Positioning Auxenochlorella protothecoides as a Chassis for the Engineering of a Pyrenoid-based Carbon-Concentrating Mechanism

Abstract

With the rapid increases in the global population predicted to occur over the coming decades, food scarcity has become a serious concern. Novel strategies to mitigate this issue must be considered, lest we risk overburdening the earth’s resources with agricultural land use. Engineering an algal carbon-concentrating mechanism (CCM), the pyrenoid, in C3 crop plants is a promising approach to enhancing crop yields and growth rates at atmospheric CO2 levels. However, the minimal set of proteins sufficient to achieve a functional pyrenoid is not known and engineering protocols in plants are time intensive. This thesis investigates the engineering of a genetically tractable and CCM-less alga, Auxenochlorella protothecoides, as a proof of concept for this reconstitution strategy. Computational modeling has identified six proteins essential for pyrenoid generation in Chlamydomonas and I have worked to assess the sufficiency of four of these candidate proteins to induce the formation of a minimal CCM in Auxenochlorella. While these initial engineering efforts have successfully replicated the characteristic Rubisco matrix condensate, there are abnormalities associated with the tubule structures, likely arising from transgene mis-expression and incomplete component stoichiometry. To address these limitations, I have developed the Auxenochlorella ToolKit (ATK). ATK comprises both a newly characterized regulatory part library with ~20 promoter/terminator pairs and a novel Modular Cloning-based cloning architecture, enabling parallelized construct assembly and expression tuning of pyrenoid-related transgenes. Collectively, this work represents a step towards the first successful reconstitution of a functional algal pyrenoid in a heterologous system while establishing a foundation for the domestication of Auxenochlorella as a programmable eukaryotic and photosynthetic chassis for synthetic biology.