Coli-plast: expressing and bioengineering higher plant Rubisco in E. coli

Buck S and Whitney S

ARC Centre of Excellence for Translational Photosynthesis. Research School of Biology. The Australian National University. Canberra, ACT 2601, Australia.

Ribulose bisphosphate carboxylase oxygenase (Rubisco) is a key enzyme in the process of carbon fixation in photosynthesis, and as such it directly or indirectly supports almost all life on earth. It is has some marred properties for such a crucial enzyme; being both slow and non-specific, catalyzing the oxygenation as well as the carboxylation of RuBP resulting in an energy intensive regeneration process. Synthetic biology and bioengineering methods for the improvement of Rubisco have been hindered by its inability to be functionally expressed in the commonly used bacterial expression host Escherichia coli. This is likely because the biogenesis requirements of plant Rubisco cannot be met in E. coli due to incompatibilities in the protein folding and assembly machinery between E. coli and chloroplasts. This study aims to develop expression capabilities in E. coli suitable for improving the catalysis of higher plant Rubisco. This involves the expression of functional tobacco Rubisco in E. coli through its co-expression with the necessary ancillary proteins needed for its biogenesis and metabolic repair. This system will complement existing Rubisco directed evolution E. coli systems that have successfully identified mutations that improve the kinetics of Archaea and cyanobacteria Rubisco - and hopefully translated to selecting improved crop Rubisco mutants useful for subsequent testing in planta.