Production of high-valued specialty chemicals using renewable resources is beneficial and is now cost-effective due to advances in metabolic engineering and fermentation techniques. Isoamyl acetate, produced via fermentation, is a natural flavor chemical with applications in food and other allied industries. A novel and cost-effective fermentation process was developed to produce isoamyl acetate using grain sorghum derived-glucose as feedstock. Two alcohol acetyltransferases from Saccharomyces cerevisiae (ATF1 and ATF2) were used in the esterification of isoamyl alcohol with acetyl coenzyme A. The respective genes were cloned and expressed in an appropriate ack-pta- mutant strain of Escherichia coli. Aerobic shake flasks experiments examined isoamyl acetate production over time, temperatures, and initial optical densities. The strain carrying the pBAD-ATF1 plasmid exhibited a high molar ester yield from glucose (1.13) after 48 hours of aerobic growth at 25¢ªC. Operational parameters have been optimized and low-cost media components, such as fusel oil, sorghum glucose and corn steep liquor was identified for high yield of isoamyl acetate. Using engineered Ecoli to produce natural ester is the first of its kind and an encouraging yield of 0.18 g isoamyl acetate/g of glucose was obtained with the low-cost fermentation medium.