Reprogramming <i>Komagataella phaffii</i> for a Robust Chassis toward Efficient <i>De Novo</i> Biosynthesis of (−)-α-Bisabolol

(-)<i>-</i>α<i>-</i>Bisabolol, a monocyclic susquiterpene alcohol, has been widely used in the fields of food, medicine, and biofuel. Recently, the rapid development of synthetic biology has offered a sustainable route for the production of (-)<i>-</i>α<i>-</i>bisabolol by microbial cell factories. Even though efficient biosynthesis of (-)<i>-</i>α<i>-</i>bisabolol has been achieved in <i>Escherichia coli</i> with the highest titer of 23.4 g/L, the possible infection risk by the bacteriophage makes <i>E. coli</i> unsuitable to act as the most robust chassis. Herein, we optimized the MVA pathway of <i>Komagataella phaffii</i> and fused farnesyl diphosphate synthase (ERG20), with bisabolol synthase for efficient production of (-)<i>-</i>α<i>-</i>bisabolol. Through the engineering of cofactor NADPH, molecular chaperone, and transcription factors, we obtained the robust (-)<i>-</i>α<i>-</i>bisabolol-producing strain, KB-30. Finally, the highly efficient production of (-)-α-bisabolol was achieved in a 5 L fed-batch fermenter, giving a titer of 32.8 g/L and a space-time yield of 283 mg/L/h. This work represents the highest production of (-)-α-bisabolol to date and provides new insights for efficient terpenoid biosynthesis.