The plant terpenoids limonene (C10H16) and α-bisabolene (C15H24) are hydrocarbon precursors to a range of industrially-relevant chemicals. We have engineered the fast-growing unicellular euryhaline cyanobacterium Synechococcus sp. PCC 7002 to photosynthetically produce yields of 4 mg L-1 limonene and 0.6 mg L-1 α-bisabolene through heterologous expression of the Mentha spicata L-limonene synthase or the Abies grandis (E)-α-bisabolene synthase genes, respectively. Titers were significantly higher when a dodecane overlay was applied during culturing, suggesting that dodecane traps large quantities of volatile limonene and α-bisabolene that would otherwise be lost to evaporation, and/or that continuous product removal in dodecane alleviates product feedback inhibition to promote higher rates of synthesis. Glycogen represents a major carbon sink under nutrient-deprived conditions in wild type Synechococcus (up to 80% biomass); therefore, we investigated limonene and bisabolene production in the glycogen-deficient ΔglgC genetic background, where carbon is repartitioned at the expense of glycogen biosynthesis. The ΔglgC mutant excreted a suite of overflow metabolites (α-ketoisocaproate, pyruvate, α-ketoglutarate, succinate and acetate) during nitrogen deprivation; however, the excreted metabolites were not effectively utilized for terpenoid metabolism. To that end, we have conducted a thorough physiological characterization of the ΔglgC mutant in an effort to understand how cyanobacteria respond to major carbon redistribution. We present fundamental new data quantifying the lipid, protein and carbohydrate sinks of ΔglgC cells over the entire growth phase, and show that sustained low levels of photosynthetic activity in ΔglgC promote continuous organic acid secretion in the absence of growth, which has important biotechnological implications. Overall, Synechococcus sp. PCC 7002 provides a highly promising platform for terpenoid biosynthetic and metabolic engineering efforts.

Photosynthetic limonene and bisabolene production in wild type and a glycogen-deficient mutant of Synechococcus sp. PCC 7002

249th National Meeting (2015)

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