Collaborative subcellular compartmentalization to improve GPP utilization and boost sabinene accumulation in Saccharomyces cerevisiae

Geranyl diphosphate (GPP) is a direct precursor in the biosynthesis of monoterpenes. Previous studies focused on the manipulation of metabolic flux to improve GPP supply in yeast. However, if the subcellular distribution of GPP with monoterpene synthase is not coordinated, the usage of GPP becomes sub-optimal. Therefore, taking sabinene production in Saccharomyces cerevisiae as an exemplar, we confirmed the location of N-truncated sabinene synthase (t34SabS1) to be primarily in the cytosol. We also revealed the existence of GPP pools in the peroxisomes and mitochondria. Combined targeting of t34SabS1 into different combinations of subcellular locations demonstrated that the highest production of sabinene was obtained when expressed simultaneously in the cytosol and mitochondria. Further overexpression of mitochondria-related genes uncovered four novel molecular targets (FIS1, LSB3, MBA1, and AIM25) associated with sabinene output, with AIM25 resulting in the highest production. Eventually, integrating all of these engineered genes into the host chromosome achieved a sabinene production of 154.9 mg/L, an almost 60-fold increase from our original titer. This research highlights the strategy of organelle engineering to improve precursor utilization and to enhance compartmentalized pathways. It also provides a good reference for the synthesis of other valuable monoterpenes and their derivatives, in a eukaryotic host.