Ester production and metabolic flux redistribution in Escherichia coli under aerobic conditions
Master of Science
Two alcohol acetyltransferases from Saccharomyces cerevisiae (ATF1 and ATF2) can catalyze the esterification of isoamyl alcohol and acetyl coenzyme A (acetyl-CoA). The respective genes were cloned from and expressed in an appropriate ack-pta strain of Escherichia coli. The new genetically engineered strains of E. coli produce isoamyl acetate, an ester, when isoamyl alcohol is added externally to the cell culture medium. Since acetyl-CoA is a substrate for this esterification reaction, the competing ackA-pta pathway at the acetyl-CoA node was inactivated to increase the intracellular acetyl-CoA pool and divert more carbon flux to the ester synthesis pathway. Experiments were carried out in aerobic shake flasks to investigate isoamyl acetate production over long periods of growth at various temperatures and starting optical densities. The ackA-pta mutant strain containing the pBAD-ATF1 plasmid exhibited the highest molar ester yield from glucose (1.13) after 48 hours of aerobic growth at 25°C.
Microbiology; Biochemistry; Chemical engineering