title: Identification and engineering of regulation-related genes toward improved kasugamycin production.
abstract: Kasugamycin, produced by Streptomyces kasugaensis and Streptomyces microaureus, is an important amino-glycoside family antibiotic and widely used for veterinary and agricultural applications. In the left flanking region of the previously reported kasugamycin gene cluster, four additional genes (two-component system kasW and kasX, MerR-family kasV, and isoprenylcysteine carboxyl methyltransferase kasS) were identified both in the low-yielding S. kasugaensis BCRC12349 and high-yielding S. microaureus XM301. Deletion of regulatory gene kasT abolished kasugamycin production, and its overexpression in BCRC12349 resulted in an increased titer by 186 %. Deletion of kasW, kasX, kasV, and kasS improved kasugamycin production by 12, 19, 194, and 22 %, respectively. qRT-PCR analysis demonstrated that the transcription of kas genes was significantly increased in all the four mutants. Similar gene inactivation was performed in the high-yielding strain S. microaureus XM301. As expected, the deletion of kasW/X resulted in a 58 % increase of the yield from 6 to 9.5 g/L. However, the deletion of kasV and over-expression of kasT had no obvious effect, and the disruption of kasS surprisingly decreased kasugamycin production. In addition, trans-complementation of the kasS mutant with a TTA codon-mutated kasS increased the kasugamycin yield by 20 %. A much higher transcription of kas genes was detected in the high-yielding XM301 than in the low-yielding BCRC12349, which may partially account for the discrepancy of gene inactivation effects between them. Our work not only generated engineered strains with improved kasugamycin yield, but also pointed out that different strategies on manipulating regulatory-related genes should be considered for low-yielding or high-yielding strains.
authors.affiliation: State Key Laboratory of Microbial Metabolism, and School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, People's Republic of China.
authors.forename: Chenchen
authors.initials: C
authors.lastname: Zhu
authors.affiliation: State Key Laboratory of Microbial Metabolism, and School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, People's Republic of China.
authors.forename: Qianjin
authors.initials: Q
authors.lastname: Kang
authors.affiliation: State Key Laboratory of Microbial Metabolism, and School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, People's Republic of China. bailq@sjtu.edu.cn.
authors.forename: Linquan
authors.initials: L
authors.lastname: Bai
authors.affiliation: School of Pharmaceutical Sciences, Wuhan University, Wuhan, 430071, People's Republic of China. lincheng2015@whu.edu.cn.
authors.forename: Lin
authors.initials: L
authors.lastname: Cheng
authors.affiliation: State Key Laboratory of Microbial Metabolism, and School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, People's Republic of China.|School of Pharmaceutical Sciences, Wuhan University, Wuhan, 430071, People's Republic of China.
authors.forename: Zixin
authors.initials: Z
authors.lastname: Deng
mesh_terms: D000617:Aminoglycosides / Q000096:biosynthesis*; D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D053898:Biosynthetic Pathways / Q000235:genetics*; D017353:Gene Deletion; D015870:Gene Expression; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D005816:Genetic Complementation Test; D056426:Genetic Loci; D060847:Metabolic Engineering; D005810:Multigene Family; D060888:Real-Time Polymerase Chain Reaction; D013302:Streptomyces / Q000235:genetics* / Q000378:metabolism*
chemical_list: D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; C100305:kasugamycin
keywords: Antibiotic; Genetic engineering; Kasugamycin; Regulation; Streptomyces