Identification and characterization of DGA2, an acyltransferase of the DGAT1 acyl-CoA:diacylglycerol acyltransferase family in the oleaginous yeast Yarrowia lipolytica. New insights into the storage lipid metabolism of oleaginous yeasts

Abstract

Triacylglycerols (TAG) and steryl esters (SE) are the principal storage lipids in all eukaryotic cells. In yeasts, these storage lipids accumulate within special organelles known as lipid bodies (LB). In the lipid accumulation-oriented metabolism of the oleaginous yeast Yarrowia lipolytica, storage lipids are mostly found in the form of TAG, and only small amounts of SE accumulate. We report here the identification of a new DAG acyltransferase gene, DGA2, homologous to the ARE genes of Saccharomyces cerevisiae. This gene encodes a member of the type 1 acyl-CoA:diacylglycerol acyltransferase family (DGAT1), which has not previously been identified in yeasts, but is commonly found in mammals and plants. Unlike the Are proteins in S. cerevisiae, Dga2p makes a major contribution to TAG synthesis via an acyl-CoA-dependent mechanism and is not involved in SE synthesis. This enzyme appears to affect the size and morphology of LB, suggesting a direct role of storage lipid proteins in LB formation. We report that the Are1p of Y. lipolytica was essential for sterol esterification, as deletion of the encoding gene (ARE1) completely abolished SE synthesis. Unlike its homologs in yeasts, YlARE1 has no DAG acyltransferase activity. We also reconsider the role and function of all four acyltransferase enzymes involved in the final step of neutral lipid synthesis in this oleaginous yeast.

Fig. 1

Schematic representation of strain constructions. The auxotrophic strain Po1d (Leu⁻ Ura⁻) was derived from wild-type strain W29. Strain JMY1201, carrying a disrupted LRO1 gene (lro1::URA3), was obtained by introducing the LRO1-PUT cassette into Po1d. Strains JMY1204 and JMY1613, carrying disruptions of DGA1 (dga1::URA3) and ARE1 (are1::URA3), respectively, were constructed similarly. The JMY1636 strain, carrying a DGA2 gene disruption (dga2::LEU2), was then obtained by transforming Po1d with the DGA2-PLT disruption cassette. Additional deletions of genes encoding acyltransferases were obtained by successive gene disruption and marker rescue as described by Fickers et al. (2003): (a) The JMY1204 marker was rescued after transformation with the replicative plasmid pRRQ2, followed by plasmid curing, yielding the strain JMY1217; (b) the LRO1 gene was deleted with the LRO1-PUT disruption cassette, yielding the strain JMY1281; (c) the marker was rescued as above, giving rise to strain JMY 1329; (d) the ARE1 gene was deleted with the ARE1-PUT disruption cassette, giving rise to strain JMY1610; and (e) the DGA2 gene was deleted with the DGA2-PLT disruption cassette, giving rise to the prototrophic JMY1631 quadruple mutant strain. Marker excision gave rise to the auxotrophic strain JMY1877 carrying quadruple deletions (Q₄). Each of the acyltransferase-coding genes under the pTEF constitutive promoter was introduced into strain JMY1877 (Q₄) giving JMY1882 (Q₄-pTEF-LRO1-URA3ex), JMY1884 (Q₄-pTEF-DGA2-URA3ex), JMY1892 (Q₄-pTEF-DGA1-URA3ex), and JMY1988 (Q₄-pTEF-ARE1-URA3ex) overexpression mutants. Similarly, the prototrophic ∆are1∆dga2 double mutant strain JMY1634 was constructed by deleting the DGA2 gene from strain JMY1613 with the DGA2-PLT disruption cassette

Fig. 2

Biosynthesis of triacylglycerols and steryl esters in Y. lipolytica. G-3-P glycerol-3- phosphate, DHAP dihydroxyacetone phosphate, LPA lysophosphatidic acid, PA phosphatidic acid, DAG diacylglycerol, TAG triacylglycerol, PL phospholipid SE steryl ester, FFA free fatty acids

