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

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P_glvc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。

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

Abstract:

Promoters with different sensitivity and response intensity are useful tools in gene expression regulation and metabolic engineering. Maltose induced promoter P_glvc was engineered to obtain promoters with different induced expression intensities. A promoter P_glvc 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.

Key words:inducible promoter;maltose;directed evolution;gradient intensity;Bacillus subtilis