The availability of the omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) is currently limited because they are produced mainly by marine fisheries that cannot keep pace with the demands of the growing market for these products. A sustainable non-animal source of EPA and DHA is needed. Metabolic engineering of the oleaginous yeast Yarrowia lipolytica resulted in a strain that produced EPA at 15% of dry cell weight. The engineered yeast lipid comprises EPA at 56.6% and saturated fatty acids at less than 5% by weight, which are the highest and the lowest percentages, respectively, among known EPA sources. Inactivation of the peroxisome biogenesis gene PEX10 was crucial in obtaining high EPA yields and may increase the yields of other commercially desirable lipid-related products. This technology platform enables the production of lipids with tailored fatty acid compositions and provides a sustainable source of EPA.

The omega-3 fatty acids, cis-5, 8, 11, 14, and 17-eicosapentaenoic acid (C20:5; EPA) and cis-4, 7, 10, 13, 16, and 19-docosahexaenoic acid (C22:6; DHA), have wide-ranging benefits in improving heart health, immune function, mental health, and infant cognitive development. Currently, the major source for EPA and DHA is from fish oil, and a minor source of DHA is from microalgae. With the increased demand for EPA and DHA, DuPont has developed a clean and sustainable source of the omega-3 fatty acid EPA through fermentation using metabolically engineered strains of Yarrowia lipolytica. In this mini-review, we will focus on DuPont’s technology for EPA production. Specifically, EPA biosynthetic and supporting pathways have been introduced into the oleaginous yeast to synthesize and accumulate EPA under fermentation conditions. This Yarrowia platform can also produce tailored omega-3 (EPA, DHA) and/or omega-6 (ARA, GLA) fatty acid mixtures in the cellular lipid profiles. Fundamental research such as metabolic engineering for strain construction, high-throughput screening for strain selection, fermentation process development, and process scale-up were all needed to achieve the high levels of EPA titer, rate, and yield required for commercial application. Here, we summarize how we have combined the fundamental bioscience and the industrial engineering skills to achieve large-scale production of Yarrowia biomass containing high amounts of EPA, which led to two commercial products, New Harvest™ EPA oil and Verlasso® salmon.

Yarrowia lipolytica is a safe and robust yeast that has a history of industrial applications. Its physiological, metabolic and genomic characteristics have made it a superior host for metabolic engineering. The results of optimizing internal pathways and introducing new pathways have demonstrated that Y. lipolytica can be a platform cell factory for cost-effective production of chemicals and fuels derived from fatty acids, lipids and acetyl-CoA. Two products have been commercialized from metabolically engineered Y. lipolytica strains producing high amounts of omega-3 eicosapentaenoic acid, and more products are on the way to be produced at industrial scale. Here we review recent progress in metabolic engineering of Y. lipolytica for production of biodiesel fuel, functional fatty acids and carotenoids.

In the oleaginous yeast Yarrowia lipolytica, de novo lipid synthesis and accumulation are induced under conditions of nitrogen limitation (or a high carbon-to-nitrogen ratio). The regulatory pathway responsible for this induction has not been identified. Here we report that the SNF1 pathway plays a key role in the transition from the growth phase to the oleaginous phase in Y. lipolytica. Strains with a Y. lipolytica snf1 (Ylsnf1) deletion accumulated fatty acids constitutively at levels up to 2.6-fold higher than those of the wild type. When introduced into a Y. lipolytica strain engineered to produce omega-3 eicosapentaenoic acid (EPA), Ylsnf1 deletion led to a 52% increase in EPA titers (7.6% of dry cell weight) over the control. Yarrowia lipolytica is one of the most extensively studied "nonconventional" yeasts with importance in multiple industrial applications (1) and has been engineered for the commercial production of omega-3 eicosapentaenoic acid (EPA) (2). Although Y. lipolytica is an oleaginous yeast capable of accumulating large amounts of lipid in the cell, lipid accumulation schemes using hydrophobic carbon sources as substrates have been reported in many cases (3-5). For de novo lipid synthesis using glucose as a carbon source, nitrogen limitation or a high carbon-to-nitrogen (C/N) ratio is the most commonly employed condition to increase intracellular lipid accumulation. However, in wild-type cells, lipids accumulate to Ͻ20% of dry cell weight (DCW) (6, 7), and it usually takes 3 to 10 days to accumulate the maximum level of lipids (7). Thus, an understanding of the regulation of lipid synthesis and accumulation will allow us to engineer this organism for increased rates of lipid accumulation, thereby significantly reducing the cost of manufacture for lipids and valuable lipid-derived compounds.Biochemical studies of various enzymes involved in de novo lipid synthesis have provided an explanation for how oleaginous microbes accumulate lipids under conditions of nitrogen limitation (reviewed in references 4 and 8). Briefly, nitrogen exhaustion in the medium results in stimulation of AMP deaminase, which breaks down AMP to IMP and ammonium in order to salvage nitrogen. The decrease in the AMP concentration inhibits isocitrate dehydrogenase, and accumulated isocitrate is equilibrated by aconitase with citrate, which then exits the mitochondria for acetyl coenzyme A (acetyl-CoA) generation by cytosolic ATPcitrate lyase (ACL). ACL activity is thought to be critical for lipid synthesis, and cytoplasmic malic enzyme was also shown to be important for lipid synthesis by supplying NADPH for fatty acid synthesis in certain oleaginous microorganisms. Although homologs of these enzymes were found in Y. lipolytica (9), there are not many biochemical studies of these enzymes in this yeast. It also remains to be examined if this mechanism is applicable to other nutritional limitations that induce lipid accumulation, such as phosphate, magnesium, or sulfur limitation.We are interested in discovering and controll...

Oleaginous yeast Yarrowia lipolytica is an important host for the production of lipidderived compounds or heterologous proteins. Selection of strong promoters and effective expression systems is critical for heterologous protein secretion. To search for a strong promoter in Y. lipolytica, activities of FBA1, TDH1 and GPM1 promoters were compared to that of TEF1 promoter by constructing GUS reporter fusions. The FBA1 promoter activity was 2.2 and 5.5 times stronger than the TDH1 and GPM1 promoters, respectively. The FBA1 IN promoter (FBA1 sequence of À826 to +169) containing an intron (+64 to +165) showed five-fold higher expression than the FBA1 promoter (À831 to À1). The transcriptional enhancement by the 5′-region within the FBA1 gene was confirmed by GPM1::FBA1 chimeric promoter construction. Using the strong FBA1 IN promoter, four different S. cerevisiae SUC2 expression cassettes were tested for the SUC + phenotype in Y. lipolytica. Functional invertase secretion was facilitated by the Xpr2 prepro-region with an additional 13 amino acids of mature Xpr2, or by the native Suc2 signal sequence. However, these two secretory signals in tandem, or the mature Suc2 with no secretory signal, did not direct secretion of functional invertase. Unlike previously reported Y. lipolytica SUC + strains, our engineered stains secreted most of invertase into the medium. Copyright

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.

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Oleaginous yeast Yarrowia lipolytica is capable of accumulating large amount of lipids. There is a growing interest to engineer this organism to produce lipid-derived compounds for a variety of applications. In addition, biosynthesis of value-added products such as carotenoid and its derivatives have been explored. In this chapter, we describe methods to integrate genes involved in lycopene biosynthesis in Yarrowia. Each bacterial gene involved in lycopene biosynthesis, crtE, crtB, and crtI, will be assembled with yeast promoters and terminators and subsequently transformed into Yarrowia through random integration. The engineered strain can produce lycopene under lipid accumulation conditions.