preparation of β-methasone,dexamethasone and other steroids.In the field of industry,sitosterol,stigmasterol,campesterol and others were often used as substrates.In general,the key enzyme,which affects biotransformation ability of phytosterone,is 3-phytosterone-9α-hydroxylase(KSH).It is made up of two subunits by 3-phytosterone-9α-hydroxylase(Ksh A) and 3-phytosterone-9α-hydroxylase reductase(Ksh B).Therefore,it affects biotransformation ability of phytosterone of 3-phytosterone-9α-hydroxylase to promote the synthesis of 9α-OH-AD.This study aims at using the National Center for Biotechnology Information(NCBI),Prime 5 and DNAMAN to design primers(Ksh A and Ksh B)in order to obtain recombinant strains of high accumulation of 9α-OH-AD product.After the amplification of PCR,Ksh A and Ksh B was cut by EcoR I,Nde I and EcoR I and Hind III,the recombinant plasmid was cloned into the vector p NIT to get the recombinant plasmid p NIT-Ksh A-Ksh B.Finally,the recombinant plasmid was transformed into Mycobacterium.The positive transformants of the strain would be chosen through minimal inhibitory concentration(MIC) of the antibiotic resistant plate and the recombinant strain is M-ks H.When the substrate is fed to the 10 g / L,the experimental results of fermentation showed that the 9α-OH-AD yield of M-ks H was higher than the original strain,which would promote 9α-OH-AD by 0.486 g / L and the transformation rate of recombinant bacteria increased by50.78%.Result provides the basis for improving the efficiency of industrial production of 9α-OH-AD.
9α-Hydroxy-4-androstene-3,17-dione(9α-OH-AD) is one of the significant intermediates for preparation of β-methasone,dexamethasone and other steroids.In the field of industry,sitosterol,stigmasterol,campesterol and others were often used as substrates.In general,the key enzyme,which affects biotransformation ability of phytosterone,is 3-phytosterone-9α-hydroxylase(KSH).It is made up of two subunits by 3-phytosterone-9α-hydroxylase(Ksh A) and 3-phytosterone-9α-hydroxylase reductase(Ksh B).Therefore,it affects biotransformation ability of phytosterone of 3-phytosterone-9α-hydroxylase to promote the synthesis of 9α-OH-AD.This study aims at using the National Center for Biotechnology Information(NCBI),Prime 5 and DNAMAN to design primers(Ksh A and Ksh B)in order to obtain recombinant strains of high accumulation of 9α-OH-AD product.After the amplification of PCR,Ksh A and Ksh B was cut by EcoR I,Nde I and EcoR I and Hind III,the recombinant plasmid was cloned into the vector p NIT to get the recombinant plasmid p NIT-Ksh A-Ksh B.Finally,the recombinant plasmid was transformed into Mycobacterium.The positive transformants of the strain would be chosen through minimal inhibitory concentration(MIC) of the antibiotic resistant plate and the recombinant strain is M-ks H.When the substrate is fed to the 10 g / L,the experimental results of fermentation showed that the 9α-OH-AD yield of M-ks H was higher than the original strain,which would promote 9α-OH-AD by 0.486 g / L and the transformation rate of recombinant bacteria increased by50.78%.Result provides the basis for improving the efficiency of industrial production of 9α-OH-AD.
引文
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[3]杨亚力,杨顺楷,吴中柳.偶发分枝杆菌发酵断甾醇侧链积累9α-羟基雄甾二酮[J].应用与环境生物学报,2015,21(2):256-262.
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[6]Bragin YE,Shtratnikova VY,Dovbnya DV,et al.Comparative analysis of genes encoding key steroid core oxidation enzymes in fast-growing Mycobacterium spp Strains[J].J Steroid Bioche Mole Biol,2013,138:41-53.
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[8]袁家代,陈贵英,程世君,等.3-甾酮-9α-羟基化酶基因在分枝杆菌中的异源表达与9α-羟基雄烯二酮的制备[J].生物工程学报,2015,31(4):523-533.
[9]Veiga-Crespo P,Feijoo-Siota L,de Miguel T,et al.Proposal of a method for the genetic transformation of Gordonia jacobaea[J].J Appl Microbiol,2006,100(3):608-614.
[10]韩冷,夏海锋,饶志明.新金色分枝杆菌中AD转化为AAD关键酶KSDD的研究[D].无锡:江南大学硕士学位论文,2010.
[11]Chen T,Zhao QJ,Li W,et al.Mycobacterium tubercμLosis PE_PGRS17 promotes the death of host cell and cytokines secretion via Erk kinase accompanying with enhanced survival of recombinant Mycobacterium smegmatis[J].J Interf Cytok Res,2013,33(8):452-458.
[12]戴海霞,张晓燕,徐开俊,等.非甾体抗炎药研究的最新进展[J].药物生物技术,2012,19(1):90-94.
[2]范书玥,魏巍,王风清,等.Mycobacterium sp.Nw IB-01 3-甾酮-9α-羟基化酶基因的克隆、异源表达及分离纯化[J].生物工程学报,2009,25(12):2014-2021.
[3]杨亚力,杨顺楷,吴中柳.偶发分枝杆菌发酵断甾醇侧链积累9α-羟基雄甾二酮[J].应用与环境生物学报,2015,21(2):256-262.
[4]Capyk JK,Angelo ID,Strynadka NC,et al.Characterization of 3-ketosteroid 9-hydroxylase,a rieske oxygenase in the cholesterol degradation pathway of Mycobacterium tubercμLosis[J].Bioch Molec Biol,2009,284(15):9937-9946.
[5]Attila A,Antonia J,David A,et al.Generation of usefu L insertionally blocked sterol degradation pathway mutants of fast-growing myceobacteria and cloning,charaeterization,and expression of the terminal oxygenase of the 3-ketosteroid 9α-hydroxylase in Mycobacterium smegmatis mc2115[J].Appl Environ Microbiol,2006,72(10):6554-6559.
[6]Bragin YE,Shtratnikova VY,Dovbnya DV,et al.Comparative analysis of genes encoding key steroid core oxidation enzymes in fast-growing Mycobacterium spp Strains[J].J Steroid Bioche Mole Biol,2013,138:41-53.
[7]van der Geize R,Hessels GI,van Gerwen R,et al.Molecular and functional characterization of ksh A and ksh B,eneoding two components of 3-ketosteroid 9α-hydroxylase,a class IA monooxygenase,in Rhodococcus erythropolis strain SQI[J].Mol Microbiol,2002,45(4):1007-1018.
[8]袁家代,陈贵英,程世君,等.3-甾酮-9α-羟基化酶基因在分枝杆菌中的异源表达与9α-羟基雄烯二酮的制备[J].生物工程学报,2015,31(4):523-533.
[9]Veiga-Crespo P,Feijoo-Siota L,de Miguel T,et al.Proposal of a method for the genetic transformation of Gordonia jacobaea[J].J Appl Microbiol,2006,100(3):608-614.
[10]韩冷,夏海锋,饶志明.新金色分枝杆菌中AD转化为AAD关键酶KSDD的研究[D].无锡:江南大学硕士学位论文,2010.
[11]Chen T,Zhao QJ,Li W,et al.Mycobacterium tubercμLosis PE_PGRS17 promotes the death of host cell and cytokines secretion via Erk kinase accompanying with enhanced survival of recombinant Mycobacterium smegmatis[J].J Interf Cytok Res,2013,33(8):452-458.
[12]戴海霞,张晓燕,徐开俊,等.非甾体抗炎药研究的最新进展[J].药物生物技术,2012,19(1):90-94.