Directed evolution of plant Rubisco with RDE (Rubisco-dependent E. coli) system

Life on Earth is energy-dependent. Autotrophic organisms, such as plants and algae, are able to convert sunlight energy into chemical energy through an apparently simple process called photosynthesis — synthesis of organic molecules from photons. The photons of sunlight are captured by photosystems and the chemical energy produced (ATP) is spent for the Calvin-Benson- Bassham (CBB) cycle for fixing atmospheric CO2 and leading to the synthesis of all necessary organic molecules. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) plays an essential role in photosynthesis, catalyzing the fixation of atmospheric CO2 into sugars by the first step of CBB cycle. Being considered a slow enzyme and due to its limiting catalysis, enhancing Rubisco performances remained one of the most promising task in order to boost photosynthesis and increasing crop yields in the last five decades (Chapter 1). In this study we present the first directed evolution approach applied on plant Rubisco that identified two single amino acid (AA) mutations on L-subunit of tobacco Rubisco that, although conserved in plants, are able to alter considerably Rubisco properties. We designed a novel screening called chloroplast compatible Rubisco-dependent Escherichia coli (ccRDE) that allowed to select for a single AA substitution XXXXXX, which increases kcatC up to ~30% compared to wild type Rubisco in E. coli and a single AA substitution XXXXX that increases notably the tobacco Rubisco biogenesis 4-fold more than wild type (up to ~14% of relative soluble protein in E. coli) suggesting a possible adaptation role with one (or more) chaperone/chaperonine interaction that facilitates the folding/assembly of its complex L8S8 active state (Chapters 2 and 3). Next we tested the two mutations in four different plant Rubiscos (Arabidopsis, carrot, potato and cassava) in E. coli, showing conserved and compatible results compared to tobacco (Chapter 3). Finally we introduced XXXXXX single mutation into tobacco chloroplasts by transplastomic transformation using cmtrL tobacco master line. Preliminary results showed an increase in height and biomass in the plant with the mutation compared to WT tobacco plant, although numerous in vivo CO2-assimilation and kinetics analyzes are still being tested. We obtained T2 transformed tobacco marker-free lines which will be subjected to numerous in vivo tests (Chapter 4). In perspective, our findings showed that plant Rubisco may further evolve, even without altering the auxiliary chaperon machine, leading us to detect new Rubisco landscapes and finally redesign an enzyme considered “untouchable” for many years.