AI Article Synopsis
- A new method called reporter-guided mutant selection (RGMS) uses reporters to help identify mutants that over-express a specific target, successfully enhancing clavulanic acid production in Streptomyces clavuligerus.
- The initial single-reporter design, using the ccaR transcriptional activator and kanamycin resistance, yielded a 51% success rate in selecting mutants with improved CA production.
- By introducing a double-reporter system, the success rate significantly increased to 90%, making this double-reporter RGMS the most effective mutant selection method to date, also offering potential for future genetic research.
Article Abstract
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 over-expression. The mutants obtained by RGMS showed great genetic diversity that could be further exploited in inverse metabolic engineering.
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| http://dx.doi.org/10.1016/j.ymben.2009.06.003 | DOI Listing |
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