Biosynthesis of a-bisabolene from low-cost renewable feedstocks by peroxisome engineering and systems metabolic engineering of the yeast Yarrowia lipolytica

Establishing efficient synthetic pathways for microbial production of biochemicals is often hampered by competing pathways and insufficient precursor supply. Compartmentalization in cellular organelles can isolate biosynthetic pathways from competing pathways, and thus provide a more compact and conducive environment for biosynthesis. Herein, the farnesyl diphosphate biosynthetic pathway and a-bisabolene synthase were compartmentalized in the yeast Yarrowia lipolytica peroxisome to enable high-level a-bisabolene production. Along with compartmentalization of the a-bisabolene biosynthesis pathway, a systems metabolic engineering approach that comprises mediating product export with an efflux pump, optimizing the gene copy numbers of rate-limiting enzymes, balancing the distribution of the common precursor acetyl-CoA between native lipid biosynthesis and heterologous a-bisabolene production, improving ATP and acetyl-CoA supply, and dynamic regulation of peroxisomes was then employed to further enhance the bisabolene production. Consequently, the optimized engineered strain produced 3028.9 mg L-1 a-bisabolene from waste cooking oil as the sole carbon source through shake flask cultivation. Finally, fed-batch fermentation was performed to accomplish a-bisabolene production at 15.5 g L-1, the highest ever reported for a-bisabolene from an engineered microbe. Taken together, this work lays the foundation for peroxisome engineering of Y. lipolytica, towards the production of a-bisabolene in a highly sustainable manner.