title: DGA1 (diacylglycerol acyltransferase gene) overexpression and leucine biosynthesis significantly increase lipid accumulation in the Deltasnf2 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.
authors: Lipid Engineering Research Group, Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566, Japan. kamisaka.y@aist.go.jp
authors: Yasushi
authors: Y
authors: Kamisaka
authors: Nao
authors: N
authors: Tomita
authors: Kazuyoshi
authors: K
authors: Kimura
authors: Kumiko
authors: K
authors: Kainou
authors: Hiroshi
authors: H
authors: Uemura
mesh_terms: D000251:Adenosine Triphosphatases; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D051048:Diacylglycerol O-Acyltransferase / Q000235:genetics / Q000378:metabolism*; D005227:Fatty Acids / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D005822:Genetic Vectors / Q000235:genetics; D007930:Leucine / Q000096:biosynthesis*; D050356:Lipid Metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; D005227:Fatty Acids; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C494880:DGA1 protein, S cerevisiae; D051048:Diacylglycerol O-Acyltransferase; D000251:Adenosine Triphosphatases; C499333:SNF2 protein, S cerevisiae; D007930:Leucine
references: Biochim Biophys Acta. 1999 May 18;1438(2):185-98
references: 10320801
references: FEBS Lett. 1999 Jun 11;452(3):145-9
references: 10386579
references: Eur J Biochem. 2000 Jan;267(1):85-96
references: 10601854
references: J Biol Chem. 2000 May 26;275(21):15609-12
references: 10747858
references: Prog Lipid Res. 1999 Sep-Nov;38(5-6):361-99
references: 10793889
references: Proc Natl Acad Sci U S A. 2000 Jun 6;97(12):6487-92
references: 10829075
references: Biochim Biophys Acta. 2000 Sep 18;1469(2):101-20
references: 10998572
references: Plant Physiol. 2001 Jun;126(2):861-74
references: 11402213
references: Prog Lipid Res. 2001 Sep;40(5):325-438
references: 11470496
references: J Biol Chem. 2001 Oct 5;276(40):37051-9
references: 11477098
references: J Biol Chem. 2001 Oct 19;276(42):38862-9
references: 11481333
references: Microbiology. 2001 Oct;147(Pt 10):2857-64
references: 11577164
references: J Biol Chem. 2002 Feb 22;277(8):6478-82
references: 11741946
references: J Bacteriol. 2002 Jan;184(2):519-24
references: 11751830
references: J Biol Chem. 2002 Mar 15;277(11):8877-81
references: 11751875
references: Adv Appl Microbiol. 2002;51:1-51
references: 12236054
references: Cell. 2002 Oct 18;111(2):155-8
references: 12408859
references: Microbiol Mol Biol Rev. 2003 Mar;67(1):1-15, table of contents
references: 12626680
references: Appl Microbiol Biotechnol. 2004 Feb;63(6):635-46
references: 14586578
references: Prog Lipid Res. 2004 Mar;43(2):134-76
references: 14654091
references: Biochem Biophys Res Commun. 2004 Jan 9;313(2):397-403
references: 14684175
references: J Microbiol Methods. 2004 Mar;56(3):331-8
references: 14967224
references: J Biochem. 2004 Feb;135(2):269-76
references: 15047730
references: Biochem Soc Trans. 2005 Nov;33(Pt 5):1174-7
references: 16246075
references: J Biol Chem. 2006 Apr 7;281(14):9210-8
references: 16467296
references: Biosci Biotechnol Biochem. 2006 Mar;70(3):646-53
references: 16556980
references: Yeast. 2006 Jun;23(8):605-12
references: 16823888
references: Lipids. 2006 Dec;41(12):1073-88
references: 17269553
references: Biochim Biophys Acta. 1989 Jul 17;1004(1):1-19
references: 2663077
references: Lipids. 1993 Jul;28(7):583-7
references: 27519880
references: Proc Natl Acad Sci U S A. 1979 Sep;76(9):4390-4
references: 41242
references: J Bacteriol. 1983 Jan;153(1):163-8
references: 6336730
references: Genetics. 1984 Dec;108(4):845-58
references: 6392017
references: J Biochem. 1995 Feb;117(2):362-8
references: 7608126
references: Trends Biochem Sci. 1995 Apr;20(4):143-6
references: 7770913
references: J Biol Chem. 1994 Jun 10;269(23):16348-56
references: 8206942
references: Prog Lipid Res. 1996;35(2):169-201
references: 8944226
references: Anal Biochem. 1976 May 7;72:248-54
references: 942051
references: Yeast. 1998 Dec;14(16):1471-510
references: 9885152