earticle

영어

Glycolysis is the most important metabolic pathway for growth and maintenance of living organisms, and it is designed highly robust by nature. However, relevant modification of glycolytic activity is significant in metabolic engineering for the product accumulation and it is considered as one of main technical challenges for the strain improvement. gapA is a one of the key glycolytic genes and encodes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) which mediates oxidative phosphorylation of glyceraldehyde-3-phosphate into 1,3-diphosphoglycerate and has vital role in glycolysis. Wild-type GAPDH has strong activity and it is not easy to control its expression level by the conventional controllable promoters. Therefore, it is required to modify GAPDH for the sensitive regulation of glycolysis in E. coli. In this study, we developed a gapA mutant for the regulationsensitive glycolysis in E. coli by directed evolution approach using the gapAdeleted mutant obtained by Red recombination system and temperature sensitive promoter system. Directed evolution of GAPDH was conducted by error-prone PCR combined with growth rate based screening system. By several round of evolution processes, several mutants with the different levels of regulation sensitivities could be obtained. From the sequence analysis of the mutants, activity and sequence relationship was also examined.