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. The relatively low
specific activity and tendency to accept O2 as a substrate
have made Rubisco an attractive but challenging target for 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 CO2 and O2 levels. We address the challenge
of designing a zero-shot input library of the Gallionella Rubisco, without prior experimental knowledge, by coupling the phylogenetically
guided model EV mutation with “in silico evolution”.
This multisite mutagenesis library of Synechocystis (n = 16) was subjected to competitive growth in
different gas feeds coupled to deep sequencing, in order to compare
Rubisco variants. We identified an amino acid exchange that increased
the thermostability of Gallionella Rubisco and conveyed
resilience to otherwise detrimental amino acid exchanges. The platform
is a first step toward 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.