Triune Engineering Approach for (+)‐valencene Overproduction in Yarrowia lipolytica

ABSTRACT The sesquiterpene (+)‐valencene, with its flavor and diverse biological functions, holds promise for applications in the food, fragrance, and pharmaceutical industries. However, the low concentration in nature and high cost of extraction limit its application. This study aimed to construct a microbial cell factory to efficiently produce (+)‐valencene. The strain Yarrowia lipolytica YL238, possessing a stronger capacity for (+)‐valencene synthesis, was selected and utilized as the chassis for further modifications. By fine‐tuning the mevalonate and squalene synthesis pathways we achieved a remarkable 13.2‐fold increase in (+)‐valencene titer compared to the original strain. Following directed evolution was employed to screen for efficient (+)‐valencene synthase, which further enhanced (+)‐valencene production by 138%. Consequently, the engineered strain overproduced 813 mg/L of (+)‐valencene in shake flasks, marking the highest titer reported in microbials to date. Furthermore, in fed‐batch fermentation, this engineered strain showed the capacity to produce 3.3 g/L of (+)‐valencene. This study offers a successful model for the application of the “strain‐pathway‐enzyme” triune strategy in the metabolic engineering of Y. lipolytica , and these methodologies could be broadly utilized for the synthesis of other natural terpenes.