Constructing an efficient synthesis pathway in peroxisomes to boost β-myrcene production in yeast

β-Myrcene is a key acyclic monoterpene that has been extensively applied in the agricultural pesticide, fragrance, and flavor industries; it also serves as a critical monoterpene precursor of high-value compounds. Our lab previously successfully achieved β-myrcene biosynthesis in the peroxisomes of Saccharomyces cerevisiae. However, the titer remained limited by the supply and utilization of precursors in peroxisomes. In this study, an efficient and stable β-myrcene biosynthetic pathway was constructed through the multicopy integration of the fusion protein MS-ERG20^ww (β-myrcene synthase) with peroxisome localization into Ty1 loci. This approach improved the supply and utilization of geranyl pyrophosphate (GPP) precursors, resulting in a 2-fold increase in the β-myrcene titer to 82.53 ± 3.78 mg/L. A new mevalonate (MVA) pathway was subsequently constructed for β-myrcene production by localizing truncated 3-hydroxy-3-methylglutaryl-CoA reductase (tHMGR) to peroxisomes, resulting in a 1.2-fold increase in the titer. In addition, KAR2 (ER chaperones) and GSH2 (reduced glutathione synthase) overexpression in chassis increased the β-myrcene titer to 176.40 ± 17.56 mg/L. Finally, 15-L fed-batch fermentation optimization increased the β-myrcene titer to 4.7 g/L, which represents the highest level yet reported. This work provides a comprehensive framework for engineering yeast cell factories, highlighting the ability of integrated metabolic engineering to enhance high-value monoterpene production.