Metabolic Engineering of Saccharomyces Cerevisiae for Production of Fatty Acid Short-Chain Alkyl Esters

Biodiesel is a mixture of fatty acid short-chain alkyl esters obtained via transesterification of vegetable oils with an alcohol. Since methanol is a cheap alcohol, biodiesels are more commonly found as methyl esters. As a more renewable and scalable biodiesel production method, microbes have been genetically engineered to produce fatty acid ethyl esters from biomass. While fatty acid (methyl and ethyl) esters are useful alternative diesel fuels, fatty acid esters with longer chain alcohol moiety, and with branching have better fuel properties. Here, we engineered yeast to produce fatty acid short-chain esters, including ethyl, isobutyl and isoamyl esters using endogenously synthesized fatty acids and alcohols. Two wax ester synthases were identified, cloned and expressed. Both enzymes were found to catalyse the formation of fatty acid short-chain esters, with different alcohol preference. In order to boost the ability of yeast in producing the aforementioned esters, multiple gene disruptions were carried out to increase flux towards fatty acyl-CoAs. In addition, as native production of isobutanol is insufficient and rate-limiting in yeast, isobutanol pathway genes targeted into the mitochondria were overexpressed. By combining these engineering strategies, we successfully engineered yeast metabolism to produce fatty acid short-chain esters, including ethyl, isobutyl and isoamyl esters