Directed evolution of rubisco and translational application in planta
Creators: name: Wilson, Robert Henry
Publisher: name: The Australian National University
Year: 2015

description: In photosynthesis Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyses the primary step in carbon assimilation by fixing CO2 to the substrate ribulose-1,5-bisphosphate (RuBP). This process occurs at a relatively slower pace (only 1 to 4 cycles per second in leaves) than would be expected for such an important selection target during evolution and as such the catalytic properties of Rubisco often limit photosynthesis rates. Current models predict that improving Rubisco function has the potential to enhance the photosynthetic efficiency of plants with a C3-physiology like wheat and rice (Parry et al. 2013). Rational and directed evolution mutagenesis of the 50kDa catalytic large subunit, and catalytically influential 15kDa small subunit, from different Rubisco isoforms has so far been unsuccessful in significantly improving overall catalytic function. In this study the decameric non-photosynthetic Rubisco from the Archaea Methanococcoides burtonii (MbR), which had previously been shown capable of supporting plant growth (Alonso et al. 2009), was improved using laboratory evolution in an E. coli selection system developed by Mueller-Cajar et al. (2007). Mutations were identified which increase the carboxylation rate of MbR by 2 to 3 fold. These evolved variants of MbR were transformed into tobacco plastids to produce tobacco progeny that were solely dependent on the evolved MbR for CO2 assimilation. Consistent with their improved Rubisco catalysis, the tobacco lines producing the evolved MbR mutants showed significant improvements in their photosynthesis and growth relative to the tobacco genotypes producing the native MbR. These findings demonstrate the potential of directed evolution for improving catalysis of Archaea Rubisco and the potential for translational integration of these improvements into plants. Selection of Rubisco assembly and function in E. coli requires the co-expression of phosphoribulokinase (PRK), but the ensuing production of RuBP that is toxic to E. coli growth which leads to PRK removal by transposon integration generating false positives during selection (Parikh et al. 2006). In this thesis a new high throughput Rubisco dependent E. coli (RDE2) selection system is presented where fusion of neomycin phophotransferase to a cyanobacterial PRK successfully diminished escape mutant selection in the presence of kanamycin. The RDE2 system successfully selected for Rubisco mutants that increase RuBP fixation rates, thus increasing E. coli viability and growth rate. Demonstrated are novel mutations selected using RDE2 which improve the functional assembly of L8S8 Rubisco from Thermosynechococcus elongatus BP-1 (a cyanobacterium) in E. coli by up to 4-fold in addition to a mutation that significantly improves the enzymes affinity for RuBP (i.e. reduces its Km for RuBP by 9-fold) - with a full characterisation of the resulting kinetic properties of these mutants. Overall this thesis uniquely demonstrates the translational potential for transplanting Rubisco mutants evolved in E. coli directly into leaf chloroplasts and the development of a highly reliable method for screening large mutant libraries of Rubisco. The outcomes provide a useful framework for directed evolution applications towards the goal of enhancing Rubisco function as a means for improving the photosynthesis and growth of plants, in particular key crop species.