A cyanobacterial screening platform for Rubisco mutant variants

Rubisco is the main entry point of inorganic carbon into the biosphere and a central player in the global carbon system. Its relatively low catalytic constant as well as its tendency to also accept O ₂ as a substrate have made it a common target of enzyme engineering. We have developed an enzyme engineering and screening platform for Rubisco using the model cyanobacterium Synechocystis sp. PCC 6803. Starting with the Form II Rubisco from Gallionella, we first show that the enzyme can replace the native Form I Rubisco in Synechocystis and that growth rates become sensitive to CO ₂ and O ₂ levels. We address the challenge of designing a zero-shot input library, without prior experimental knowledge, by coupling the phylogenetically-guided model EVmutation with “ in silico evolution”. Starting with this targeted mutagenesis library, we used competitive growth coupled to deep sequencing to compare the properties of Rubisco protein variants under different cultivation conditions. We identified an amino acid exchange which increased the thermostability of Gallionella Rubisco and conveyed resilience to detrimental exchanges. 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 Calvin-Benson-Bassham cycle in cyanobacteria and possibly chloroplasts.