Cyanobacterial Form II Rubisco screening platform

Flux control in the Calvin-Benson-Bessham (CBB) cycle is distributed over several reactions operating far from equilibrium. In particular Rubisco has significant control over carbon fixation for growth or biosynthesis. We have developed an enzyme engineering platform with the ultimate goal of increasing carbon fixation and conversion by the model cyanobacterium Synechocystis sp. PCC 6803. Starting with targeted mutagenesis libraries, we used competitive growth coupled to deep sequencing to compare the properties of Rubisco protein variants under different cultivation conditions. As the type I Rubisco of cyanobacteria, green algae and land plants may be phylogenetically constrained regarding mutations and catalytic parameters, we used a type II Rubisco, as these show more sequence diversity and lower folding requirements. We show that the platform identifies enzyme variants of the type II Rubisco that affect cell growth in different conditions. The establishment of this platform is a first step towards high-throughput screening of Rubisco variants in Synechocystis and creating optimized enzyme variants to accelerate the CBB cycle in cyanobacteria and possibly chloroplasts.