YALI0E32769g (DGA1) and YALI0E16797g (LRO1) encode major triacylglycerol synthases of the oleaginous yeast Yarrowia lipolytica

Abstract

The oleaginous yeast Yarrowia lipolytica has an outstanding capacity to produce and store triacylglycerols resembling adipocytes of higher eukaryotes. Here, the identification of two genes YALI0E32769g (DGA1) and YALI0E16797g (LRO1) encoding major triacylglycerol synthases of Yarrowia lipolytica is reported. Heterologous expression of either DGA1 or LRO1 in a mutant of the budding yeast Saccharomyces cerevisiae defective in triacylglycerol synthesis restores the formation of this neutral lipid. Whereas Dga1p requires acyl-CoA as a substrate for acylation of diacylglycerol, Lro1p is an acyl-CoA independent triacylglycerol synthase using phospholipids as acyl-donor. Growth of Yarrowia lipolytica strains deleted of DGA1 and/or LRO1 on glucose containing medium significantly decreases triacylglycerol accumulation. Most interestingly, when oleic acid serves as the carbon source the ratio of triacylglycerol accumulation in mutants to wild-type is significantly increased in strains defective in DGA1 but not in lro1Δ. In vitro experiments revealed that under these conditions an additional acyl-CoA dependent triacylglycerol synthase contributes to triacylglycerol synthesis in the respective mutants. Taken together, evidence is provided that Yarrowia lipolytica contains at least four triacylglycerol synthases, namely Lro1p, Dga1p and two additional triacylglycerol synthases whereof one is acyl-CoA dependent and specifically induced upon growth on oleic acid.

Fig. 1

Mechanisms of triacylglycerol synthesis. Triacylglycerols (TAG) are either formed via an acyl-CoA dependent (light gray) or acyl-CoA independent (dark gray) mechanism. Abbreviations: DAG: diacylglycerol; MAG: monoacylglycerol; PL: phospholipid.

Fig. 2

Sequence alignments and hydropathy plots of triacylglycerol synthases. Multiple alignments of acyl-CoA:diacylglycerol acyltransferases (A) and the lecithin:cholesterol acyltransferase (LCAT) related proteins (B) of the budding yeast Saccharomyces cerevisiae (SeqA), the fission yeast Schizosaccharomyces pombe (SeqB) and the oleaginous yeast Yarrowia lipolytica (SeqC). “*”: identical amino acid in all proteins; “:” identical amino acid in two polypeptides; “.” homologous amino acid. Program: http://www.ch.embnet.org/. Hydropathy plots of Dga1p and Lro1p of Yarrowia lipolytica are shown in panel C. Dga1p contains an insert of 113 (155) amino acids (marked by the black line) after the N-terminal transmembrane domain which is not present in the respective counterparts of the budding yeast and the fission yeast. Program: http://fasta.bioch.virginia.edu/.

Fig. 2

Sequence alignments and hydropathy plots of triacylglycerol synthases. Multiple alignments of acyl-CoA:diacylglycerol acyltransferases (A) and the lecithin:cholesterol acyltransferase (LCAT) related proteins (B) of the budding yeast Saccharomyces cerevisiae (SeqA), the fission yeast Schizosaccharomyces pombe (SeqB) and the oleaginous yeast Yarrowia lipolytica (SeqC). “*”: identical amino acid in all proteins; “:” identical amino acid in two polypeptides; “.” homologous amino acid. Program: http://www.ch.embnet.org/. Hydropathy plots of Dga1p and Lro1p of Yarrowia lipolytica are shown in panel C. Dga1p contains an insert of 113 (155) amino acids (marked by the black line) after the N-terminal transmembrane domain which is not present in the respective counterparts of the budding yeast and the fission yeast. Program: http://fasta.bioch.virginia.edu/.

Fig. 2

Sequence alignments and hydropathy plots of triacylglycerol synthases. Multiple alignments of acyl-CoA:diacylglycerol acyltransferases (A) and the lecithin:cholesterol acyltransferase (LCAT) related proteins (B) of the budding yeast Saccharomyces cerevisiae (SeqA), the fission yeast Schizosaccharomyces pombe (SeqB) and the oleaginous yeast Yarrowia lipolytica (SeqC). “*”: identical amino acid in all proteins; “:” identical amino acid in two polypeptides; “.” homologous amino acid. Program: http://www.ch.embnet.org/. Hydropathy plots of Dga1p and Lro1p of Yarrowia lipolytica are shown in panel C. Dga1p contains an insert of 113 (155) amino acids (marked by the black line) after the N-terminal transmembrane domain which is not present in the respective counterparts of the budding yeast and the fission yeast. Program: http://fasta.bioch.virginia.edu/.

