Snf1 Is a Regulator of Lipid Accumulation in Yarrowia lipolytica

Abstract

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...

Combinatorial Engineering of Yarrowia lipolytica as a Promising Cell Biorefinery Platform for the de novo Production of Multi-Purpose Long Chain Dicarboxylic Acids

“…SNF2 deletion has been found to enhance lipid accumulation [76]. Inactivation of the SNF1 gene led to an enhanced constitutive fatty acid accumulation and citric acid production in Y. lipolytica [66,77]. An alternative strategy to enhance carbon flux re-direction from citric acid production to fatty acid biosynthesis is to overexpress ACC1 or use hyperactive Acc1p [67,78].…”

Lipid accumulation from glucose and xylose in an engineered, naturally oleaginous strain of Saccharomyces cerevisiae

“…Engineering of the S. cerevisiae D5A strain for xylose utilization has been previously described (Hector et al, 2011), however, our strategy differed in that we targeted integration of our three-gene expression cassette to knock-out a negative regulator of a key pentose transporter (Gal2p) to ensure effective expression of this transporter in the presence of glucose. We then further engineered BFY692 for increased lipid accumulation by deleting the global regulator SNF1 as it has previously been shown to play an important role in lipid accumulation in both S. cerevisiae and Y. lipolytica (Chumnanpuen et al, 2012;Seip et al, 2013). This required a two-step process as D5A is a diploid yeast (Bailey et al, 1982).…”

“…In S. cerevisiae, SNF1 encodes an ADP-activated serine/threonine kinase (Mayer et al, 2011) that, in addition to its role in regulating carbon homeostasis and general stress responses, regulates genes involved in lipid synthesis and nitrogen metabolism (Usaite et al, 2009;Chumnanpuen et al, 2012) and has been shown to regulate the first committed step of fatty acid synthesis by directly phosphorylating and thus inactivating acetyl-CoA carboxylase (Mitchelhill et al, 1994;Woods et al, 1994). Snf1 -mutants have also been shown to be defective in glycogen synthesis (Thompson-Jaeger et al, 1991; Hardie et al, 1998) and β-oxidation of lipids (Zaman et al, 2008;Usaite et al, 2009), and have shown up-regulation of genes involved in lipid biosynthesis particularly those leading to an increase in the acetyl-CoA pool and the downstream fatty acid synthases and glycerol-3-phosphate dehydrogenase that work in concert to turn that pool into neutral lipids (Seip et al, 2013). In addition, a SNF1 knock-out in the oleaginous yeast Yarrowia lipolytica showed increased fatty acid accumulation over that of wild type in both nitrogen replete and deplete conditions (Seip et al, 2013).…”

“…Snf1 -mutants have also been shown to be defective in glycogen synthesis (Thompson-Jaeger et al, 1991; Hardie et al, 1998) and β-oxidation of lipids (Zaman et al, 2008;Usaite et al, 2009), and have shown up-regulation of genes involved in lipid biosynthesis particularly those leading to an increase in the acetyl-CoA pool and the downstream fatty acid synthases and glycerol-3-phosphate dehydrogenase that work in concert to turn that pool into neutral lipids (Seip et al, 2013). In addition, a SNF1 knock-out in the oleaginous yeast Yarrowia lipolytica showed increased fatty acid accumulation over that of wild type in both nitrogen replete and deplete conditions (Seip et al, 2013).…”

Yarrowia lipolytica as an Oleaginous Cell Factory Platform for Production of Fatty Acid-Based Biofuel and Bioproducts

“…In addition, the maximum level of intracellular lipid accumulation in resting cells is usually achieved over several days (Seip et al, 2013), making this process time-consuming. Therefore, any attempt to enhance the growth and accumulation rate will result in a higher overall productivity.…”