title: Characterization of negative regulatory genes for the biosynthesis of rapamycin in Streptomyces rapamycinicus and its application for improved production.
abstract: Sequence analysis of the rapamycin biosynthetic gene cluster in Streptomyces rapamycinicus ATCC 29253 identified several putative regulatory genes. The deduced product of rapY, rapR, and rapS showed high sequence similarity to the TetR family transcription regulators, response regulators and histidine kinases of two-component systems, respectively. Overexpression of each of the three genes resulted in a significant reduction in rapamycin production, while in-frame deletion of rapS and rapY from the S. rapamycinicus chromosome improved the levels of rapamycin production by approximately 4.6-fold (33.9 mg l(-1)) and 3.7-fold (26.7 mg l(-1)), respectively, compared to that of the wild-type strain. Gene expression analysis by semi-quantitative reverse transcription-PCR (RT-PCR) in the wild-type and mutant strains indicated that most of the rapamycin biosynthetic genes are regulated negatively by rapS (probably through its partner response regulator RapR) and rapY. Interestingly, RapS negatively regulates the expression of the rapY gene, and in turn, rapX encoding an ABC-transporter is negatively controlled by RapY. Finally, overexpression of rapX in the rapS deletion mutant resulted in a 6.7-fold (49 mg l(-1)) increase in rapamycin production compared to that of wild-type strain. These results demonstrate the role of RapS/R and RapY as negative regulators of rapamycin biosynthesis and provide valuable information to both understand the complex regulatory mechanism in S. rapamycinicus and exploit the regulatory genes to increase the level of rapamycin production in industrial strains.
authors.affiliation: Department of Chemistry and Nano Science, Ewha Global Top5 Research Program, Ewha Womans University, Seoul, 120-750, Republic of Korea.
authors.forename: Young Ji
authors.initials: YJ
authors.lastname: Yoo
authors.forename: Jae-Yeon
authors.initials: JY
authors.lastname: Hwang
authors.forename: Hea-Luyung
authors.initials: HL
authors.lastname: Shin
authors.forename: Heqing
authors.initials: H
authors.lastname: Cui
authors.forename: Jinwon
authors.initials: J
authors.lastname: Lee
authors.forename: Yeo Joon
authors.initials: YJ
authors.lastname: Yoon
mesh_terms: D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005809:Genes, Regulator; D005810:Multigene Family; D020123:Sirolimus / Q000378:metabolism*; D013302:Streptomyces / Q000235:genetics* / Q000378:metabolism
chemical_list: D020123:Sirolimus