Development of a Synthetic Oxytetracycline-Inducible Expression System for Streptomycetes Using de Novo Characterized Genetic Parts.
Precise control of gene expression using exogenous factors is of great significance. To develop ideal inducible expression systems for streptomycetes, new genetic parts, oxytetracycline responsive repressor OtrR, operator otrO, and promoter otrBp from Streptomyces rimosus, were selected de novo and characterized in vivo and in vitro. OtrR showed strong affinity to otrO (KD = 1.7 × 10(-10) M) and oxytetracycline induced dissociation of the OtrR/DNA complex in a concentration-dependent manner. On the basis of these genetic parts, a synthetic inducible expression system Potr* was optimized. Induction of Potr* with 0.01-4 μM of oxytetracycline triggered a wide-range expression level of gfp reporter gene in different Streptomyces species. Benchmarking Potr* against the widely used constitutive promoters ermE* and kasOp* revealed greatly enhanced levels of expression when Potr* was fully induced. Finally, Potr* was used as a tool to activate and optimize the expression of the silent jadomycin biosynthetic gene cluster in Streptomyces venezuelae. Altogether, the synthetic Potr* presents a new versatile tool for fine-tuning gene expression in streptomycetes.
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences , No.1 West Beichen Road, Chaoyang District, Beijing 100101, People's Republic of China.
Weishan
W
Wang
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences , No.1 West Beichen Road, Chaoyang District, Beijing 100101, People's Republic of China.|University of Chinese Academy of Sciences , No.19A Yuquan Road, Beijing 100049, People's Republic of China.
Tongjian
T
Yang
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences , No.1 West Beichen Road, Chaoyang District, Beijing 100101, People's Republic of China.
Yihong
Y
Li
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences , No.1 West Beichen Road, Chaoyang District, Beijing 100101, People's Republic of China.
Shanshan
S
Li
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences , No.1 West Beichen Road, Chaoyang District, Beijing 100101, People's Republic of China.
Shouliang
S
Yin
School of Life Sciences and Department of Chemistry, University of Warwick , Coventry CV4 7AL, United Kingdom.
Kathryn
K
Styles
School of Life Sciences and Department of Chemistry, University of Warwick , Coventry CV4 7AL, United Kingdom.
Christophe
C
Corre
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences , No.1 West Beichen Road, Chaoyang District, Beijing 100101, People's Republic of China.
Keqian
K
Yang
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D017930:Genes, Reporter; D016680:Genome, Bacterial; D007546:Isoquinolines / Q000378:metabolism; D005810:Multigene Family; D010118:Oxytetracycline / Q000494:pharmacology*; D061065:Polyketides / Q000378:metabolism; D011401:Promoter Regions, Genetic; D013302:Streptomyces / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D007546:Isoquinolines; D061065:Polyketides; C081750:jadomycin B; D010118:Oxytetracycline
biosynthetic gene cluster; genetic parts; inducible expression system; natural product; streptomycetes; wide-range strength