| Mortierella alpina is an oleaginous fungus that can produce a variety of polyunsaturated fatty acids(PUFAs)with physiological activity.The fatty acids produced by M.alpina mainly exist in the form of triacylglycerol(TAG),which has higher stability and bioavailability than free and ethyl forms.As a key enzyme involved in TAG synthesis,Diacylglycerol acyltransferase(DGAT)catalyzes the combination of acyl-Co A and Diacylglycerol(DAG)to form TAG in organisms.The selectivity of DGAT for substrates containing specific fatty acids directly affects the composition of fatty acids in TAG,which is of great significance for the metabolic engineering for producing TAG containing specific fatty acids.There are various DGATs in M.alpina,but comparative analysis of the functional properties and substrate selectivity of these different DGATs is currently lacking.In this study,the sequences of three DGAT1 subtypes(Ma DGAT1A/1B/1C)and two DGAT2 subtypes(Ma DGAT2A/2B)were analyzed.And five Ma DGATs were heterologous expressed in TAG synthesis-deficient Saccharomyces cerevisiae H1246,the functional properties and substrate selectivity of Ma DGATs were analyzed by measuring the TAG synthesis in yeast recombinants without exogenous PUFAs,exogenous feeding PUFAs and microsomal reactions in vitro.This research aimed to provide a certain reference for improving the yield of TAG in oleaginous microorganisms and producing TAG rich in specific PUFAs.The following were the essential contents and findings of the study:(1)S.cerevisiae H1246-Ma DGATs was successfully constructed.And Western blot and relative gray scale analysis were used to identify the optimal time for induction of protein expression and screen out the transformants with the highest expression.The results showed that the ratios of the relative gray levels of expression level of Ma DGAT1A/1B/1C at 24 h,48 h and 72 h were: Ma DGAT1A(0.09 : 0.73 : 1),Ma DGAT1B(0.12 : 0.22 : 1),Ma DGAT1C(0.89 : 0.97 : 1),indicated that the optimal induction time was 72 h;the transformant with the highest expression of target protein was selected for subsequent experiments;sequence analysis showed that Ma DGAT1 C and Ma DGAT1A/1B had significant differences in hydrophobicity and phylogenetic evolution,and Ma DGAT1 s may have more complex advanced structures than Ma DGAT2 s,and thus exhibit different functional properties.(2)The content and composition of TAG and total lipids produced by recombinant yeasts were analyzed by thin-layer chromatography(TLC)and gas chromatography-mass spectrometry(GC-MS).The results showed that Ma DGAT1A/1B/2A/2B could restore TAG synthesis in S.cerevisiae H1246 and showed DGAT activity,while Ma DGAT1 C had no DGAT activity;the TAG content of H1246-Ma DGAT1A/1B/2A/2B(of dry cell weight)were2.92±0.42%,3.62±0.22%,0.86±0.34%,and 0.18±0.09%,respectively,indicating that Ma DGAT1A/1B has the better effect in promoting TAG synthesis in yeast recombinants than Ma DGAT2s;In S.cerevisiae H1246,Ma DGAT1 A preferred C16:1 in MUFAs,Ma DGAT1 B preferred C16:0 in SFAs,and Ma DGAT2A/2B preferred C18:0 in SFAs,indicating that Ma DGAT1A/1B/2A/2B had different selectivity for substrates with different chain length and unsaturation.(3)The selectivity of Ma DGAT1A/1B/2A/2B to PUFAs-Co A and DAG was systematically evaluated by analyzing TAG synthesis of yeast recombinants with exogenous PUFAs and TAG production in microsomal reactions in vitro.The results showed that: In S.cerevisiae H1246,both Ma DGAT1 A and Ma DGAT2 A preferred to assemble linoleic acid(C18:2,LA)into TAG when PUFAs was added externally,Ma DGAT2 B showed a preference for eicosapentaenoic acid(C20:5,EPA)and LA in ω-6 PUFAs,while Ma DGAT1 B showed no obvious substrate selectivity;in the microsomal reaction system in vitro;Ma DGAT1 A showed no obvious acyl-Co A selectivity,and Ma DGAT1 B preferred C20:5-Co A,for DAG,Ma DGAT1A/1B preferred C18:1/C18:1-DAG compared with C20:4/C20:4-DAG.In conclusion,Ma DGATs has the potential to be used to produce TAG rich in PUFAs(LA and EPA). |
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