P. Chang, J. Summer, C. E. Blaby-Haas
Lawrence Berkeley National Laboratory,
United States
Keywords: CO2, Rubisco, Algae, Biocondensate
Summary:
Rubisco is the primary enzyme responsible for global carbon fixation, yet it is notoriously limited by a slow catalytic rate and poor substrate specificity. To overcome these limitations under declining atmospheric CO2 levels, many photosynthetic organisms—including cyanobacteria and algae—have evolved Rubisco biocondensates, such as carboxysomes and pyrenoids. While these structures are known to function alongside complex Carbon Concentrating Mechanisms (CCMs) to elevate local CO2 concentrations, it remains unclear whether the biocondensate state itself inherently enhances Rubisco’s enzymatic efficiency independent of auxiliary CCM components. In this study, we establish two complementary strategies to decouple the effects of protein condensation from auxiliary CCM components. First, we utilize the minimalist green alga Auxenochlorella protothecoides, which naturally lacks a pyrenoid, as a model for the synthetic introduction of rudimentary Rubisco condensates. By measuring the photoautotrophic growth rates of these engineered strains, we aim to determine if the formation of a protein condensate provides a direct fitness benefit under CO2-limiting conditions. Second, we developed a high-throughput microbial assay in Escherichia coli. By co-expressing phosphoribulokinase (PRK) to generate the inhibitory metabolite RuBP and recombinant Rubisco to facilitate its clearance, we utilize bacterial growth kinetics as a sensitive proxy for Rubisco activity in vivo. This dual-platform approach aims to determine whether phase separation alone provides a kinetic advantage. Our work establishes a robust framework for investigating the evolutionary drivers of protein condensation and evaluates the feasibility of using synthetic condensates to engineer more efficient carbon fixation pathways.