DID2 Gene Enhances ?-carotene Production In An Engineered Strain Of Yarrowia Lipolytica Through The Protein Sorting Complex (ESCRT) Component

?-carotene,precursor of vitamin A,has been widely used in food,pharmaceutical industries,and animal husbandry,has great commercial value.?-carotene synthetic pathway has been strictly reg?lated by cell?lar metabolism networks.Optimization the pathway that are not directly related to ?-carotene synthesis pathway is a new stategy for the production of ?-carotene.The Yarrowia lipolytica engineering strain has the potential to synthesize ?-carotene as having independent lipid droplets and redundant ?-carotene synthesis precursor acetyl-CoA.This study explored the effect of the MVB sorting pathway,related to protein degradation in cells,on ?-carotene synthesis by Y.lipolytica.The main research res?lts obtained are as follows:(1)Promotion of the component in ESCRT complex increased the production of ?-carotene in Y.lipolytica engineering strain by 1.58 fold.After overexpressing the Did2 gene,the content of?-carotene in Y.lipolytica engineered strain was increased form 9.89 mg/g DCW to 15.63 mg/g DCW.(2)Overexpression of the Did2 gene increased protein level of key enzymes in ?-carotene synthesis pathway which lead to the improvement of ?-carotene.We tasted the transcription levels of key gene in ?-carotene synthesis pathway,Thmg,Ggsl,CarRA and CarB,the utilization of key precursors in ?-carotene synthesis,FPP and lycopene,cofactor content of ATP and NADPH,the growth of cells to explore the reason that overexpressing Did2 gene increased the production of?-carotene.The res ? Its show that overexpression of one copy of the Did2 gene increased the level of the key enzymes protein in ?-carotene synthesis pathway,the protein level of tHMG and carB increased by 37%and 17%,respectively.(3)The ESCRT complex not just responsible for protein degradation by the MVB sorting pathway,but has a crucial reg?lating effect on transcriptional reg?lation in cells.Overexpression of ESCRT components causes degradation of proteins in cells through the MVB sorting pathway.Moreover,the effect of ESCRT on nuclear transcriptional reg?lation accelerates protein synthesis by increasing the transcription level of corresponding genes.Compared with the overexpression of one copy of the Did2 gene,the integration of two copies of the Did2 gene into the Y.lipolytica engineered strain further increased the rate of protein degradation leading to the transcription of?-carotene key genes Thmg,Ggsl,CarRA and CarB increased by 63%,57%,54%and 85%,but not enough to compensate for protein degradation.The protein level of key enzymes of ?-carotene synthesis pathway(tHMG,carB)decreased,respectively.Moreover,the content of ?-carotene synthesis precursor,FPP and lycopene,decreased by 25%and 35%,and the content of ?-carotene decreased by 25%.Optimation of metabolic pathways that not directly associated with ?-carotene synthesis increased the production of ?-carotene in Y.lipolytica.Provide a theoretical basis for optimizing metabolic pathways not directly related to ?-carotene to increase ?-carotene production of Y.lipolytica engineering strain.Provide reference for optimizing the cell?lar metabolic network in the future to increase the output of other high value-added natural products.