Metabolic engineering ofSaccharomyces cerevisiaeto produce a reduced viscosity oil from lignocellulose.
BACKGROUND
Acetyl-triacylglycerols (acetyl-TAGs) are unusual triacylglycerol (TAG) molecules that contain ansn-3 acetate group. Compared to typical triacylglycerol molecules (here referred to as long chain TAGs; lcTAGs), acetyl-TAGs possess reduced viscosity and improved cold temperature properties, which may allow direct use as a drop-in diesel fuel. Their different chemical and physical properties also make acetyl-TAGs useful for other applications such as lubricants and plasticizers. Acetyl-TAGs can be synthesized byEaDAcT, a diacylglycerol acetyltransferase enzyme originally isolated fromEuonymus alatus(Burning Bush). The heterologous expression ofEaDAcT in different organisms, includingSaccharomyces cerevisiae, resulted in the accumulation of acetyl-TAGs in storage lipids. Microbial conversion of lignocellulose into acetyl-TAGs could allow biorefinery production of versatile molecules for biofuel and bioproducts.


RESULTS
In order to produce acetyl-TAGs from abundant lignocellulose feedstocks, we expressedEaDAcT inS. cerevisiaepreviously engineered to utilize xylose as a carbon source. The resulting strains were capable of producing acetyl-TAGs when grown on different media. The highest levels of acetyl-TAG production were observed with growth on synthetic lab media containing glucose or xylose. Importantly, acetyl-TAGs were also synthesized by this strain in ammonia fiber expansion (AFEX)-pretreated corn stover hydrolysate (ACSH) at higher volumetric titers than previously published strains. The deletion of the four endogenous enzymes known to contribute to lcTAG production increased the proportion of acetyl-TAGs in the total storage lipids beyond that in existing strains, which will make purification of these useful lipids easier. Surprisingly, the strains containing the four deletions were still capable of synthesizing lcTAG, suggesting that the particular strain used in this study possesses additional undetermined diacylglycerol acyltransferase activity. Additionally, the carbon source used for growth influenced the accumulation of these residual lcTAGs, with higher levels in strains cultured on xylose containing media.


CONCLUSIONS
Our results demonstrate thatS. cerevisiaecan be metabolically engineered to produce acetyl-TAGs when grown on different carbon sources, including hydrolysate derived from lignocellulose. Deletion of four endogenous acyltransferases enabled a higher purity of acetyl-TAGs to be achieved, but lcTAGs were still synthesized. Longer incubation times also decreased the levels of acetyl-TAGs produced. Therefore, additional work is needed to further manipulate acetyl-TAG production in this strain ofS. cerevisiae, including the identification of other TAG biosynthetic and lipolytic enzymes and a better understanding of the regulation of the synthesis and degradation of storage lipids.
Department of Biochemistry and Molecular Biophysics, Kansas State University, 141 Chalmers Hall, Manhattan, KS 66506 USA.
Tam N T
TNT
Tran
DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI 53726 USA.
Rebecca J
RJ
Breuer
DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI 53726 USA.
Ragothaman
R
Avanasi Narasimhan
DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI 53726 USA.
Lucas S
LS
Parreiras
DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI 53726 USA.
Yaoping
Y
Zhang
DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI 53726 USA.
Trey K
TK
Sato
Department of Biochemistry and Molecular Biophysics, Kansas State University, 141 Chalmers Hall, Manhattan, KS 66506 USA.
Timothy P
TP
Durrett
AFEX corn stover hydrolysate; Acetyl-TAGs; Metabolic engineering; Saccharomyces cerevisiae