红球菌启动子识别及b-半乳糖苷酶报告基因表达

Promoter recognition and b-galactosidase reporter gene expression in Rhodococcus

摘要:

红球菌属在生物降解、生物修复、生物转化和生物表面活性剂等领域得到了日益广泛的应用。本研究以红球菌-大肠杆菌穿梭质粒pNV18.1为载体, 以腈水合酶为模式酶, 研究了大肠杆菌tac、lacZ启动子和红球菌酰胺酶启动子(ami-1/ami-2)在红球菌中的启动效率。结果表明, 启动子Ptac、Pami-1(7 bp SD-ATG间隔)、Pami-2(13 bp SD-ATG间隔)和PlacZ在紫红红球菌ATCC 33278中启动表达腈水合酶的比酶活分别是野生菌株的7.5、6.3、5.3和1.8倍, 表明这些启动子都可以被红球菌的RNA聚合酶所成功识别。采用PlacZ启动子在红球菌宿主中启动b-半乳糖苷酶报告基因(lacZ), 结果表明, lacZ能够在红球菌中高效表达, 是优选的红球菌报告基因。

Abstract:

The genus Rhodococcus is of considerable interest in recent years, stemming from their diverse applications in biodegradation, bioremediation, biotransformation and biosurfactant. Using Nocardia/Rhodococcus-Escherichia coli shuttle plasmid pNV18.1 as the backbone vector, we tested the driven efficiency of promoters Ptac and PlacZ of E. coli and Pami-1/Pami-2 of R. ruber in host R. rhodochrous ATCC 33278 by overexpression of nitrile hydratase. Results showed that the specific activity of nitrile hydratase per dry cell weight in engineered Rhodococcus strains driven by Ptac, Pami-1, Pami-2 and PlacZ was 7.5, 6.3, 5.3 and 1.8 times of that in the wild, respectively. It indicated that these promoters could be well recognized by RNA polymerase of Rhodococcus. We further expressed the β-galactosidase reporter gene (lacZ) in R. ruber driven by promoter PlacZ. Results indicated that lacZ was an appropriate reporter gene for genetic or metabolic engineering research of Rhodococcus.