Improving plant carbon fixation through engineering Rubisco in E. coli

Zhou Y, Conlan B and Whitney S

Research School of Biology, Australian National University.

Enhancing the light and carbon capture reactions of photosynthesis are seen as prime opportunities for improving crop productivity and yield. A significant research focus has been on identifying strategies to enhance the CO2-fixing potential of the photosynthetic enzyme Ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco). Identifying mutations that enhance the carboxylation properties of plant Rubisco has proven particularly difficult. A significant obstacle is the extensive cocktail of ancillary proteins (chaperonin/chaperones) needed to assemble the 8 large and 8 small subunits of the 520 kDa plant L8S8 holoenzyme. Fortunately, the cocktail of ancillary proteins required for producing bacterial L8S8 Rubisco isoforms is less than plant Rubisco. This enables some bacterial Rubiscos to be expressed in both E. coli and plant chloroplasts. My project seeks to take advantage of the “E. coli & chloroplast expression capability” of a Rubisco sourced from a photosynthetic bacterium. In the first research stage, the kinetics of the bacterial Rubisco are being engineered by directed evolution using a novel Rubisco Dependent E. coli (RDE) screen re-designed from that published (1). The new RDE3 screen incorporates features tailored for selecting Rubisco mutants with improved carboxylation properties. The second research stage of the project involves transplanting the evolved bacterial Rubiscos by plastome transformation into leaf chloroplasts to translationally test their capacity to improve photosynthesis and plant growth. Towards this objective tobacco lines producing compatible components for maximising the assembly and metabolic regulation of the bacterial Rubisco are being generated. An update on progress with both research stages will be presented. (1) Wilson R.H., et al. (2018). JBC, 293, 18-27.