Enhancing myrcene biosynthesis in yeast through nuclear compartmentalization

Myrcene is a high-value monoterpene extensively applied in the fragrance, flavor, and agricultural industries, yet its efficient microbial production remains challenging due to pathway competition and limited metabolic flux. Compartmentalization offers a unique strategy to spatially organize heterologous metabolic pathways in Saccharomyces cerevisiae , enabling improved pathway efficiency through physical separation from competing cytosolic metabolism. In this study, we engineered the S. cerevisiae nucleus as a synthetic metabolic compartment for myrcene biosynthesis. Screening of myrcene synthases identified two highly active enzymes from Snapdragon Oc15 and Picea abies that function efficiently in S. cerevisiae . Myrcene production was detected only when myrcene synthase and the engineered GPP synthase mERG20p were co-localized to the nucleus, whereas cytosolic expression failed to yield detectable myrcene under their co-expression. Reconstruction of the complete mevalonate (MVA) pathway in the nucleus further increased myrcene titers. By identifying and optimizing rate-limiting steps, we substantially enhanced metabolic flux toward myrcene, achieving a final titer of 23.4 mg L −1 in flask-shaking fermentation. This work demonstrates the feasibility of repurposing the yeast nucleus for myrcene efficient biosynthesis and provide a new strategy for further improving microbial production of myrcene.