title: Engineering of the LysR family transcriptional regulator FkbR1 and its target gene to improve ascomycin production.
abstract: Ascomycin (FK520), a macrocyclic polyketide natural antibiotic, displays high anti-fungal and immunosuppressive activity. In this study, the LysR family transcriptional regulator FkbR1 was characterized, and its role in ascomycin biosynthesis was explored by gene deletion, complementation, and overexpression. Inactivation of fkbR1 led to 67.5% reduction of ascomycin production, which was restored by complementation of fkbR1. Overexpression of fkbR1 resulted in a 33.5% increase in ascomycin production compared with the parent strain FS35. These findings indicated that FkbR1 was a positive regulator for ascomycin production. Quantitative RT-PCR analysis revealed that the expressions of fkbE, fkbF, fkbS, and fkbU were downregulated in the fkbR1 deletion strain and upregulated in the fkbR1 overexpression strain. Electrophoretic mobility shift assays (EMSAs) in vitro and chromatin immunoprecipitation (ChIP)-qPCR assays in vivo indicated that FkbR1 bound to the intergenic region of fkbR1-fkbE. To investigate the roles of the target genes fkbE and fkbF in ascomycin production, the deletion and overexpressions of fkbE and fkbF were implemented, respectively. Overexpression of fkbE resulted in a 45.6% increase in ascomycin production, but little change was observed in fkbF overexpression strain. To further enhance ascomycin production, the fkbR1 and fkbE combinatorial overexpression strain OfkbRE was constructed with the ascomycin yield increased by 69.9% to 536.7 mg/L compared with that of the parent strain. Our research provided a helpful strategy to increase ascomycin production via engineering FkbR1 and its target gene.
authors.affiliation: Key Laboratory of System Bioengineering (Tianjin University), Ministry of Education, Tianjin, 300072, People's Republic of China.|SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, People's Republic of China.
authors.forename: Kejing
authors.initials: K
authors.lastname: Song
authors.affiliation: Key Laboratory of System Bioengineering (Tianjin University), Ministry of Education, Tianjin, 300072, People's Republic of China.|SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, People's Republic of China.
authors.forename: Liang
authors.initials: L
authors.lastname: Wei
authors.affiliation: Key Laboratory of System Bioengineering (Tianjin University), Ministry of Education, Tianjin, 300072, People's Republic of China.|SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, People's Republic of China.
authors.forename: Jiao
authors.initials: J
authors.lastname: Liu
authors.affiliation: Key Laboratory of System Bioengineering (Tianjin University), Ministry of Education, Tianjin, 300072, People's Republic of China.|SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, People's Republic of China.
authors.forename: Junhua
authors.initials: J
authors.lastname: Wang
authors.affiliation: Key Laboratory of System Bioengineering (Tianjin University), Ministry of Education, Tianjin, 300072, People's Republic of China.|SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, People's Republic of China.
authors.forename: Haishan
authors.initials: H
authors.lastname: Qi
authors.affiliation: Key Laboratory of System Bioengineering (Tianjin University), Ministry of Education, Tianjin, 300072, People's Republic of China. jpwen@tju.edu.cn.|SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, People's Republic of China. jpwen@tju.edu.cn.
authors.forename: Jianping
authors.initials: J
authors.lastname: Wen
mesh_terms: D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D047369:Chromatin Immunoprecipitation; D021901:DNA, Intergenic; D024202:Electrophoretic Mobility Shift Assay; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D060888:Real-Time Polymerase Chain Reaction; D013302:Streptomyces / Q000235:genetics* / Q000378:metabolism; D016559:Tacrolimus / Q000031:analogs & derivatives* / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D021901:DNA, Intergenic; D014157:Transcription Factors; C073470:LysR protein, Bacteria; C058028:immunomycin; D016559:Tacrolimus
keywords: Ascomycin; FkbR1; Streptomyces hygroscopicus var. ascomyceticus; Target gene; Transcriptional regulator