title: An engineered strong promoter for streptomycetes.
abstract: Well-characterized promoters are essential tools for metabolic engineering and synthetic biology. In Streptomyces coelicolor, the native kasOp is a temporally expressed promoter strictly controlled by two regulators, ScbR and ScbR2. In this work, first, kasOp was engineered to remove a common binding site of ScbR and ScbR2 upstream of its core region, thus generating a stronger promoter, kasOp3. Second, another ScbR binding site internal to the kasOp3 core promoter region was abolished by random mutation and screening of the mutant library to obtain the strongest promoter, kasOp* (where the asterisk is used to distinguish the engineered promoter from the native promoter). The activities of kasOp* were compared with those of two known strong promoters, ermEp* and SF14p, in three Streptomyces species. kasOp* showed the highest activity at the transcription and protein levels in all three hosts. Furthermore, relative to ermEp* and SF14p, kasOp* was shown to confer the highest actinorhodin production level when used to drive the expression of actII-ORF4 in S. coelicolor. Therefore, kasOp* is a simple and well-defined strong promoter useful for gene overexpression in streptomycetes.
authors.affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, People's Republic of China.
authors.forename: Weishan
authors.initials: W
authors.lastname: Wang
authors.forename: Xiao
authors.initials: X
authors.lastname: Li
authors.forename: Juan
authors.initials: J
authors.lastname: Wang
authors.forename: Sihai
authors.initials: S
authors.lastname: Xiang
authors.forename: Xiaozhou
authors.initials: X
authors.lastname: Feng
authors.forename: Keqian
authors.initials: K
authors.lastname: Yang
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005818:Genetic Engineering; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D048372:Streptomyces coelicolor / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C446304:ScbR protein, Streptomyces coelicolor