[SCAN] Opposing effects of Folding and Assembly Chaperones on RuBisCO Evolution

SCAN

Title: Opposing effects of Folding and Assembly Chaperones on RuBisCO Evolution

Speaker: Paulo Durão

Affiliation: Gulbenkian Institute for Science, ITQB alumni

Abstract:

The enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) catalyzes the fixation of CO2 in photosynthesis. Despite its pivotal role, RuBisCO is an inefficient enzyme and thus is a key target for directed evolution. RuBisCO biogenesis depends on auxiliary factors, including the GroEL/ES-type chaperonin for folding and the chaperone RbcX for assembly. We have performed directed evolution of cyanobacterial form I RuBisCO using a RuBisCO-dependent Escherichia coli strain. Overexpression of GroEL/ES enhanced RuBisCO solubility and tended to expand the range of permissible mutations. In contrast, the specific assembly chaperone RbcX had a negative effect on evolvability by preventing a subset of mutants from forming holoenzyme. Mutation F140I in the large RuBisCO subunit, isolated in the absence of RbcX, increased carboxylation efficiency approximately threefold without reducing CO2 specificity. The F140I mutant resulted in a ~55% improved photosynthesis rate in cyanobacteria Synechocystis PCC6803. Our results suggest that the requirement of specific biogenesis factors downstream of chaperonin may have slowed down the natural evolution of RuBisCO.