Control of lipid accumulation in the yeast Yarrowia lipolytica

Abstract

A genomic comparison of Yarrowia lipolytica and Saccharomyces cerevisiae indicates that the metabolism of Y. lipolytica is oriented toward the glycerol pathway. To redirect carbon flux toward lipid synthesis, the GUT2 gene, which codes for the glycerol-3-phosphate dehydrogenase isomer, was deleted in Y. lipolytica in this study. This Delta gut2 mutant strain demonstrated a threefold increase in lipid accumulation compared to the wild-type strain. However, mobilization of lipid reserves occurred after the exit from the exponential phase due to beta-oxidation. Y. lipolytica contains six acyl-coenzyme A oxidases (Aox), encoded by the POX1 to POX6 genes, that catalyze the limiting step of peroxisomal beta-oxidation. Additional deletion of the POX1 to POX6 genes in the Delta gut2 strain led to a fourfold increase in lipid content. The lipid composition of all of the strains tested demonstrated high proportions of FFA. The size and number of the lipid bodies in these strains were shown to be dependent on the lipid composition and accumulation ratio.

FIG. 1.

Overview of different pathways involved in FA synthesis and storage and degradation of neutral lipids. Synthesis of FA (Acyl-CoA) is catalyzed by the FA synthesis complex from the basic blocks acetyl-CoA and malonyl-CoA. Acyl-CoA can be stored either as SE or as TAG. The synthesis of SE is catalyzed by SE synthases homologous to the human acyl-CoA:cholesterol acyltransferase and their mobilization by the SE hydrolases, releasing sterol and FFA. The synthesis of TAG require acyl-CoA and G3P. G3P could be produced from glycerol or from DHAP. GUT1 encodes a glycerol kinase that converts glycerol to G3P in the cytosol. The product of this reaction can be oxidized to DHAP by the G3PDH encoded by the GUT2 gene. DHAP can enter either glycolysis or gluconeogenesis. G3P could also be used as a skeleton for TAG synthesis. Three acyls are added to the G3P backbone to give TAG, and this process requires four enzymatic steps. First, an acyl is added at the sn-1 position of G3P by a G3P acyltransferase to produce LPA, and then a second acyl is added at the sn-2 position by a 1-acyl G3P acyltransferase to produce PA, which is then dephosphorylated by PAP, yielding DAG. Finally, the third acyl can be added at the sn-3 position either by the acetyl-CoA-dependent pathway (directly from acyl-CoA) or by the acetyl-CoA-independent pathway (from a glycerophospholipid). TAG can be mobilized by the conversion to FFA and DAG upon hydrolysis by TAG lipase. The FFA can then be degraded in the β-oxidation pathway. This pathway requires four enzymatic steps (for a review, see reference 11). In Y. lipolytica, six genes (POX1 to POX6) that code for acyl-CoA oxidases are involved in the second step of β-oxidation (11, 29). Proteins encoded by the genes in parentheses were found to be associated with lipid particles (LB) in Y. lipolytica.

FIG. 2.

Schematic representation of strain construction. Auxotrophic strain Po1d (Leu⁻ Ura⁻) was derived from WT strain W29. Strain JMY1202, carrying a disrupted G3PDH gene (gut2::URA3), was obtained by transformation of the PUTgut2 cassette into Po1d. Strain MTLY40, which contains four deletions of POX genes that code for acyl-CoA oxidases, was constructed by successive gene disruptions (21, 29). Additional deletions of the two remaining POX genes (POX1 and POX6) were introduced by successive gene disruptions and marker rescue according to Fickers and coworkers (12); i.e., (i) a deletion of the LEU2 gene (leu2::hyg) was introduced into MTLY40; (ii) the marker was rescued after transformation with replicative plasmid pRRQ2, followed by plasmid loss; (iii) the POX1 gene was deleted with the PHTpox1 disruption cassette; (iv) the marker was rescued as in step ii; (v) the POX6 gene was deleted with the PHTpox6 disruption cassette; (vi) the marker was rescued as for step ii; and finally (vii) the G3PDH gene was deleted. Strain JMY1351 contained complete deletions of the six POX genes (Δpox1-6), together with the GUT2 deletion. Control strain JMY330 was obtained by transformation with the p62-URA3 cassette containing the URA3 marker from plasmid JMP62.

FIG. 3.

Growth (A) and total lipid accumulation (B) in the WT strain and in mutant strains with altered GUT2 and POX genotypes in YNBD_0.5O₃. The growth of the WT and mutant strains listed in Table 2 was monitored over time (A). TFA accumulation, expressed as a percentage of cell DW, is shown in panel B. Symbols: circles, WT strain; squares, Δgut2 mutant strain; triangles, Δgut2 Δpox1-6 mutant strain. Closed symbols correspond to growth expressed as optical density at 600 nm. Open symbols correspond to TFA content. The results are mean values from three independent experiments. The standard deviations were <10% of the respective values.

FIG. 4.

TFA content at 24 h. The TFA contents of the WT and mutant strains are expressed as percentages of cell DW at the end of cell cultivation (24 h). Strains were grown on YNBD, YNBD_0.5O₃, and YNBD_0.5O₃up. The results represent mean values from three independent experiments. Open bars, WT strain; hatched bars, Δgut2 strain; black bars, Δgut2 Δpox1-6 strain; pur, ultrapure (>98% purity).

FIG. 5.

LB phenotypes of mutant strains. LB morphologies of the WT (A, B), Δgut2 (C, D), and Δgut2 Δpox1-6 (E, F) strains are shown. Light microscopy images are shown in panels A, C, and E, and fluorescence microscopy images of Nile red-stained cells are shown in panels B, D, and F. Strains were grown for 24 h on YNBD_0.5O₃ medium.

FIG. 6.

Changes in LB number and cell area occupied by LB over time. The mean LB counts over time in the WT and mutant strains are shown in panel A. Panel B represents the percentage of the cell area occupied by LB. Strains were grown on YNBD_0.5O₃ medium. Standard deviations of measurements are also shown. Open bars, WT strain; hatched bars, Δgut2 strain; black bars, Δgut2 Δpox1-6 strain.

FIG. 7.

TAG and FFA contents as fractions of total lipids. The graph represents the total lipid accumulation over time (11 and 24 h) as a percentage of the DW of cells grown on YP₂D₄O₃ medium. Lipids were fractionated into TAG and FFA by solid-phase extraction, and quantification was done by GC.