PMID- 29370801 OWN - NLM STAT- MEDLINE DCOM- 20190108 LR - 20190108 IS - 1475-2859 (Electronic) IS - 1475-2859 (Linking) VI - 17 IP - 1 DP - 2018 Jan 25 TI - Expression of cocoa genes in Saccharomyces cerevisiae improves cocoa butter production. PG - 11 LID - 10.1186/s12934-018-0866-2 [doi] LID - 11 AB - BACKGROUND: Cocoa butter (CB) extracted from cocoa beans (Theobroma cacao) is the main raw material for chocolate production, but CB supply is insufficient due to the increased chocolate demand and limited CB production. CB is mainly composed 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). In general, Saccharomyces cerevisiae produces TAGs as storage lipids, which consist of C16 and C18 fatty acids. However, cocoa butter-like lipids (CBL, which are composed of POP, POS and SOS) are not among the major TAG forms in yeast. TAG biosynthesis is mainly catalyzed by three enzymes: glycerol-3-phosphate acyltransferase (GPAT), lysophospholipid acyltransferase (LPAT) and diacylglycerol acyltransferase (DGAT), and it is essential to modulate the yeast TAG biosynthetic pathway for higher CBL production. RESULTS: We cloned seven GPAT genes and three LPAT genes from cocoa cDNA, in order to screen for CBL biosynthetic gene candidates. By expressing these cloned cocoa genes and two synthesized cocoa DGAT genes in S. cerevisiae, we successfully increased total fatty acid production, TAG production and CBL production in some of the strains. In the best producer, the potential CBL content was eightfold higher than the control strain, suggesting the cocoa genes expressed in this strain were functional and might be responsible for CBL biosynthesis. Moreover, the potential CBL content increased 134-fold over the control Y29-TcD1 (IMX581 sct1Delta ale1Delta lro1Delta dga1Delta with TcDGAT1 expression) in strain Y29-441 (IMX581 sct1Delta ale1Delta lro1Delta dga1Delta with TcGPAT4, TcLPAT4 and TcDGAT1 expression) further suggesting cocoa GPAT and LPAT genes functioned in yeast. CONCLUSIONS: We demonstrated that cocoa TAG biosynthetic genes functioned in S. cerevisiae and identified cocoa genes that may be involved in CBL production. Moreover, we found that expression of some cocoa CBL biosynthetic genes improved potential CBL production in S. cerevisiae, showing that metabolic engineering of yeast for cocoa butter production can be realized by manipulating the key enzymes GPAT, LPAT and DGAT in the TAG biosynthetic pathway. FAU - Wei, Yongjun AU - Wei Y AD - Department of Biology and Biological Engineering, Chalmers University of Technology, 41296, Gothenburg, Sweden. AD - Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, 41296, Gothenburg, Sweden. AD - CAS-Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200032, China. FAU - Bergenholm, David AU - Bergenholm D AD - Department of Biology and Biological Engineering, Chalmers University of Technology, 41296, Gothenburg, Sweden. AD - Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, 41296, Gothenburg, Sweden. FAU - Gossing, Michael AU - Gossing M AD - Department of Biology and Biological Engineering, Chalmers University of Technology, 41296, Gothenburg, Sweden. AD - Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, 41296, Gothenburg, Sweden. FAU - Siewers, Verena AU - Siewers V AD - Department of Biology and Biological Engineering, Chalmers University of Technology, 41296, Gothenburg, Sweden. AD - Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, 41296, Gothenburg, Sweden. FAU - Nielsen, Jens AU - Nielsen J AD - Department of Biology and Biological Engineering, Chalmers University of Technology, 41296, Gothenburg, Sweden. nielsenj@chalmers.se. AD - Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, 41296, Gothenburg, Sweden. nielsenj@chalmers.se. AD - Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, 2800 Kgs., Lyngby, Denmark. nielsenj@chalmers.se. LA - eng GR - DE-SC0008744/the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research, Genomic Science program/ PT - Journal Article DEP - 20180125 PL - England TA - Microb Cell Fact JT - Microbial cell factories JID - 101139812 RN - 0 (Dietary Fats) RN - 0 (Fatty Acids) RN - 0 (Triglycerides) RN - 512OYT1CRR (cocoa butter) RN - EC 2.3.1.20 (Diacylglycerol O-Acyltransferase) SB - IM MH - Biosynthetic Pathways/genetics/physiology MH - Cacao/*genetics MH - Diacylglycerol O-Acyltransferase/metabolism MH - Dietary Fats/analysis/*metabolism MH - Fatty Acids/biosynthesis/genetics/metabolism MH - Lipid Metabolism MH - Metabolic Engineering/methods MH - Saccharomyces cerevisiae/enzymology/*genetics MH - Synthetic Biology/methods MH - Triglycerides/*biosynthesis/genetics PMC - PMC5784687 OTO - NOTNLM OT - Cocoa butter-like lipid OT - Metabolic engineering OT - Synthetic biology OT - TAG biosynthetic genes OT - Theobroma cacao OT - Yeast cell factories EDAT- 2018/01/27 06:00 MHDA- 2019/01/09 06:00 PMCR- 2018/01/25 CRDT- 2018/01/27 06:00 PHST- 2017/10/20 00:00 [received] PHST- 2018/01/19 00:00 [accepted] PHST- 2018/01/27 06:00 [entrez] PHST- 2018/01/27 06:00 [pubmed] PHST- 2019/01/09 06:00 [medline] PHST- 2018/01/25 00:00 [pmc-release] AID - 10.1186/s12934-018-0866-2 [pii] AID - 866 [pii] AID - 10.1186/s12934-018-0866-2 [doi] PST - epublish SO - Microb Cell Fact. 2018 Jan 25;17(1):11. doi: 10.1186/s12934-018-0866-2.