Metabolic reprogramming and computation-aided protein engineering for high-level de novo biosynthesis for 2-phenylethanol in Pichia pastoris

2-Phenylethanol (2-PE), an aromatic compound with a characteristic rose fragrance, is extensively used in the food and cosmetic industries as a flavoring and fragrance agent. Due to limitations in obtaining 2-PE from natural plant sources, microbial cell factories offer a promising alternative for sustainable biosynthesis. In this study, <i>Pichia pastoris</i> was engineered to efficiently synthesize 2-PE. Using computer-assisted predictions of interactions between the key phenylpyruvate decarboxylase KDC2 and its substrates or products, an optimal enzyme variant was rationally designed to boost 2-PE production. Additionally, the shikimic acid pathway was enhanced, and a dynamic regulation promoter was employed to reduce competition from alternative pathways. These strategies significantly increased metabolic flux toward 2-PE production, achieving a titer of 2.81 g/L and 45.8-fold improvement over the non-engineered strain. By integrating controlled carbon feeding and in situ extraction to alleviate acetic acid inhibition and product toxicity, the recombinant strain achieved a final 2-PE titer of 7.10 g/L and a yield of 0.14 g/g glucose, the highest reported microbial production to date. This study highlights the significant potential of <i>P. pastoris</i> as a versatile cell factory for the green biosynthesis of 2-PE and other natural products.