Increasing cocoa butter-like lipid production of Saccharomyces cerevisiae by expression of selected cocoa genes

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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-017-0333-1) contains supplementary material, which is available to authorized users.

Modulation of saturation and chain length of fatty acids in Saccharomyces cerevisiae for production of cocoa butter‐like lipids

“…The increase in TAG species with saturated fatty acids (e.g., 50:1, 52:1, and 54:1) indicates that an increased availability of C16:0 and C18:0 leads to incorporation of these fatty acids into the sn ‐3 position. Expression of TAG biosynthetic genes from T. cacao in the strains constructed here could increase levels of cocoa butter‐like TAGs further by preferentially incorporating saturated fatty acids into TAGs (Wei, Gossing, Bergenholm, Siewers, & Nielsen, ). Overexpression of TAG biosynthetic acyltransferases (e.g., LPAT and DGAT) also typically increases fatty acid and TAG content (Kamisaka, Tomita, Kimura, Kainou, & Uemura, ; Silverman, Qiao, Xu, & Stephanopoulos, ).…”

“…Expression of TAG biosynthetic genes from T. cacao in the strains constructed here could increase levels of cocoa butter-like TAGs further by preferentially incorporating saturated fatty acids into TAGs (Wei, Gossing, Bergenholm, Siewers, & Nielsen, 2017).…”

Identification of genes involved in shea butter biosynthesis from Vitellaria paradoxa fruits through transcriptomics and functional heterologous expression

“…Previous genomic analyses revealed several cocoa genes of glycerol-3-phosphate acyltransferase (GPAT), lysophospholipid acyltransferase (LPAT), and diacylglycerol acyltransferase (DGAT) participating in TAG biosynthetic pathway (Argout et al 2011 ; Chapman and Ohlrogge 2012 ; Motamayor et al 2013 ; Napier et al 2014 ). Further expressing some of them in Saccharomyces cerevisiae increased yeast lipid or TAG production (Wei et al 2018 ; Wei et al 2017a ). As shea butter contains higher SOS than cocoa butter (Chaiseri and Dimick 1989 ; Di Vincenzo et al 2005 ; Jahurul et al 2013 ), shea tree might harbor more efficient TAG biosynthetic genes than cocoa corresponding genes for SOS production.…”

Quantitative mapping of triacylglycerol chain length and saturation using broadband CARS microscopy

“…Before imaging, all cells were fixed in 4 vol.% paraformaldehyde, crosslinking proteins without effect on lipid chemistry. CENPK 113-11C Saccharomyces cerevisiae and Yarrowia lipolytica, TAG producing yeast strains, were cultured as previously described (8,44) and fixed in paraformaldehyde. Nannochloropsis sp.…”

“…Similar to mammals, prokaryotic yeast have lipid accumulationslargely full of TAGsthat allow cells to deal with lipotoxicity (7). TAGs in yeast can also be a desired product in the search for replacement of plant-based resources such as cocoa butter (8). Not only cocoa seeds, but many other plant seeds store TAGs in oil bodies (3), which are analogous to the lipid droplets in mammalian cells in that they are coated with amphipathic lipids and regulatory proteins (9).…”