Reprogramming Komagataella phaffii for a Robust Chassis towardEfficient <i>De Novo</i> Biosynthesisof (−)-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 Escherichia coli with the highest titer of 23.4 g/L, the possible infection risk by the bacteriophage makes E. coli unsuitable to act as the most robust chassis. Herein, we optimized the MVA pathway of Komagataella phaffii 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.