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. Other components of the Y. lipolytica SNF1 pathway were also identified, and their function in limiting fatty acid accumulation is suggested by gene deletion analyses. Deletion of the gene encoding YlSnf4, YlGal83, or YlSak1 significantly increased lipid accumulation in both growth and oleaginous phases compared to the wild type. Furthermore, microarray and quantitative reverse transcription-PCR (qRT-PCR) analyses of the Ylsnf1 mutant identified significantly differentially expressed genes during de novo lipid synthesis and accumulation in Y. lipolytica. Gene ontology analysis found that these genes were highly enriched with genes involved in lipid metabolism. This work presents a new role for Snf1/AMP-activated protein kinase (AMPK) pathways in lipid accumulation in this oleaginous yeast.

Fig 1

Growth of Y. lipolytica strains on different carbon sources. Wild-type strain ATCC 20362 and the isogenic Ylsnf1 deletion mutant were grown on synthetic medium plates with 2% glucose (A), 2% glycerol (B), 1% ethanol (C), or 0.1% oleate (D) as the sole carbon source. In each panel, the streak at the top is ATCC 20362, and that at the bottom is the Ylsnf1 mutant. Cells were grown at 30°C and photographed after 2 days for glucose, 3 days for glycerol and ethanol, and 5 days for oleate plates.

Fig 2

Total fatty acid (TFA) and EPA accumulation and organic acid excretion of strain Y4184 and the Y4184 Ylsnf1 mutant. (A and B) Cells were grown for 2 days in SD medium (A) or in FM medium (B) for the samples at day 0. In both panels A and B, subsequent time points (day 1 to day 5) were sampled from HGM cultures. Plots for Y4184 and the Y4184 Ylsnf1 mutant are represented by circles and squares, respectively. Filled symbols with solid lines are total fatty acid content (TFA% DCW), open symbols with solid lines are EPA weight percentage of total fatty acids (EPA% TFA), and filled symbols with dotted lines indicate DCW. Values are the averages of two independent experiments, with standard errors of <15%. (C) Amount of organic acids excreted into the culture medium (g/liter), measured after cells were grown in SD medium for 2 days followed by HGM for 5 days. αKG, α-ketoglutarate. Values are averages from at least two different isolates. Standard deviations are shown as error bars.

Fig 3

TFA accumulation of mutants in the SNF1 complex or pathway. All strains are from the Y4184 background. Filled bars indicate total fatty acid content (TFA% DCW) from cells grown in SD growth medium for 2 days, and open bars indicate data from cells grown in HGM for 5 days. The values plotted are the averages of replicate experiments with 2 to 10 independent isolates for each strain. Standard deviations are shown as error bars. Asterisks indicate that the TFA content of the mutant is statistically significantly different from that of wild-type (WT) strain Y4184 under a given culture condition (P value of <0.05, determined by paired, two-tailed Student's t test).

Fig 4

Metabolic pathway for triacylglycerol (TAG) synthesis. Based on microarray and qRT-PCR analyses (Table 6), genes that were differentially upregulated in the Ylsnf1 mutant are shown. FBP, fructose 1,6-bisphosphate; GAD-3-P, glycerol aldehyde 3-phosphate; DHAP, dihydroxyacetone phosphate.