Decreasing protein biotinylation background in a diatom facilitates proximity labeling of the periplastidial compartment proteome

SUMMARY Diatoms are ecologically and industrially significant microalgae, crucial for global carbon fixation and biotechnological applications. Their complex plastid membrane structures, resulting from secondary endosymbiosis, remain poorly characterized, particularly the periplastidial compartment (PPC). Proximity labeling techniques, such as TurboID and ascorbate peroxidase 2 (APEX2)‐based labeling, are powerful tools for identifying protein–protein interactions and spatial proteomes, but their application in diatoms is hindered by unknown factors. In this study, we identified and characterized the high biotinylation background in diatoms, including Phaeodactylum tricornutum and other microalgae, which significantly impairs the effectiveness of proximity labeling. We also characterized the biotin synthase (BIOB) in P. tricornutum , a key enzyme in biotin biosynthesis. By using a biob mutant to deplete biotin, we successfully decreased the biotinylation background, enhancing the sensitivity and quality of proximity labeling. Applying this approach to the PPC, we identified several proteins previously undetectable through bioinformatics and confocal microscopy. Our results demonstrate that inhibiting biotin synthesis improves TurboID‐based proximity labeling methods for studying protein interactions and spatial proteomics in diatoms. The case study of the improved proximity labeling system in PPC also increased our understanding of the complex plastids derived from higher‐order endosymbiosis.