Study On Synthesis Regulation Of Medium Chain Fatty Acid Ethyl Esters In Saccharomyces Cerevisiae

Medium chain fatty acid ethyl esters are important flavour compounds that impart the aromas to Chinese liquor.Medium chain acyl-CoAs in Saccharomyces cerevisiae are vital precursor of medium chain fatty acid ethyl esters synthesis.In this study,several metabolic engineering strategies which could improve acyl-CoAs production and further increase the productivity of the medium chain fatty acid ethyl esters were explored,such as co-overexpression of FAS1 and FAS2 of fatty acid synthesis pathway,constructing butyryl-CoA synthetic biology approach from Clostridium acetobutylicum or Clostridium butyricun,overexpressing heterologous thioesterases from Lactobacillus brevis,Lactobacillus plantarum or Cuphea hookeriana.The main contents and conclusions were as follows:On the basis of previous research,the effects of MFAEEs by co-overexpression of FAS1 and FAS2 were examined.The 154.3%and 65.2%improvement of ethyl caproate or ethyl octanoate production and 23.5%increasement of ethyl caprate accumulation in the co-overexpression of FAS1 and FAS2 with FAA1 integration engineered strain FF1F2 compared to strains a5F-FAS1 demonstrate the enhancement on MFAEEs by the fatty acid synthesis pathway strengthing.Consistent with this effect,the co-overexpression strain OF1F2 achieve 17.3%,30.7%and 27.1%increasement of the three substances mentioned above and 28.1%improvement of ethyl dodecanoate compared to strains ?5O-FAS 1,respectively.It means that co-overexpression of FAS1 and FAS2 could further improve the capacity of MFAEEs synthesis.In this study,heterologous thioesterases from L.brevis,L.plantarum or Cuphea hookeriana were overexpressed to explore the influence on MFAEEs.The mutant OF1-LBTE,OF1-LPTE and mutant OF1-ChTE were constructed.The production of ethyl octanoate by OF1-LBTE and OF 1-LPTE were 68.2%and 58.5%higher than that of parental strain ?50-FAS1 respectively,the ethyl caprate production were improved by 53%and 79%.However,the production of the the production of other MFAEEs(C6:0,C12:0,C14:0)remain unchanged in OF1-LBTE and OF1-LPTE.The production of MFAEEs(C6:0,C8:0,C10:0,C12:0 and C14:0)by OF1-ChTE were 16%,78%,97.8%,29.1%and 63.8%higher than those of parental strain ?50-FAS1 respectively.It means that overexpressing heterologous thioesterases can enhance the capacity of MFAEEs synthesis,particularly of ethyl octanoate and ethyl caprate.The potential capacity of S.cerevisiae in ethyl butyrate biosynthesis was explored by dual construction of a synthetic pathways comprising three genes from Clostridium acetobutylicum or Clostridium butyricum and overexpression of EHT1 or ATF1.With no ethyl butyrate synthesized by ?5,Mutant M-cbB-E and E-caB-E could produce ethyl butyrate to 7.3mg/L and 5.78mg/L respectively.However,There are no ethyl butyrate be detected in the Mutant strains M-cbB-A and E-caB-A.The results indicate that the synthetic pathways from anaerobic bacteria could produce butyryl-CoA effectively which then be catalyzed by EHT1 and not by ATF1 to synthesize ethyl butyrate.