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.