The Role Of Thioesterases In Lipid Accumulation Of Mucor Circinelloides

Microorganism that accumulates more than 20% of lipid in cells is called oil-producing microorganisms.Most of them are bacteria,yeast,fungi and microalgae.The lipid produced by them is called microbial oil.Microbial oils are rich in polyunsaturated fatty acids,such as ?-linolenic acid(GLA),arachidonic acid(ARA),eicosapentaenoic acid(EPA)and docosahexaenoic acid(DHA),which can promote human health and are known as functional oils.On the other hand,microbial oils are highly similar to the fatty acid composition of animal and vegetable oils,and potential sources of biodiesel.There are a lot of lipids in the cells of oilproducing filamentous fungus Mucor circinelloides,which contains a large number of healthcare GLA,giving it high economic value.It is the first strain in the world to be used for commercial production of microbial oil.The genetic background of M.circinelloides is clear,and the genetic manipulation tools are perfect.In terms of lipid accumulation,the methods of regulating gene expression by overexpression,gene silencing or knockout are more mature and convenient,making it a model organism for studying the lipid accumulation mechanism of filamentous fungi.Thioesterases(TE)play a key role in the process of lipid accumulation and metabolism.It widely exists in prokaryotic and eukaryotic organisms,which can hydrolyze the thioester bond in a variety of substrates.It is an important processor of various products,and is favored by many researchers for genetic transformation to build a cell factory with high yield microbial oil.According to the specificity of catalytic substrates,thioesterases are widely classified into25 TE families.In this study,six different thioesterase genes were found from the genome of M.circinelloides WJ11.Through amino acid sequence alignment and phylogenetic tree analysis of thioesterase,it was found that the thioesterases in WJ11 were members of four families: three belonged to the TE4 family,named as TE4 a,TE4b and TE4 c,and the other three belonged to the TE6,TE20 and TE21 families,and named them on their family respectively.According to its classification,TE4a-c and TE6 are acyl-Co A thioesterase,TE20 is palmitoyl-protein thioesterase,and TE21 is acyl-protein thioesterase,they have special functions in lipid synthesis and metabolism.The strain Mc2075,which complements the WJ11 uracil deficient gene,was cultured in a 1.5 L fermentor.The consumption of carbon and nitrogen sources were monitored,the intracellular lipid content and the transcription levels of six thioesterases were measured.It was found that the expression levels of TE4 b and TE4 c were relatively high during the rapid lipid synthesis period,and TE4 c,TE6 and TE20 were still active during the stable lipid accumulation period,indicating that they may play different roles in different stages of lipid synthesis and metabolism.In order to study the role of thioesterase in the lipid synthesis and metabolism of M.circinelloides,six genes of thioesterases were overexpressed by homologous recombination method,and two transformants were retained after color screening and PCR verification.These transformants were cultured in shake flasks to determine their biomass and total fatty acid(TFA)content,and it was found that the overexpression of thioesterase increased the TFA content in cells.The transformants with high TFA content were selected for fermentation in a 1.5 L fermentor.The consumption of carbon and nitrogen sources by the thioesterase overexpression strains was investigated,and their biomass,TFA content and profile were determined.It was found that the growth status of the overexpression strains was not significantly different from that of the control strains,but the TFA content of almost all the overexpression strains increased.The TFA content increased by 16 % to 34 % except TE6.The highest TFA content is Mc TE20-2,which reached 34.7 % of the cell dry weight.In terms of fatty acid profiles,except for Mc TE21-1,the other fatty acid profiles changed.Mc TE4a-1 and Mc TEb-2 increased the content of unsaturated fatty acids in C18,and Mc TE4a-1,Mc TE4c-2,Mc TE6-2 and Mc TE20-2increased the content of C16:0 in fatty acids.The fatty acid composition of the strain was analyzed by thin layer chromatography.The proportion of triacylglycerol and monoacylglycerol in the lipids of all transformants was relatively reduced,and the proportion of free fatty acids was relatively increased to 1.3 to 2.1 times of the control strain Mc2075.Among them,Mc TE6-2 was the most abundant free fatty acid,accounting for 29.1 % of the total lipid.M. circinelloides always tends to store intracellular lipids in the form of triglycerides,and the function of thioesterase is more like a “ cargo sorting station ”.It hydrolyzes the thioester bond in the ester acyl-Co A / acyl carrier protein / protein / glutathione bound together,releasing free fatty acids and carrier groups to make them enter the downstream reactions,respectively.Free fatty acids are stored in cells or synthesized by ester acylation to diacylglycerol or triglycerides,and stored in cells.In this study,the endogenous thioesterases in WJ11 were classified and their functions were discussed,which broadened the research on key enzymes in the lipid synthesis and metabolic pathway of WJ11,enriched the gene pool of thioesterase family,and provided a theoretical basis for future research on WJ11.Thioesterase provides the possibility for the specific production of some fatty acid products,and further theoretical basis for the construction of the cell factory of M. circinelloides.