Systematic Identification of a Panel of Strong Constitutive Promoters from Streptomyces albus.
Actinomycetes are important organisms for the biosynthesis of valuable natural products. However, only a limited number of well-characterized native constitutive promoters from actinomycetes are available for the construction and engineering of large biochemical pathways. Here, we report the discovery and characterization of 32 candidate promoters identified from Streptomyces albus J1074 by RNA-seq analysis. These 32 promoters were cloned and characterized using a streptomycete reporter gene, xylE, encoding catechol 2,3-dioxygenase. The strengths of the identified strong promoters varied from 200 to 1300% of the strength of the well-known ermE*p in MYG medium, and the strongest of these promoters was by far the strongest actinomycete promoter ever reported in the literature. To further confirm the strengths of these promoters, qPCR was employed to determine the transcriptional levels of the xylE reporter. In total, 10 strong promoters were identified and four constitutive promoters were characterized via a time-course study. These promoters were used in a plug-and-play platform to activate a cryptic gene cluster from Streptomyces griseus, and successful activation of the target pathway was observed in three widely used Streptomyces strains. Therefore, these promoters should be highly useful in current synthetic biology platforms for activation and characterization of silent natural product biosynthetic pathways as well as the optimization of pathways for the synthesis of important natural products in actinomycetes.
Institute for Genomic Biology, University of Illinois at Urbana-Champaign , 1206 West Gregory Drive, Urbana, Illinois 61801, United States .
Yunzi
Y
Luo
Lu
L
Zhang
Katherine W
KW
Barton
Institute for Genomic Biology, University of Illinois at Urbana-Champaign , 1206 West Gregory Drive, Urbana, Illinois 61801, United States .
Huimin
H
Zhao
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D060847:Metabolic Engineering; D005810:Multigene Family; D011401:Promoter Regions, Genetic; D011549:Pseudomonas / Q000235:genetics / Q000378:metabolism; D012329:RNA, Bacterial / Q000235:genetics; D017423:Sequence Analysis, RNA; D013302:Streptomyces / Q000235:genetics* / Q000378:metabolism; D059467:Transcriptome
D001426:Bacterial Proteins; D012329:RNA, Bacterial
RNA-seq; Streptomyces promoters; XylE assay; actinomycetes; qPCR; synthetic biology