Repurposing the Diatom Periplastidial Compartment for Heterologous Terpenoid Production

Diatoms are promising microorganisms to provide sustainable routes for photosynthetic terpenoid production from CO₂, yet their potential for compartmentalized engineering remains largely unexplored. Here, we systematically profiled the biosynthetic capacity of Phaeodactylum tricornutum by targeting representative synthases for hemi-, mono-, sesqui-, and tetraterpenoids to the cytosol, chloroplast, and periplastidial compartment (PPC). This comprehensive analysis revealed that all major prenyl phosphate precursors, DMAPP, GPP, FPP, and GGPP, are accessible in all compartments, including in the PPC, and can sustain heterologous flux without major physiological penalties, although production efficiency varies across compartments and product classes. By determining precursor availability, we propose the diatom PPC as an engineerable intracellular space directly integrated with a eukaryotic complex chloroplast, establishing a foundation for further compartmentalized terpenoid biosynthesis engineering strategies. Moreover, we highlight its utility as a unique interface to investigate the metabolic exchange between the MVA and MEP pathways. These findings provide a systematic framework for compartment-specific terpenoid engineering in diatoms and open new opportunities for modular pathway assembly and synthetic biology in photosynthetic eukaryotes.