Application of a double-reporter-guided mutant selection method to improve clavulanic acid production in Streptomyces clavuligerus.
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.
Xiang
Si-Hai
SH
Joint Laboratory of Biopharmaceutical Research, IMCAS-HISUN (Beijing), Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, People's Republic of China.
Li
Jia
J
Yin
Hua
H
Zheng
Jian-Ting
JT
Yang
Xu
X
Wang
Hai-Bin
HB
Luo
Jia-Li
JL
Bai
Hua
H
Yang
Ke-Qian
KQ
D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D019818:Clavulanic Acid / Q000096:biosynthesis*; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000379:methods*; D009154:Mutation; D010957:Plasmids; D013302:Streptomyces / Q000235:genetics / Q000378:metabolism*; D014169:Transformation, Bacterial
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D019818:Clavulanic Acid