Construction of Acyltransferase Overexpression Mutants in Yarrowia lipolytica

For the construction of the single acyltransferase overexpression mutants (LRO1, ARE1, DGA1, DGA2), the genes were placed under the control of the yeast TEF constitutive promoter (Muller et al. 1998 (link)). Acyltransferase genes were amplified by PCR using Y. lipolytica W29 genomic DNA as the template and the corresponding primer pairs (Table 2) as described in the previous section. Acyltransferase-coding genes were then inserted between the BamHI-AvrII sites of the expression vector pTEF, JMP62 (JME 803) containing the selective marker URA3 (Nicaud et al. 2002 (link)). Plasmids were digested with NotI and used to transform the quadruple mutant Y. lipolytica strain JMY1877 by the lithium acetate method (Barth et al. 2003 ). Transformants were selected on YNBcasa medium. A schematic representation of the strains constructed is depicted in Fig. 1.

Table 3

Genes involved in storage lipid synthesis in Y. lipolytica and S. cerevisiae. S. cerevisiae gene names and Enzyme Commission numbers, Y. lipolytica orthologs (gene name), and corresponding functions

Gene	SC name	YL name	EC number	Function
SCT1	YBL011w	YALI0C00209g	EC 2.3.1.15	Glycerol-3-phosphate acyltransferase
SLC1	YDL052c	YALI0E18964g	EC 2.3.1.51	1-acyl-sn-glycerol-3-phosphate acyltransferase
PAP	YMR165C		EC 3.1.3.4	Phosphatidate phosphatase
DGA1	YOR245c	YALI0E32769g	EC 2.3.1.20	Diacylglycerol acyltransferase (DGAT2)
LRO1	YNR008w	YALI0E16797g	EC 2.3.1.158	Phospholipid:diacylglycerol acyltransferase
TGL3	YMR313c	YALI0D17534g	EC 3.1.1.3	Triacylglycerol lipase
TGL4	YKR089c	YALI0F10010g	EC 3.1.1.3	Triacylglycerol lipase
TGL5	YOR081c		EC 3.1.1.3	Triacylglycerol lipase
ARE1	YCR048w	YALI0F06578g	EC 2.3.1.26	Acyl-CoA:sterol acyltransferase
ARE2	YNR019w		EC 2.3.1.26	Acyl-CoA:sterol acyltransferase
DGA2		YALI0D07986g		Diacylglycerol acyltransferase (DGAT1)
TGL1	YKL140w	YALI0E32035g	EC 3.1.1.13	Cholesterol esterase

Bioinformatic data were obtained from the Saccharomyces Genome Database (http://www.yeastgenome.org/) and the Genolevures database (http://cbi.labri.fr/Genolevures/)

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Beopoulos A., Haddouche R., Kabran P., Dulermo T., Chardot T, & Nicaud J.M. (2011). 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. Applied Microbiology and Biotechnology, 93(4), 1523-1537.

Publication 2011

Acyltransferase Diacylglycerol Enzyme Gene Genome Glycerol Lipid synthesis Lithium acetate Phosphate acyltransferase Plasmids Primer S cerevisiae Saccharomyces Sterol Strains Vector Y lipolytica

Corresponding Organization :

Other organizations : Centre National de la Recherche Scientifique, Institut National de la Recherche Agronomique

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Variable analysis

independent variables

Overexpression of acyltransferase genes (LRO1, ARE1, DGA1, DGA2)

dependent variables

Not explicitly mentioned

control variables

Yeast TEF constitutive promoter used to control expression of acyltransferase genes
Selective marker URA3 used in the expression vector pTEF, JMP62 (JME 803)
Quadruple mutant Y. lipolytica strain JMY1877 used as the host strain

controls

No positive or negative controls were explicitly mentioned in the protocol.

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