Engineering Saccharomyces Cerevisiae for the Production of Hexadecanol and Octadecanol

The fatty alcohols hexadecanol and octadecanol are widely used compounds in the cosmetic industry and find extensive application as platform chemicals. These fatty alcohols occur in their free form rarely in nature and are produced in very low abundance by a few plants and algae where they are incorporated into waxes. The primary source of hexadecanol and octadecanol is palm and coconut oil. The use of these food oils for industrial chemical and cosmetic production has lead to extensive deforestation in Asia and competition with the food industry for supply. 

We have engineered yeast as a microbial cell factory to produce hexadecanol and octadecanol from sugar, starch, or cellulosic hydrolysates. Production of free fatty alcohols was achieved by over expressing key enzymes in the fatty acid biosynthetic pathway. This was coupled with deletion of the POX1 gene encoding fatty acyl-CoA oxidase, the first step in degradation of fatty acyl-CoA molecules. These modifications increased fatty acid accumulation in the cells. Conversion of fatty acids and fatty acyl-CoA to fatty alcohol was achieved by expression of a thioesterase from E. coli and a fatty acyl reductase from mouse. Surprisingly, we found that production could be further improved by over expression of FAA3 a fatty acyl-CoA synthetase. These manipulations yielded strains capable of producing up to 194 mg/L hexadecanol and octadecanol in rich medium at a ratio of 47.8% hexadecanol to 52.2% octadecanol. This yield was further increased by up to 91% in nitrogen poor fermentation medium. In screening for strains with greater yields of hexadecanol we identified mutants in the SNF2 gene. Snf2 is part of a chromatin-remodeling complex that influences the expression of many genes so the mechanism by which snf2 mutation increases fatty alcohol synthesis is not clear. Interestingly in this strain background the unsaturated C18-1 and C16-1 are the most abundant fatty acids yet the major fatty alcohols produced are C18-OH and C16-OH, likely reflecting specificity of the fatty acyl reductase expressed in the cells. We identified mutants that could shift the balance of alcohol produced to an increase in octadecanol over hexadecanol. In addition to producing fatty alcohols from refined sugar we show that the strains are capable of using cellulosic hydrolysates as a feedstock.