Abstract
<jats:p> To overcome the enzymatic limitations of Rubisco, algae operate CO <jats:sub>2</jats:sub> -concentrating mechanisms (CCMs) that deliver concentrated CO <jats:sub>2</jats:sub> to Rubisco tightly packaged in a specialized microcompartment called a pyrenoid. Pyrenoids are globally important biomolecular condensates, but their convergent evolution means that their molecular composition and emergent architecture cannot be inferred across clades. Here we characterize the pyrenoid of the Trebouxiophyceae alga, <jats:italic>Chlorella sorokiniana</jats:italic> . Using cryo-electron tomography, we provide an architectural overview of the pyrenoid and visualize pyrenoid-specific protein complexes. Quantitative proteomics and Rubisco co-immunoprecipitation followed by mass spectrometry demonstrate that inorganic carbon delivery machinery is conserved across green algae but the pyrenoid structural components are not. In vitro reconstitution supports the role of two previously undescribed proteins, one in assembly of pyrenoid traversing thylakoids (putative matrix thylakoid tether; PMTT) and another in starch tethering to the Rubisco matrix (putative matrix starch tether; PMST). In <jats:italic>Nicotiana benthamiana</jats:italic> , PMTT localized to the thylakoid stromal lamellae and PMST to chloroplast starch granules. Our findings provide insights into the molecular logic of pyrenoid assembly; how proteins mediate condensate-membrane and condensate-starch interactions; and expands the pyrenoid plant engineering toolkit, setting the stage for engineering a <jats:italic>Chlorella</jats:italic> pyrenoid into plants. </jats:p>