Metabolic pathway engineering for fatty acid ethyl ester production in Saccharomyces cerevisiae using stable chromosomal integration

Fatty acid ethyl esters are fatty acid derived molecules similar to first generation biodiesel (fatty acid methyl esters; FAMEs) which can be produced in a microbial cell factory. Saccharomyces cerevisiae is a suitable candidate for microbial large scale and long term cultivations, which is the typical industrial production setting for biofuels. It is crucial to conserve the metabolic design of the cell factory during industrial cultivation conditions that require extensive propagation. Genetic modifications therefore have to be introduced in a stable manner. Here, several metabolic engineering strategies for improved production of fatty acid ethyl esters in S. cerevisiae were combined and the genes were stably expressed from the organisms' chromosomes. A wax ester synthase (ws2) was expressed in different yeast strains with an engineered acetyl-CoA and fatty acid metabolism. Thus, we compared expression of ws2 with and without overexpression of alcohol dehydrogenase (ADH2), acetaldehyde dehydrogenase (ALD6) and acetyl-CoA synthetase (acs(SE)(L641P)) and further evaluated additional overexpression of a mutant version of acetyl-CoA decarboxylase (ACC1(S1157A,) (S659A)) and the acyl-CoA binding protein (ACB1). The combined engineering efforts of the implementation of ws2, ADH2, ALD6 and acs(SE)(L641P), ACC1(S1157A, S659A) and ACB1 in a S. cerevisiae strain lacking storage lipid formation (are1 Delta, are2 Delta, dga1 Delta and lro1 Delta) and beta-oxidation (pox1 Delta) resulted in a 4.1-fold improvement compared with sole expression of ws2 in S. cerevisiae.