Design And Construction Of Saccharomyces Cerevisiae Strains For Cocoa Butter Equivalent Production

Cocoa butter is extracted from cocoa beans,and it is mainly used as the raw material for the production of chocolate and cosmetics.The cocoa tree can only grow at tropical place,and the planting area is limited.The increased demands and insufficient cocoa plants led to a shortage of cocoa butter supply and price increase.Therefore,there is much interesting in finding an alternative cocoa butter supply.The cocoa butter is mainly composed with three different kinds of triacylglycerols(TAGs)including 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),and1,3-distearoyl-2-oleoyl-glycerol(SOS,C18:0-C18:1-C18:0),and the main fatty acids in the TAGs are 16 and 18 carbon fatty acids.Saccharomyces cerevisiae is a widely used model microbital species for plant natural product biosynthesis,and the main fatty acids in biomass are 16 and 18 carbon fatty acids.S.cerevisiae has been proved to be used in cocoa butter equivalent(CBE)production.However,the contents of POP,POS and SOS in S.cerevisiae are low.Therefore,synthetical biology and metabolic engineering strategies should be applied to design and construct efficient yeast cell factories for CBE production.In plant,the major biochemical reaction for TAG biosynthesis includes plastid fatty acid synthesis step,acyl editing step and TAG synthesis step,which mainly occur in plastid,mitochondria and endoplasmic reticulum(ER),and the TAGs are stored in plant oil body.The precursors for plant fatty acid synthesis are Acetyl-Co A and malonyl-ACP,which are converted to fatty acids-ACP through catalysis by several enzymes in plastid and mitochondria.The fatty acid-ACPs are further converted to fatty acids via ACP thioesterase(FATA and FATB).The long-chain acyl-Co A synthetase catalyzes fatty acids to acyl-Co A in ER.The acyl-Co A are further elongated or desaturated with several enzymes.The diverse TAG products are formed via the enzymes of glycerol-sn-3-phosphate acyl-transferase(GPAT),lysophophatidate acyl-transferase(LPAT),and acyl-Co A:diacylglycerol acyl-transferase(DGAT)in ER.Different with plants,the TAGs are mainly synthesized in ER of S.cerevisiae.The precursors for TAG production in S.cerevisiae are acetyl-Co A and malonyl-Co A,and these precursors can form fatty acyl-Co A with the catalysis of several enzymes.With the help of GPAT,LPAT,DGAT and other enzymes,the fatty acyl-Co A and other substrate were used for TAG biosynthesis.Therefore,based on the proper fatty acyl-Co A,the key enzymes to determine TAG types and contents are GPAT,LPAT and DGAT.Among them,overexpression of DGAT genes can increase yeast TAG production ability.There are diverse oleaginous plants and yeast in nature.Thus,it might recover some efficient CBE-producing genes from the oleaginous yeasts and plants.We download predicted GPAT,LPAT,and DGAT genes from Uniprot database,and potential efficient genes for high-level lipid production were screened using phylogenetic analysis strategy.We cloned three potential efficient DGAT genes from oleaginous plants of Arachis hypogaea,Glycine max,and Ricinus communis(genes of Ah DGAT,Gm DGAT,and Rc DGAT),and some genes of oleaginous yeast of Trichosporon oleaginosus(Cutaneotrichosporon oleaginosus).We added high-strengthen promoters and terminators to these genes,and transformed these genes into five different S.cerevisiae strains of IMX581,Y29,YJZ45,Y&Z019 and Y&Z036,generating 27 different S.cerevisiae strains.The growth characterization of the obtained 27 yeast strains was analyzed,and11 S.cerevisiae strains were selected for lipidomics analyses.The TAG and fatty acid analysis results showed that the expression of these genes improved potential cocoa butter production in these strains.Moreover,compared with other known cocoa butter producing yeast strains,the strains obtained in this study is higher.The potential cocoa butter compositions of some strains,such as Y&Z019-To DGAT,were as high as more than 20% of the total lipids.Among the 11 strains,YJZ45-Gm DGAT can produce the highest level of CBE.The CBE production of YJZ45-Gm DGAT increase several folds,and it is higher than S.cerevisiae CBE production in previous reports.Further engineering of the YJZ45-Gm DGAT and some efficient CBE biosynthetic genes might lead to efficient CBE production in the future.In this study,we identified some potential genes for CBE production using efficient gene recovering strategies,and several S.cerevisiae strains with CBE production ability were obtained.It is necessary to apply synthetic biology strategies to rewire S.cerevisiae and oleaginous yeasts for higher CBE production,in order to realize CBE production using engineered yeasts with high titer,rate and yield,which might help realize the industrial application of yeast CBE production.