Heterologous expression of alcohol acetyltransferase genes in Escherichia coli and Clostridium acetobutylicum for the production of esters
Horton, Catherine Emily
Rudolph, Frederick B.; Bennett, George N.
Doctor of Philosophy
This thesis focuses on the heterologous expression of alcohol acetyltransferase (AATase) genes in Escherichia coli and Clostridium acetobutylicum for the production of the esters isoamyl acetate, butyl acetate and butyl butyrate. Isoamyl acetate, butyl acetate and butyl butyrate are esters that confer a fruity aroma and taste to the materials in which they are found. AATases are a class of enzymes that have been found to enzymatically catalyze the reaction between alcohols and acyl-CoAs to produce the corresponding ester. Butanol, acetyl-CoA, and butyryl-CoA, the substrates necessary for butyl acetate and butyl butyrate production, are produced in high concentrations by C. acetobutylicum, making it an ideal host for the expression of genes for AATases. Previous studies have characterized AATases in yeast, and expressed them in E. coli, but this is the first report of ester production by AATase activity in C. acetobutylicum . The genes ATF1 and ATF2, encoding AATase I and AATase II from the yeast Saccharomyces cerevisiae, and SAAT encoding a strawberry alcohol acetyltransferase, Saatp, have previously been sequenced. Biochemical studies have shown them to possess AATase activity. For this thesis, ATF1, ATF2, and SAAT were subcloned and expressed in E. coli. Ester production was determined by head-space gas analysis on a gas chromatagraph. AATase I in E. coli cultures and cell-free extracts produced more ester than AATase II did with each alcohol substrate that was investigated. Saatp produced less ester than AATase I, but more than AATase II. The ester butyl butyrate was detected in cell-free extracts of E. coli expressing SAAT, but not in cell-free extracts expressing ATF1 or ATF2 . ATF2 was also expressed in C. acetobutylicum for the production of isoamyl acetate and butyl acetate. Ester concentrations in C. acetobutylicum strains expressing ATF2 were lower than ester concentrations in E. coli strains expressing ATF2. The expression of ATF2 in C. acetobutylicum demonstrates the viability of using this organism for natural ester production via microbial fermentation.
Molecular biology; Microbiology