Directed evolution of a plant Rubisco chaperone with altered client recognition.

Abstract

Improving the Calvin-Benson-Bassham cycle enzyme Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) has the potential to increase crop productivity. However, the selectivity of the chaperones mediating Rubisco assembly in vascular plants toward their cognate Rubisco presents a substantive roadblock to both Rubisco protein engineering and transgenic expression of heterologous Rubisco orthologs, both of which necessitate changes to the Rubisco sequence. Here, we ask whether a plant Rubisco chaperone can be reprogrammed by directed evolution to accommodate a nonnative client. We developed a selection strategy to assess Rubisco assembly factor activity in high-throughput and used this selection to identify mutants of the chaperone Raf1 from <i>Arabidopsis thaliana</i> (<i>At</i>Raf1) that assemble <i>Nicotiana tabacum</i> Rubisco, for which wild-type <i>At</i>Raf1 has minimal activity. We show that directed evolution can generate <i>At</i>Raf1 variants that enable significantly increased <i>N. tabacum</i> Rubisco assembly compared to wild-type <i>At</i>Raf1. Evaluation of evolved <i>At</i>Raf1s indicates that they retain the ability to assemble their native client and can assemble other dicot Rubisco orthologs that they were not evolved to recognize. This work may provide a strategy for addressing the constraints chaperone selectivity impose upon Rubisco-centric efforts to improve plant photosynthesis.