What is true for plants may not be true for Phaeodactylum tricornutum : The case of Vanilla planifolia vanillin synthase ( Vp VAN) targeted to four subcellular compartments of the diatom

Vanillin is the major organoleptic compound of the vanilla flavor. Since the extraction process from its natural source ( Vanilla planifolia ) is limited by a low yield, the market demand for vanillin is met by producing it through chemical synthesis or biotechnologically using microorganisms to convert ferulic acid, lignin, and guaiacol into vanillin. Our aim was to use the diatom Phaeodactylum tricornutum , a photosynthetic model organism already used to produce eukaryotic proteins and high-value compounds, to characterize V. planifolia vanillin synthase ( Vp VAN). This enzyme is the subject of conflicting results and was reported to be localized in the plastid of V. planifolia cells, which may play a role in its activity. For this purpose, we targeted Vp VAN linked by an in vitro cleavable sequence (Xa) to the enhanced green fluorescent protein (eGFP) to the cytoplasm, peroxisomes, plastid, and Golgi apparatus. We used an episomal expression system transforming P. tricornutum with VpVAN:Xa:eGFP by bacterial conjugation. Positive cell lines were selected by fluorescence and characterized by sequencing the episomes, confirming stable replication in the diatom, as well as assessing the subcellular localization in the targeted cell compartments. Strategies such as enriching GFP-positive cells by cell sorting, protein purification, and organelle isolation did not yield detectable levels of the fusion protein. A better understanding of the diatom expression system is needed to complete the characterization of this enzyme and reveal whether the subcellular localization could be a missing aspect of Vp VAN expression in microorganisms.