With a growing population, there is a growing concern for food security in the 21^st century. It is thought the output of global food production must increase by more than 50% by 2050 to reach demand. This increase in biomass is ultimately limited by plant growth rates - with the key, yet sluggish, photosynthetic CO₂-fixing enzyme Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) considered central to this limitation. For this reason, much attention has been dedicated to characterising the spectrum of Rubisco structure and kinetics to identify key regions for improvement by rational design. To a far lesser degree, irrational design using directed enzyme evolution has been used to directly elucidate some of these key residues¹ .

Given the reluctance of Rubisco to evolve beyond a narrow spectrum of catalytic diversity, it is postulated that a better mutational starting point might be an ancient Rubisco unconstrained by modern photosynthetic architecture. We have manipulated the Rubisco gene from Methanococcoides burtonii - a non-photosynthetic methanogenic archaea within a clade thought to contain some of the most primitive ancestors to extant Rubiscos² . In vitro evolution of M. burtonii Rubisco (MbR) was able to identify mutations that improve its carboxylation rate as much as 3-fold. The most promising MbR mutant genes were codon modified and introduced into tobacco chloroplasts by plastome transformation. Promisingly, in the transplastomic lines generated the improvements in MbR catalysis correlate with the enhanced rates in leaf photosynthesis and plant growth. This research demonstrates the feasibility of using in vitro evolution to improve the CO₂-fixation capacity of a non-photosynthetic Rubisco in E. coli and its translation to increasing leaf photosynthesis. This work proves the versatility of artificial evolution as a tool for engineering phylogenetically diverse Rubisco genes towards improving the photosynthetic capability of plants.