Fig. 3

Lipid particle formation in Yarrowia lipolytica mutants defective in triacylglycerol synthesis. Fluorescence microscopic inspection of wild-type cells (A), the single deletion mutants lro1Δ (Β), dga1Δ (C) and the double deletion mutant dga1Δlro1Δ (D) grown to the stationary phase (24 h) on glucose containing media. Cells were stained with the fluorescent lipophilic dye NileRed®. Size bar: 5 μm.

Fig. 4

Triacylglycerol accumulation in strains defective in triacylglycerol synthesizing enzymes. Accumulation of triacylglycerols (TAG) in the single deletion mutants lro1Δ , dga1Δ and the double deletion mutant lro1Δdga1Δ grown on glucose containing media (white bars) and on oleic acid containing media (gray bars). The value for wild-type (glucose: 0.71 ± 0.07% TAG/CDW; oleic acid: 17.9 ± 2.1% TAG/CDW) was set to 100%. Values are mean values of three independent experiments. *: P < 0.05; **: P < < 0.0005; (Student's-t-test).

Fig. 5

Heterologous expression of DGA1 and LRO1, respectively, restores the formation of triacylglycerols. (A) Fluorescence microscopic inspection reveals that lipid particles are formed upon heterologous expression of DGA1 (c) and LRO1 (d) in the quadruple mutant lro1Δdga1Δare1Δare2Δ of the budding yeast Saccharomyces cerevisiae otherwise lacking lipid particles (b). Panel a shows lipid particle formation in the corresponding wild-type strain of the quadruple mutant bearing the empty vector. Size bar: 5 μm. (C) Analysis of the lipid pattern of the quadruple mutant heterologously expressing either LRO1 (lane 4) or DGA1 (lane 5) and the quadruple mutant bearing the empty vector (lane 3). Lane 1: standard containing ergosterol (E), ergosteryl oleate (STE) and triolein (TAG); Lane 2: lipid extract of total cells of the corresponding wild-type strain bearing the empty vector. Solvent system: petroleum ether-diethyl ether-acetic acid 70:30:2 (per vol.). (B) To verify the absence of steryl esters in strains with quadruple mutant background lipids were additionally separated with a solvent system allowing the separation of squalene (S) and STE. Solvent systems: petroleum ether-diethyl ether-acetic acid (25:25:1; per vol.; development of the TLC plate to 1/3 of the distance) and petroleum ether-diethyl ether (49:1; vol./vol.; development to the full distance). Post-chromatographic staining was performed as described in Materials and methods. Note: The amounts of lipids separated by thin-layer chromatography were extracted from equal amounts of homogenates based on the protein content.

Fig. 6

Mechanism of triacylglycerol synthesis. Homogenates (A) or microsomes (B) of Saccharomyces cerevisiae wild-type cells bearing the empty vector (WT + pYES2) and the quadruple mutants lro1Δdga1Δare1Δare2Δ heterologously expressing DGA1 (QM + DGA1) and LRO1 (QM + LRO1), respectively, were used as enzyme sources in TAG synthase assays containing either oleoyl-CoA (A) or phospholipids (B) as a substrate. Activity of wild-type fractions was set to 1 (A_spec “WT + pYES2” with oleoyl-CoA as substrate (A): 10.2 ± 0.3 pmol/mg ∗ min; A_spec “WT + pYES2” with phospholipids as substrate (B): 11.8 ± 2.4 pg/mg ∗ min). Enrichment data were obtained from measurements of three independent data-sets. Note: In contrast to acyl-CoA independent TAG synthase activity (B), acyl-CoA dependent TAG synthase activity (A) is not restricted to microsomes (see Discussion). Thus, homogenates instead of microsomes were chosen as enzyme source in (A). No enzymatic activity was detected with the respective fractions of the quadruple mutant bearing the empty plasmid in the acyl-CoA dependent and acyl-CoA independent assay, which is in line with the lack of neutral lipids and lipid particles (see Fig. 5).

Fig. 7

In vitro triacylglycerol synthase activities of homogenates of cells defective in DGA1 and/or LRO1. Homogenates of Yarrowia lipolytica cells grown either on glucose (A) or oleic acid (B) containing media were used as an enzyme source in triacylglycerol synthase assays specific for acyl-CoA independent (black bars) and acyl-CoA dependent (white bars) acylation of diacylglycerol (see Materials and Methods). The specific activity of wild-type samples was set to 100% (A_spec “wt” with oleoyl-CoA as substrate (white bars): 10.3 ± 1.8 pmol/mg ∗ min (glucose; A); 29.7 ± 1.5 pmol/mg min (oleic acid; B); A_spec “wt” with phospholipids as substrate (black bars): 1.1 ± 0.05 pg/mg min (glucose; A); 0.74 ± 0.1 pg/mg ∗ min (oleic acid; B); mean values of at least 3 independent experiments).