DGA1 (diacylglycerol acyltransferase gene) overexpression and leucine biosynthesis significantly increase lipid accumulation in the Δsnf2 disruptant of Saccharomyces cerevisiae

Abstract

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 Deltasnf2 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 Deltasnf2 disruptant. First, expression of LEU2 (a gene encoding beta-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 Deltasnf2 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 (beta-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 Deltasnf2 disruptant. Overexpression of DGA1 significantly increased lipid accumulation, especially in the Deltasnf2 disruptant, whereas LRO1 overexpression decreased lipid accumulation in the Deltasnf2 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 Deltasnf2 disruptant, but did enhance lipid accumulation in the presence of exogenous fatty acids. Lastly, the total lipid content in the Deltasnf2 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 Deltasnf2 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 Deltasnf2 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.

Combinatorial Engineering of Yarrowia lipolytica as a Promising Cell Biorefinery Platform for the de novo Production of Multi-Purpose Long Chain Dicarboxylic Acids

“…In addition to that, our preliminary bioreactor fermentation using YNB-Ura, without additional leucine supplementation, resulted in less biomass and LCDCA production from the M strain (Biomass up to 7 g/L and maximum LCDCA of 3 g/L). Similalrly, addition of leucine was reported to increase the growth and lipid accumulation in S. cerevisiae devoid of active SNF2 [105].…”

Lipid accumulation from glucose and xylose in an engineered, naturally oleaginous strain of Saccharomyces cerevisiae

“…Total lipid production from glycerol was less than 12% dcw but represented nearly a doubling over empty vector control (Yu et al, 2013), and up to 17% dcw lipids, a four-fold increase by over-expression of FAS1, FAS2, and ACC1 with small amounts (up to 4 g/L) of ethanol being produced (Runguphan and Keasling, 2014). Previous work has shown that a SNF2 delete strain with over-expression of DGA1 or a DGA1 variant lacking the N-terminal 29 amino acids could accumulate lipids up to 45% and 50% dcw on 5% and 10% glucose, respectively (Kamisaka et al, 2007;Kamisaka et al, 2013). The gene SNF2 codes for an ATPase subunit of the SWI-SNF chromatin remodeling complex (Peterson and Tamkun, 1995).…”

Bioengineering Recombinant Diacylglycerol Acyltransferases

“…Animals with reduced DGAT activity are resistant to diet-induced obesity Smith et al, 2000) and lack milk production (Smith et al, 2000). Over-expression of DGAT enzymes increases TAG content in plants (Andrianov et al, 2010;Bouvier-Nave et al, 2000;Burgal et al, 2008;Durrett et al, 2010;Jako et al, 2001;Lardizabal et al, 2008;Xu et al, 2008), animals (Kamisaka et al, 2010;Liu et al, 2009;Liu et al, 2007;Roorda et al, 2005), and yeast (Kamisaka et al, 2007). DGATs have nonredundant functions in TAG biosynthesis in species such as mice (Stone et al, 2004) and tung tree (Vernicia fordii) (Shockey et al, 2006).…”