With the expanding global population and the associated growing demand for food and energy, researchers have been looking into ways to improve plants productivity, and more particularly the way they convert carbon dioxide to biomass during photosynthesis. Targeting carbon capture efficiency seems a good opportunity to raise crop productivity both for food and biomass-based biofuels. Most approaches have focused on optimizing the CBB cycle and more particularly the key carboxylating enzyme, Rubisco, because of its rather low catalytic rate and poor substrate specificity. Here we propose an alternative approach by engineering an orthogonal artificial carbon fixation pathway into photosynthetic organism. We choose cyanobacteria, a model system for phototrophic eukaryotes, while also implementing the new pathway into the model plant organism, Arabidopsis thaliana. The new CO2 fixation cycle would not rely on Rubisco and would fix carbon with only 30% of the energy requirement compared to the native CBB cycle. Over the course of this study we encountered a number of logistical problems that thwarted the implementation of the orthogonal carbon fixation cycle. First, the Rubisco mutant strain that could have served as a platform for efficient screening of our pathway was too sick to be used as such. Secondly, our pathway relied on a pyruvate ferredoxin:oxidoreductase for one of its carboxylation step, which is known to be oxygen sensitive. Efforts to evolve an oxygen tolerant mutant of this enzyme were unsuccessful. Lastly, a possible kinetic trap that could prevent the cycle from running was identified. The accumulation of setbacks forced us to modify our initial pathway cycle design. The final pathway, termed rGS2, does not use pyruvate ferredoxin:oxidoreductase for carbon fixation and avoids the kinetic trap. It does lose any energy efficiency gain over the CBB cycle but could nevertheless still increase carbon fixation efficiency as it does not solely depend on Rubisco. Implementation of the rGS2 in cyanobacteria increased the intracellular acetyl-CoA pool and led to secretion of α- ketoisocaproate. The engineered strain was genetically unstable, which was probably the result of toxic intermediates being built up from the rGS2 pathway. In the future, we hope to optimize the rGS2 pathway by testing new homologues enzymes or alternative steps to improve the rGS2 related toxicity.
In plants, the original synthetic carbon fixation pathway, the rGS cycle, was engineered in both wild type Arabidopsis and in two mutant strains defective in their endogenous CBB cycle. In the three different backgrounds, mutants with enhance biomass and/or height were identified, suggesting that the rGS pathway may be functional and could be working as an optional pathway for CO2 fixation. But like in cyanobacteria, genetic instability was observed, and genotyping of the T3 mutant plants could not confirm the presence of all heterologous rGS genes, possibly the results of DNA recombination between homologous regions on the T-DNA causing some of the fragment to loop out.
Numerous obstacles were encountered throughout the course of this work, but the results and lessons learned could later be applied to build more robust orthogonal carbon fixation cycles, and improve production of downstream pathways.
