C4 photosynthesis is a specialised carbon concentrating mechanism that achieves higher rates of photosynthesis. Engineering C4 photosynthesis into C3 plants could improve agronomic features of staple C3 crops. Introducing the biochemistry of C4 photosynthesis is considered a key step in engineering the C3-to-C4 transition. Herein, a reverse C4 photosynthesis system that aimed to install C4 biochemistry in the opposite cell types from those used by C4 species was investigated. This approach was hypothesised to replicate the spatial patterning of the C4 biochemical pathway but make use of the native leaf anatomy of C3 species.

The main objective of this thesis was to test if introducing a reverse C4 photosynthetic pathway in C3 Arabidopsis thaliana could improve its photosynthetic efficiency. Two subtypes of the C4 biochemical pathway, the PEPCK and NAD-ME subtypes were tested. A. thaliana genes most suitable for assembling these subtypes were selected based on homology to C4 genes and relative transcript abundance. Transgenic plants carrying a reverse PEPCK C4 pathway displayed no differences in carbon isotope composition or Photosystem II activity, and in a genetic background with less RuBisCO showed lower CO2 assimilation. Genes for a reverse NAD-ME C4 pathway were assembled into two large multigene constructs containing 9 and 10 transcription units. Homozygous lines carrying each construct were obtained and showed overexpression of most transgenes. Although plants carrying bundle sheath genes of the pathway showed no visible phenotypes and those carrying mesophyll genes showed retarded growth, the latter phenotype seemed to be alleviated in F1 crosses containing genes encoding a full reverse NAD-ME C4 pathway. Work is also reported that aimed to explore two alternative approaches to accelerate the main objective of C4 engineering. First, clustering enzymes of a metabolic pathway via protein scaffold was tested. Three enzymes for the biosynthesis of betalain were transiently expressed in tobacco in the presence of scaffold proteins of various structures. However, betalain production was inhibited. Varying the number of domains on scaffold proteins or attaching them to the vacuole membrane did not generate significant impact on pathway efficiency. Second, a microdroplet-based cell-free protein expression system was built to mimic plant metabolic pathways in vitro. Fluorescent proteins and enzymes could be directly synthesised from DNA templates in microdroplets. Fluorescence corresponding to betalains was observed when part of the pathway was expressed and substrate provided. A potential future application of this system was to mimic multicellular physiological processes such as the C4 biochemical pathway.