Characterization of Engineered Pyrenoid Structural and CO2-Delivery Components in Auxenochlorella protothecoides

Abstract

The enzyme Rubisco, which catalyzes carbon fixation in photosynthesis, is inherently inefficient and limits crop productivity. Some algae, including the model alga Chlamydomonas reinhardtii (Chlamydomonas), overcome this limitation through carbon-concentrating mechanisms (CCMs) centered on an organelle called the pyrenoid, which concentrates CO2 around Rubisco. Engineering a pyrenoid-based CCM into crop plants could substantially enhance agricultural yield. As a proof-of-concept, members of the Jonikas laboratory have reconstituted a protopyrenoid, a condensate of Rubisco and its linker protein EPYC1, in Auxenochlorella protothecoides (Auxenochlorella), a green alga that naturally lacks a pyrenoid. However, this protopyrenoid lacks the CO2 delivery machinery required for a functional CCM. This thesis advances the protopyrenoid toward a minimal functional CCM by adapting expansion microscopy for Auxenochlorella and using it to characterize its structural and CO2-delivery components. Using expansion microscopy, I found that MITH1, a protein required for the formation of membrane tubules that traverse the Chlamydomonas pyrenoid, localizes to similar traversing structures in the protopyrenoid. I further showed that the bicarbonate transporter BST1 can be expressed in engineered Auxenochlorella and displays thylakoid-associated localization consistent with its native pattern in Chlamydomonas. I then identified a transmembrane fusion construct of the carbonic anhydrase CAH3 that retains function in Chlamydomonas. Together, these results demonstrate that key pyrenoid structural and CO2-delivery components can be localized or introduced into a heterologous host, laying the groundwork for reconstituting a minimal CCM in Auxenochlorella.