Engineering of fatty acid production and secretion in Saccharomyces cerevisiae

Abstract

Production of renewable liquid biofuels that can substitute fossil fuel, has emerged as a major challenge for applied biology. Biodiesel, in the form of fatty acid esters, produced by oleaginous microorganisms could be an attractive alternative, since the utilization of diesel fuel is more efficient than for example ethanol. Oleaginous algae and yeasts may accumulate very high (60%) levels of intracellular lipids but two drawbacks are the relatively complicated extraction process and the subsequent transesterification with the accompanying glycerol by-product formation. The objective of this work is to apply metabolic engineering of fatty acid synthesis and secretion in the model yeast S. cerevisiae in order to create a microorganism able to produce and secrete free fatty acids or fatty acid esters. S. cerevisiae is a proper model, since lipid metabolism has been studied extensively and all genes encoding enzymes directly involved in lipid synthesis are known. This organism has also been reported to acquire oleaginous properties by no more than three genetic modifications (1). In the yeast S. cerevisiae, activation of exogenous long-chain fatty acids to coenzyme A derivatives, prior to metabolic utilization, proceeds through the fatty acyl-CoA synthetases Faa1p and Faa4p. It has been shown that free fatty acids are secreted from a FAA1,4 double mutant (2). This modification will be combined with modifications of core fatty acid elongation, such as overexpression of acetyl-CoA synthetase Acs1p in an attempt to improve fatty acid production rate. Essential for this work is to gain a deeper understanding of the dynamics of fatty acid synthesis and export, and hopefully facilitate a biological platform for efficient fatty acid or lipid production. In this work, the “delitto perfetto” method (3) is applied to delete these two fatty acyl-CoA synthetases generating genetically clean strains without markers or bacterial DNA. Results show that this technique can be used to generate multiple knockouts by recycling the marker gene.

1. Kamisaka Y et al. Biochem J. 2007 Nov 15;408(Pt 1):61–68.

2. Michinaka Y et al. Journal of Bioscience and Bioengineering. 2003 ;95(5):435-440.

3. Storici F, Resnick MA. Methods Enzymol. 2006 ;409329-45.