Thermodynamics Plays an Important Role in Enhancing Terpene Productivity in Cyanobacteria

The terpene family is very diverse with more than 50,000 unique chemical structures. Terpenes are condensed from two C₅ precursor isomers, isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). Limonene, a C₁₀ monoterpene, has been tested to use as a drop-in fuel additive for combustion engines and as the kerosene substitute for aviation fuels. Photosynthesis-driven CO₂ conversion to terpenes is particularly attractive for biofuel applications due to its low carbon footprint but is challenged with far lower productivities compared to heterotrophic hosts. We hypothesize that the low terpene yield in phototrophs is due to the low driving force determined by thermodynamics as it readily does not favor efficient photosynthetic CO₂ conversion. To bypass this limitation, we used the sigma factor engineering approach. An essential sigma factor, RpoD, encoding was overexpressed in photosynthetic cyanobacteria and showed a 1.7-fold improvement in limonene productivity. Further investigations on the engineered strains revealed a higher photosynthetic rate stimulating enhanced carbon fixation. Computational modeling and wet lab analyses showed an increased flux toward both native carbon sink glycogen synthesis and the non-native limonene synthesis from photosynthate output. Interestingly, our proteomics result shows the decreased abundance of terpene biosynthesis enzymes indicating their limited role in determining terpene flux. Thus, increased abundance of RpoD illustrates interesting metabolic rewiring that supports robust direct CO₂ to limonene conversion. This study reveals thermodynamics as the key determinant to boost low flux metabolites like terpene and demonstrates the merits of sigma factor engineering for efficient chemical productions.