山梨糖脱氢酶基因在酮古龙酸菌中的过表达  

Clone and overexpression of sorbose dehydrogenase in Ketogulonigenium

作　　者：王威勋 李燕[1] 李野[1,2] 张天园[1] 张怡轩[1] 

机构地区：[1]沈阳药科大学生命科学与生物制药学院,辽宁沈阳110016 [2]东北制药集团公司,辽宁沈阳110032

出　　处：《沈阳药科大学学报》2016年第10期821-825,共5页Journal of Shenyang Pharmaceutical University

基　　金：国家自然科学基金资助项目(31370125)

摘　　要：目的在酮古龙酸菌中克隆并过表达山梨糖脱氢酶基因,以便提高单菌发酵产2-酮基-L-古龙酸的能力。方法从酮古龙酸菌DSM4025基因组中克隆获得山梨糖脱氢酶基因(sorbose dehydrogenase,sdh),酶切连接到含有双亲本接合转移关键基因即可移动基因(mobilization,mob)的p BBR1MCS-2质粒上,构建p BBR1MCS-2-sdh重组质粒;再将p BBR1MCS-2、p BBR1MCS-2-sdh分别转入供体菌大肠杆菌(Escherichia coli,E.coli)S17-1中,以酮古龙酸菌(Ketogulonigenium sp.)Rif-B-003为受体菌进行双亲本接合转移。挑取利福霉素(Rifamycin,Rif)和卡那霉素双抗平板上的接合子进行聚合酶链式反应(polymerase chain reaction,PCR)、双酶切和测序验证。取重组菌进行摇瓶发酵,用菌体蛋白的十二烷基硫酸钠聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis,SDS-PAGE)电泳结果来验证阳性克隆的过表达成功,并检测发酵终点2-酮基-L-古龙酸的产量。结果构建的重组质粒p BBR1MCS-2-sdh成功转入酮古龙酸菌(Ketogulonigenium sp.)Rif-B-003中,SDS-PAGE检测到,发酵终点菌体蛋白在58 ku(目标蛋白)处条带有加深,重组菌株发酵终点2-酮基-L-古龙酸产量相比于对照菌株酮古龙酸菌(Ketogulonigenium sp.)Rif-B-003提高了20.86%。结论山梨糖脱氢酶基因在酮古龙酸菌Rif-B-003里过表达成功,可以提高发酵终点2-酮基-L-古龙酸的产量。Objective To clone and overexpress the sorbose dehydrogenase（ SDH） in Ketogulonigenium sp.Rif-B-003 and improve the ability of producing 2-keto-L-gulonic acid. Methods The recombined plasmid pBBR1MCS2-sdh was constructed by enzyme digestion and ligating the sdh gene which cloned from the Ketogulonigenium sp. DSM4025 with the plasmid pBBR1MCS-2 containing the key gene also named mobilization（ mob） gene in diparental conjugation transfer. Plasmid pBBR1MCS-2 and pBBR1MCS-2-sdh were introduced into Escherichia coli（ E. coli） S17-1 as the donor strain, respectively,by the method of chemical conversion. Then the plasmids pBBR1MCS-2 and pBBR1MCS-2-sdh were separately transferred into the recipient bacterium Ketogulonigenium sp. Rif-B-003 by diparental conjugation. The transformed clone that resistant to kanamycin and rifamycin was identified by polymerase chain reaction（ PCR）,restriction enzyme digestion and gene sequencing. The successful expression of SDH was verified by the SDH enzyme activity determination、sodium dodecyl sulfate polyacrylamide gel electrophoresis（ SDS-PAGE）of mycoprotein extract from recombined strain fermentation liquor in shake flask, subsequently, the production of 2-keto-L-gulonic acid was detected. Results The pBBR1MCS-2-sdh was successfully introduced into Ketogulonigenium sp. Rif-B-003,compared with the control group Ketogulonigenium sp. Rif-B-003, the production of 2-keto-L-gulonic acid of recombinant strain improved 20. 86%and it＇s target protein SDH band（ 58 ku） got darker in SDS-PAGE. Conclusions The recombinant plasmid vector is successfully constructed and overexpressed in Ketogulonigenium sp. Rif-B-003. It can increase the production of 2-keto-L-gulonic acid at the end of fermentation.

关 键 词：酮古龙酸菌 山梨糖脱氢酶 接合转移 2-酮基-L-古龙酸 

分 类 号：Q786[生物学—分子生物学]