麦芽糖诱导梯度强度启动子的定向进化改造  

Directed evolution of maltose induced promoters with expanded gradient intensity

作　　者：张国强 滕茂放 刘松 周景文 李江华 堵国成[1,3] ZHANG Guoqiang;TENG Maofang;LIU Song;ZHOU Jingwen;LI Jianghua;DU Guocheng(Science Center for Future Foods,Jiangnan University,Wuxi 214122,Jiangsu,China;National Engineering Research Center for Cereal Fermentation and Food Biomanufacturing,Jiangnan University,Wuxi 214122,Jiangsu,China;School of Biotechnology,Jiangnan University,Wuxi 214122,Jiangsu,China)

机构地区：[1]江南大学未来食品科学中心,江苏无锡214122 [2]江南大学粮食发酵与食品生物制造国家工程研究中心,江苏无锡214122 [3]江南大学生物工程学院,江苏无锡214122

出　　处：《生物工程学报》2022年第7期2606-2617,共12页Chinese Journal of Biotechnology

基　　金：国家重点研发计划(2019YFA09004900);国家自然科学基金(32172153)。

摘　　要：不同灵敏度与响应强度的启动子在基因表达调控与代谢工程改造中应用广泛。为筛选不同诱导表达强度的启动子元件,本研究以麦芽糖诱导启动子Pglvc为对象,通过易错PCR方法对麦芽糖诱导型启动子进行突变获得启动子突变体库,然后基于四环素筛选的细胞生长偶联方法对突变体进行高效筛选,获得了不同响应范围和强度的启动子突变体,最终得到的诱导型启动子突变体(MT2、MT3、MT4、MT6)对麦芽糖诱导剂的响应范围从0–3 g/L扩展至0–15 g/L,其中最高诱导表达强度菌株(MT8)较原始启动子菌株的绿色荧光蛋白表达水平提高约3.15倍,有利于进一步拓展梯度强度启动子在枯草芽孢杆菌代谢工程和合成生物学中的应用。Promoters with different sensitivity and response intensity are useful tools in gene expression regulation and metabolic engineering.Maltose induced promoter Pglvc was engineered to obtain promoters with different induced expression intensities.A promoter Pglvc mutant library was built by error-prone PCR,and screened by a growth-associated method using tetracycline resistance as an indicator.A library of promoter mutants with different sensitivity and intensity was obtained,and the maltose-induced response threshold range of promoter mutants(MT2,MT3,MT4,MT6)was extended from 0–3 g/L to 0–15 g/L.Among them,the highest induced expression intensity(MT8)was about 3.15 times higher than that of the original promoter for eGFP expression,which would be useful for its application in metabolic engineering and synthetic biology.

关 键 词：诱导启动子 麦芽糖 定向进化 梯度强度 枯草芽孢杆菌 

分 类 号：Q78[生物学—分子生物学]