Microbial production of fatty acid derived chemicals and fuels is currently of great interest due to the limited resources and increasing prices of petroleum and petroleum-based products. The development of Saccharomyces cerevisiae as a fatty acid ethyl ester (FAEE) cell factory would represent an opportunity for biodiesel production due to its successful history in the biotechnology area. However, fatty acid (FA) biosynthesis is highly regulated and usually not high enough for developing an efficient production process. In S. cerevisiae, FAs are degraded by beta-oxidation and a large fraction is utilized to synthesize steryl esters (SEs) and triacylglycerols (TAGs), which are not essential for the cell. Here, by eliminating nonessential FA utilization pathways, we developed a metabolic engineering strategy resulting in a S. cerevisiae strain that can overproduce FAs and in turn use these for producing FAEEs (biodiesel). Compared to the wild-type, there is an about 3-fold increase in free FA content in a strain devoid of both TAG and SE formation, a 4-fold increase in free FA content in a strain that is incapable of beta-oxidation, and a 5-fold increase of free FAs in a strain lacking all of these non-essential FA utilization pathways. It is also demonstrated that there are similar positive effects on FAEE production in these deletion strains. The highest production of FAEEs is 17.2 mg/l in the strain in which all these pathways were blocked. The results of this study serve as a basis for further strategies to improve the production of FA derivatives in S. cerevisiae. (C) 2013 The Authors. Published by Elsevier Ltd. All rights reserved.