Improved production of fatty acids bySaccharomyces cerevisiaethrough screening a cDNA library from the oleaginous yeastYarrowia lipolytica

Biological production of fatty acid (FA)-derived products has gained increasing attention to replace petroleum-based fuels and chemicals. FA biosynthesis is highly regulated, and usually it is challenging to design rational engineering strategies. In addition, the conventional 'one sample at a time' method for lipid determination is time consuming and laborious, and it is difficult to screen large numbers of samples. Here, a method for detecting free FAs in viable cells using Nile red staining was developed for use in large-scale screening. Following optimization of the method, it was used for screening a cDNA library from the oleaginous yeast Yarrowia lipolytica for identification of genes/enzymes that were able to enhance free FA accumulation in Saccharomyces cerevisiae. Several novel enzymes resulting in increasing FA accumulation were discovered. These targets include a GPI anchor protein, malate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, FA hydroxylase, farnesyltransferase, anoctamin, dihydrolipoamide dehydrogenase and phosphatidylethanolamine-binding protein. The best enzyme resulted in a 2.5-fold improvement in production of free FAs. Our findings not only provide a novel method for high-throughput evaluation of the content of free FAs, but also give new insight into how enzymes from Y. lipolytica may increase the production of fatty acids in S. cerevisiae.