원문정보
초록
영어
Laboratory evolution has become a commonly used approach to address many of the fundamental long-standing questions about evolution as well as to provide an experimental basis for delineating the mechanistic basis of adaptive evolution, such as antibiotic resistance. We have got 7 independent glycerol evolved strains from wild type E. coli K-12 MG1655 by laboratory evolution. We have fully resequenced 5 independent glycerol evolved strains using the microarray-based Comparative Genome Sequencing service provided by Nimblegen Systems Inc. Surprisingly, one of them has only three mutations of whole genome in the endpoint. We added two mutations out of them into the wild type strain by site-directed mutagenesis. The double knock-in mutant, which had mutations in glpK and rpoC, had similar specific growth rate of the evolved strain. The two mutations thus seem to fully be responsible for the growth rate change during adaptive evolution. For intracellular metabolome analysis, we used capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). The double knock-in mutant and the evolved endpoint strain showed very similar intracellular metabolite profile at mid exponential phase. Especially, concentrations of N-Carbamoyl-L-aspartate and N-Acetyl-L-aspartate, which participate in alanine and aspartate metabolism, were significantly decreased.