Modeling-Guided Engineering Enables Efficient Limonene Production in Cyanobacteria

Terpenes are plant-derived secondary metabolites with diverse industrial applications. Many terpene molecules are potential candidates for pharmaceutical, nutraceutical, and biofuel applications. To produce high titer of terpenes in microbes, high carbon flux toward terpene pathway is desired. In cyanobacteria, terpene precursors are synthesized from the MEP (2-C-methyl-D-erythritol 4-phosphate) pathway. The inherent low carbon flux toward this secondary metabolic pathway, and the still elusive regulation of carbon partition among carbon catabolic pathways are key limitations impeding high titer of terpene production. In our study, computational modeling-guided metabolic engineering led to efficient limonene production in the cyanobacterium Synechococcus elongatus. The downstream terpene synthase was determined as a key metabolic flux controlling node in the MEP-derived terpene biosynthesis. Through synthetic biology design, we achieved >100 fold increase in limonene productivity, and a significant productivity increase compared to that achieved through stepwise metabolic engineering. The strong limonene carbon sink led to enhanced carbon flux into the MEP pathway, and potentially saturated the native MEP flux. The limonene productivity change along bacterial growth also indicates that MEP pathway is highly regulated, and constantly competing carbon source with sugar metabolism. Through various ‘-omics’ studies, we also determined flux controlling points in the MEP pathway. Further identifying upstream regulation point that controls MEP enzyme expression will bring important insight into carbon partition between primary and secondary metabolic pathways, and provide valuable targets for metabolic engineering efforts.