Construction and analysis of a cell factory for terpenoid biosynthesis in Pichia pastoris via metabolic engineering and metabolomics

Terpenoids are widely distributed in nature and have various applications in health products, pharmaceuticals, and fragrances. Despite the tremendous potential of terpenoids, traditional production methods such as plant extraction and chemical synthesis face challenges in meeting current market demands. With the developments in synthetic biology and metabolic engineering, it has become feasible to construct efficient microbial cell factories for large-scale production for terpenoids. In this work, using the yeast <i>Pichia pastoris</i> as the host cell, a "plug-and-play" cell factory for universal terpenoid production was constructed by enhancing the expression of the MVA pathway for common precursor synthesis and reducing branch pathway diversion strategies. We have successfully and efficiently synthesized β-elemene, β-farnesene, (+)-valencene, (-)-α-bisabolol by this cell factory. Furthermore, by analyzing metabolites in different engineered strains in terms of system biology, it was discovered that an increase in key protein copy numbers enhanced the synthesis of arginine and other metabolic pathways. The robustness of the strains and the tightly regulated metabolic network constrain rational metabolic engineering transformations. These data provide important clues for the modification and optimization of production strains.