Multidimensional metabolic engineering of Yarrowia lipolytica for highly efficient biosynthesis of betulinic acid

Abstract

Engineered yeast has emerged as a promising platform for the production of anticancer drug betulinic acid (BA), yet its efficiency is often hampered by low cytochrome P450 enzyme activity, inadequate carbon utilization, and metabolic mismatches with heterologous pathways. To overcome these bottlenecks, a multidimensional metabolic engineering strategy, including rational design of CYP716A155, pathway engineering, cofactor engineering, subcellular engineering, and carbon source optimization, was developed to reconstruct a highly efficient BA biosynthetic pathway. Two key precursor modules, the heterologous non-oxidative glycolysis (NOG) pathway and the isoprenol utilization pathway (IUP) were introduced, significantly enhancing the supply of crucial precursors, acetyl-CoA and IPP. Protein engineering of CYP716A155 by introducing the E120Q mutation enhanced its catalytic activity and facilitated downstream metabolism. Importantly, organelle engineering, including subcellular compartmentalization of the key enzyme and enhanced membrane contact sites (MCSs), was employed to accelerate downstream carbon flux. To increase NADPH availability, redox engineering was implemented by introducing NADP+-dependent enzymes GPD1 and MCE2 for the conversion of cytosolic NADH to NADPH. Furthermore, global cellular metabolic rewiring was conducted, including mobilization of lipid metabolism pathways, down-regulation of the competing sterol pathways, and fine-tuning of the glycolysis pathway, to redirect carbon flux distribution and enhance carbon source utilization efficiency. The engineered cell factory, optimized through multidimensional metabolic strategies, achieved a BA titer of 271.3 mg/L in shake-flask culture—the highest reported to date. Utilizing cost-effective mannitol derived from third-generation algal biomass as the primary carbon source in a 3 L bioreactor, the process achieved a final BA titer of 657.8 mg/L.