Abstract
Cocoa butter (CB) extracted from cocoa beans mainly consists of three different kinds of triacylglycerols (TAGs), 1,3-dipalmitoyl-2-oleoyl-glycerol (POP, C16:0–C18:1–C16:0), 1-palmitoyl-3-stearoyl-2-oleoyl-glycerol (POS, C16:0–C18:1–C18:0) and 1,3-distearoyl-2-oleoyl-glycerol (SOS, C18:0–C18:1–C18:0), but CB supply is limited. Therefore, CB-like lipids (CBL, which are composed of POP, POS and SOS) are in great demand. Saccharomyces cerevisiae produces TAGs as storage lipids, which are also mainly composed of C16 and C18 fatty acids. However, POP, POS and SOS are not among the major TAG forms in yeast. TAG synthesis is mainly catalyzed by three enzymes: glycerol-3-phosphate acyltransferase (GPAT), lysophospholipid acyltransferase (LPAT) and diacylglycerol acyltransferase (DGAT). In order to produce CBL in S. cerevisiae, we selected six cocoa genes encoding GPAT, LPAT and DGAT potentially responsible for CB biosynthesis from the cocoa genome using a phylogenetic analysis approach. By expressing the selected cocoa genes in S. cerevisiae, we successfully increased total fatty acid production, TAG production and CBL production in some S. cerevisiae strains. The relative CBL content in three yeast strains harboring cocoa genes increased 190, 230 and 196% over the control strain, respectively; especially, the potential SOS content of the three yeast strains increased 254, 476 and 354% over the control strain. Moreover, one of the three yeast strains had a 2.25-fold increased TAG content and 6.7-fold higher level of CBL compared with the control strain. In summary, CBL production by S. cerevisiae were increased through expressing selected cocoa genes potentially involved in CB biosynthesis.
Highlights
Cocoa butter (CB) extracted from cocoa seeds (Theobroma cacao) mainly contains three different kinds of triacylglycerols (TAGs)—1,3-dipalmitoyl-2-oleoyl-glycerol (POP, C16:0–C18:1–C16:0), 1-palmitoyl-3-stearoyl2-oleoyl-glycerol (POS, C16:0–C18:1–C18:0) and 1,3-distearoyl-2-oleoyl-glycerol (SOS, C18:0–C18:1– C18:0), which are composed of C16 and C18 fatty acids (Jahurul et al 2013)
Among all the cocoa genes annotated as glycerol-3-phosphate acyltransferase (GPAT), lysophospholipid acyltransferase (LPAT) and diacylglycerol acyltransferase (DGAT) genes, the ones responsible for CB biosynthesis are unknown (Argout et al 2011; Motamayor et al 2013)
As GPAT, LPAT and DGAT genes of S. cerevisiae had been characterized and their function had been determined before, cocoa genes most similar to GPAT, LPAT and DGAT genes of S. cerevisiae might be functional in S. cerevisiae and they were prioritized for expression (Benghezal et al 2007; Oelkers et al 2002; Zheng and Zou 2001)
Summary
Cocoa butter (CB) extracted from cocoa seeds (Theobroma cacao) mainly contains three different kinds of triacylglycerols (TAGs)—1,3-dipalmitoyl-2-oleoyl-glycerol (POP, C16:0–C18:1–C16:0), 1-palmitoyl-3-stearoyl2-oleoyl-glycerol (POS, C16:0–C18:1–C18:0) and 1,3-distearoyl-2-oleoyl-glycerol (SOS, C18:0–C18:1– C18:0), which are composed of C16 and C18 fatty acids (Jahurul et al 2013). S. cerevisiae contains two GPATs (Gpt2p and Sct1p), two LPATs (Slc1p and Slc4p) and one DGAT (Dga1p). Another phospholipid:diacylglycerol acyltransferase, PDAT (Lro1p), can synthesize TAG using diacylglycerol (DAG) and phospholipid as substrates (Fig. 1) (Coleman and Lee 2004; de Kroon et al 2013; Ratledge 2002; Zheng and Zou 2001). Though C16:0, C16:1, C18:0 and C18:1 are the four main fatty acids in the total fatty acid composition of S. cerevisiae (Khoomrung et al 2012), only small amounts of CBL (POP, POS, SOS) have been identified among the TAGs in wild-type S. cerevisiae cells (Ejsing et al 2009), suggesting that its GPAT, LPAT and DGAT enzymes might not be optimal for CBL production
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