期刊
METABOLIC ENGINEERING
卷 11, 期 4-5, 页码 310-318出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ymben.2009.06.003
关键词
Strain improvement; Reporter; Metabolic engineering; Clavulanic acid
资金
- Hisun Pharmaceutical Co. Ltd [KSCX2-YW-G-013]
- Chinese Academy of Sciences [2006AA020302]
- Chinese Ministry of Science and Technology
A reporter-guided mutant selection (RGMS) method has been developed wherein reporters are used to facilitate selection of target over-expressing mutants. It was applied to improve clavulanic acid (CA) production in Streptomyces clavuligerus. In a single-reporter design, the transcriptional activator ccaR of CA biosynthesis was chosen as the over-expressing target, and neo (resistance to kanamycin) as the reporter; 51% of the selected mutants produced higher CA titer than the starting strain. To reduce the high false positive rate of single-reporter method, a double-reporter RGMS vector was configured, in which an xylE-neo double-reporter cassette was used to monitor ccaR expression; 90% of mutants selected by the modified method showed improvement in CA titer. Double-reporter RGMS is the most efficient tool for mutant selection reported to date and is also an alternative method for target overexpression. The mutants obtained by RGMS showed great genetic diversity that could be further exploited in inverse metabolic engineering. (C) 2009 Elsevier Inc. All rights reserved.
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