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Anticipated future population growth necessitates that more food is produced using the same, or less, area of land than we use today. One potential way to achieve this is through the improvement of photosynthesis in the crop plants that feed the world. Recently, there has been several successful attempts to improve photosynthesis in C3 crop plants. However, not all crop plants conduct C3 photosynthesis, and comparatively little effort has been focussed on improving alternative photosynthetic pathways such as C4 photosynthesis and CAM. Correspondingly, this DPhil project had two parallel aims. The first was to develop a novel approach to improve photosynthesis in C4 crop plants. The premise of the approach is that there is substantial natural variation in the catalytic properties of enzymes of the C4 cycle between different species, and if one could understand the molecular basis for this variation then one could engineer enhanced flux through the C4 cycle through simple sequence changes. The novel developed in this thesis is divided into three stages. 1) Identification of target sites for enzyme engineering though comparative genomics. 2) Use bacterial systems to assess the impact of variation at these target sites on enzyme function. 3) Engineer the changes required to produce the best performing variant directly in crop plants using genome editing technologies. I achieved step one and came close to achieving step two in during the course of this project. The second overarching aim was to identify the dicarboxylate and monocarboxylate transporters that facilitate malate import and pyruvate export from the bundle sheath cell chloroplast in C4 plants. The lack of knowledge about the identity of these transporters represents the largest gap in our understanding of C4 photosynthesis and hinders efforts to engineer the C4 pathway into C3 plants. This part of the DPhil project resulted in the identification of a single gene which provided both the malate import and pyruvate export functions of the bundle sheath cell chloroplast in the C4 plant Setaria italica, thus completing the first biochemical map for a C4 cycle in any species. Together, the results from this thesis provide a novel platform for potentially enhancing C4 photosynthesis, and for the first time define a complete parts list required to engineer a complete C4 pathway into C3 plants.