We previously found that SNF2, a gene encoding a transcription factor forming part of the SWI/SNF (switching/sucrose non-fermenting) chromatin-remodelling complex, is involved in lipid accumulation, because the Δsnf2 disruptant of Saccharomyces cerevisiae has a higher lipid content. The present study was conducted to identify other factors that might further increase lipid accumulation in the Δsnf2 disruptant. First, expression of LEU2 (a gene encoding β-isopropylmalate dehydrogenase), which was used to select transformed strains by complementation of the leucine axotroph, unexpectedly increased both growth and lipid accumulation, especially in the Δsnf2 disruptant. The effect of LEU2 expression on growth and lipid accumulation could be reproduced by adding large amounts of leucine to the culture medium, indicating that the effect was not due to Leu2p (β-isopropylmalate dehydrogenase) itself, but rather to leucine biosynthesis. To increase lipid accumulation further, genes encoding the triacylglycerol biosynthetic enzymes diacylglycerol acyltransferase (DGA1) and phospholipid:diacylglycerol acyltransferase (LRO1) were overexpressed in the Δsnf2 disruptant. Overexpression of DGA1 significantly increased lipid accumulation, especially in the Δsnf2 disruptant, whereas LRO1 overexpression decreased lipid accumulation in the Δsnf2 disruptant. Furthermore, the effect of overexpression of acyl-CoA synthase genes (FAA1, FAA2, FAA3 and FAA4), which each supply a substrate for Dga1p (diacylglycerol acyltransferase), was investigated. Overexpression of FAA3, together with that of DGA1, did not further increase lipid accumulation in the Δsnf2 disruptant, but did enhance lipid accumulation in the presence of exogenous fatty acids. Lastly, the total lipid content in the Δsnf2 disruptant transformed with DGA1 and FAA3 overexpression vectors reached approx. 30%, of which triacylglycerol was the most abundant lipid. Diacylglycerol acyltransferase activity was significantly increased in the Δsnf2 disruptant strain overexpressing DGA1 as compared with the wild-type strain overexpressing DGA1; this higher activity may account for the prominent increase in lipid accumulation in the Δsnf2 disruptant with DGA1 overexpression. The strains obtained have a lipid content that is high enough to act as a model of oleaginous yeast and they may be useful for the metabolic engineering of lipid production in yeast.
Nucleotide Sequence of the Gene Encoding β-Isopropylmalate Dehydrogenase (leuB) fromBacteroides fragilis