Fig. 3

Dendrogram of the DGAT2 and ACAT membrane-bound families. The protein sequences deduced from the various full-length cDNAs of species belonging to three different kingdoms were aligned and bootstrapped with the ClustalX program (www.clustal.org). The tree was then generated using the NJplot program (Perrière and Gouy 1996). The proteins sequences used were DGAT1 (NP03611.2), DGAT2 (NP115953.1), and ACAT1 (NP003092.4) sequences from humans (Homo sapiens); DGAT1 (NP034176.1), DGAT2 (NP080660.1), and ACACT (NP033256.2) from the mouse (Mus musculus); the DGAT1 (XP002514132.1) and DGAT2 (XP002528531.1) sequences from the castor bean (Ricinus communis); the DGAT1 (NP179535.1) and DGAT2 (NM115011.3) sequences from Arabidopsis (Arabidopsis thaliana); the DGAT1 (AAG23696.1) sequences from the plant P. frutescens; the DGA1 (NP014888.1), ARE1 (CAY78257.1), and ARE2 (NP014416.1) sequences from the baker’s yeast (S. cerevisiae); and the DGA1 (XP504700.1), DGA2 (XP502557.1), and ARE1 (XP505086.1) sequences from Y. lipolytica. GenBank accession numbers are provided in parentheses. Note that sterol-related ACAT plant proteins were omitted due insufficient annotation

Fig. 4

A TAG and FFA lipid fractions of wild-type (Po1d) and single acyltransferase gene overexpression strains in the Q₄ background: I beginning of the stationary phase (11 h); II late stationary phase (48 h). B TAG and FFA lipid fractions from wild-type, single, double, triple, and quadruple mutant strains in I beginning of the stationary phase (11 h) and II late stationary phase (48 h). The presence or absence of a particular gene in each strain is indicated in the table at the bottom of the figure. Light gray bars indicate the amount of FFA; dark gray bars indicate the amount of TAG. The first row indicates the amount of lipids (FFA and TAG) as a percentage of yeast dry weight. The second row shows the amount of TAG found in mutant strains as a percentage of the TAG found in the wild-type strain. The third row shows the amount of FFA found in mutant strains as a percentage of the FFA found in the wild-type strain. Lipid quantification data in A and B represent mean values from three independent experiments

Fig. 5

Neutral lipids separated by TLC. Plates were overloaded for visualization of the minor SE fraction, which accounts for less than 5% of total lipids in Y. lipolytica. Lipids were extracted using the Folch method from the following strains: Po1d; JMY1613 (Δare1); JMY1636 (Δdga2); JMY1634 (Δare1Δdga2); JMY1610 (Δdga1Δlro1Δare1). A, B, and C deposits correspond to the lipid extracts at 11, 24, and 48 h of culture, respectively. M TLC marker containing S sterols, FFA free fatty acids, TAG triacylglycerols, FAME fatty acid methyl esters, and SE steryl esters. FAME are artifacts produced during the Folch extraction procedure. The box is used to highlight the SE fraction

Fig. 6

Expression of the acyltransferase genes at 7 and 24 h of culture in media containing 2% glucose or 3% oleic acid (O.A.). Total RNA was isolated from the Po1d wild-type strain. Reverse transcription-quantitative PCR was carried out, with gene-specific primers (Table 2), for expression analysis. Three independent cDNA preparations obtained in independent experiments were used for the analysis

Fig. 7

Observation of the phenotypes of the disrupted mutants after 24 h of culture in media containing 3% oleic acid. Fluorescence microscopy (left) with BodiPy neutral lipid staining, visual microscopy (middle), and combined photomicrographs (right) of A Po1d, B quadruple mutant, C ∆are1, D ∆dga1∆lro1, E ∆dga1∆lro1∆are1, F ∆dga2, and G ∆are1∆dga2 disrupted strains