ω-3 fatty acid desaturase is a key enzyme for the biosynthesis of ω-3 polyunsaturated fatty acids via the oxidative desaturase/elongase pathways. Here we report the identification of three ω-3 desaturases from oomycetes, Pythium aphanidermatum, Phytophthora sojae, and Phytophthora ramorum. These new ω-3 desaturases share 55 % identity at the amino acid level with the known Δ-17 desaturase of Saprolegnia diclina, and about 31 % identity with the bifunctional Δ-12/Δ-15 desaturase of Fusarium monoliforme. The three enzymes were expressed in either wild-type or codon optimized form in an engineered arachidonic acid producing strain of Yarrowia lipolytica to study their activity and substrate specificity. All three were able to convert the ω-6 arachidonic acid to the ω-3 eicosapentanoic acid, with a substrate conversion efficiency of 54-65 %. These enzymes have a broad ω-6 fatty acid substrate spectrum, including both C18 and C20 ω-6 fatty acids although they prefer the C20 substrates, and have strong Δ-17 desaturase activity but weaker Δ-15 desaturase activity. Thus, they belong to the Δ-17 desaturase class. Unlike the previously identified bifunctional Δ-12/Δ-15 desaturase from F. monoliforme, they lack Δ-12 desaturase activity. The newly identified Δ-17 desaturases could use fatty acids in both acyl-CoA and phospholipid fraction as substrates. The identification of these Δ-17 desaturases provides a set of powerful new tools for genetic engineering of microbes and plants to produce ω-3 fatty acids, such as eicosapentanoic acid and docosahexanoic acid, at high levels.

Numerous clinical studies have demonstrated that the omega-3 fatty acids in fish oil significantly reduce the risk of cardiovascular disease in adults. This chapter discusses the approach to introduce the genes encoding an omega-3 fatty acid biosynthesis pathway into an oleaginous yeast that synthesizes and stores triglycerides as an energy reserve when starved for nitrogen in the presence of an excess carbon source, such as glucose. It explains the development of a clean and sustainable source of omega-3 fatty acids by fermentation, which uses a metabolically engineered strain of the oleaginous yeast Y. lipolytica. While certain strains of Y. lipolytica can accumulate oil up to 40% of the dry cell weight, the only Polyunsaturated Fatty Acid (PUFA) normally synthesized by the organism is Linoleic Acid (LA). Coordinate expression of desaturase genes and elongase genes comprising a "delta6 pathway" was sufficient to demonstrate the synthesis of Eicosapentaenoic Acid (EPA). However, only an integrated strategy, based on the use of strong promoters, an increase in gene copy numbers, the push and pull of carbon into the engineered pathway, and the use of oleaginous condition, resulted in the generation of a high EPA production strain.

ABSTRACT The yeast Saccharomyces cerevisiae senses glucose, its preferred carbon source, through multiple signal transduction pathways. In one pathway, glucose represses the expression of many genes through the Mig1 transcriptional repressor, which is regulated by the Snf1 protein kinase. In another pathway, glucose induces the expression of HXT genes encoding glucose transporters through two glucose sensors on the cell surface that generate an intracellular signal that affects function of the Rgt1 transcription factor. We profiled the yeast transcriptome to determine the range of genes targeted by this second pathway. Candidate target genes were verified by testing for Rgt1 binding to their promoters by chromatin immunoprecipitation and by measuring the regulation of the expression of promoter lacZ fusions. Relatively few genes could be validated as targets of this pathway, suggesting that this pathway is primarily dedicated to regulating the expression of HXT genes. Among the genes regulated by this glucose signaling pathway are several genes involved in the glucose induction and glucose repression pathways. The Snf3/Rgt2-Rgt1 glucose induction pathway contributes to glucose repression by inducing the transcription of MIG2, which encodes a repressor of glucose-repressed genes, and regulates itself by inducing the expression of STD1, which encodes a regulator of the Rgt1 transcription factor. The Snf1-Mig1 glucose repression pathway contributes to glucose induction by repressing the expression of SNF3 and MTH1, which encodes another regulator of Rgt1, and also regulates itself by repressing the transcription of MIG1. Thus, these two glucose signaling pathways are intertwined in a regulatory network that serves to integrate the different glucose signals operating in these two pathways.