Application Research Of Compartmentalization Of Yarrowia Lipolytica For The Production Of Bisabolene

Bisabolene(C₁₅H₂₄)has three structural isomers namedα-bisabolene,β-bisabolene andγ-bisabolene.As one of the sesquiterpenes found in plant,bisabolene has been traditionally utilized as fragrances,food additives and therapeutic agents,moreover,in recent years,with its favorable fuel properties,bisabolene was demonstrated to be potential fuel alternatives in some reports.Yarrowia lipolytica,a non-conventional oleaginous yeast,has been investigated and modified for biotechnological applications with rising interest.In particularly,Y.lipolytica is well suited for mitochondrial biosynthesizing bisabolene as wildtype strains naturally possess higher mitochondrial acetyl-Co A flux and higher tricarboxylic acid（TCA）cycle flux for supplying the precursor and cofactors of the MVA pathway over Saccharomyces cerevisiae.Moreover,the distinguished capabilities of hydrophobic substrates assimilation and high cell-density fermentation render Y.lipolytica to be potential industrial strain for the large-scale production of bisabolene.To meet the rapidly rising market demand for bisabolene and derivatives,metabolic engineering microbes for bisabolene production is becoming an attractive alternative approach.However,the low conversion efficiency and high cost in fermentation platform renders this approach non-economically viable.Herein,the farnesyl diphosphate synthetic pathway andα-bisabolene synthase were compartmentalized into the Y.lipolytica mitochondria to enable high-levelα-bisabolene production.Through comprehensive metabolic engineering approaches,we exploited the potential and capability of the mitochondria as a subcellular factory to achieve 257.40 mg/Lα-bisabolene production,the highest titre from glucose reported to date.By combining mitochondrial and cytoplasmic engineering,we further boosted theα-bisabolene titre to765.09 mg/L by utilizing waste cooking oil as the sole carbon source.Overall,our study has provided valuable insights into mitochondrial engineering of Y.lipolytica for sustainable and green production of valuable compounds.