Introduction of a condensed, reverse tricarboxylic acid cycle for additional CO2 fixation in plants

Plants employ the Calvin-Benson cycle (CBC) to fix atmospheric CO2 for the production of biomass. The flux of carbon through the CBC is limited by the activity and selectivity of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). Alternative pathways that do not use RuBisCO to fix CO2 exist but occur only in anaerobic microorganisms. Rather than modifying existing routes of carbon metabolism in plants, we have developed a synthetic carbon fixation cycle that does not exist in nature, but is inspired by metabolisms of bacterial autotrophs. This synthetic cycle uses endogenous plant metabolites to fix CO2 and yield glyoxylate as a product. In this work, we build and characterize a condensed, reverse tricarboxylic acid (crTCA) cycle in vitro and in planta. We demonstrate that a simple, synthetic cycle can be used to fix carbon in vitro under aerobic and mesophilic conditions and that these enzymes retain activity when expressed transiently in planta. We then evaluate stable transgenic lines of Camelina sativa that have both phenotypic and physiologic changes. Transgenic C. sativa are shorter than controls with increased rates of photosynthetic CO2 assimilation and changes in photorespiratory metabolism. This first iteration of a build-test-learn phase of the crTCA cycle provides promising evidence that this pathway can be used to increase photosynthetic capacity in plants.