The marine diatom Phaeodactylum tricornutum is an emerging platform for metabolic engineering and recombinant protein production. However, heterologous protein accumulation often shows strong cell-to-cell variability and decreases over successive generations in bioengineered diatoms. In this study, we investigated whether overexpression of an endogenous cysteine protease inhibitor, cystatin ( Pt CYS), could stabilize heterologous protein production in P. tricornutum . We generated extrachromosomal expression strains co-expressing the yellow fluorescent protein ( yfp ) gene with Pt CYS. The anti-protease activity of Pt CYS was verified in vitro . Co-expression of YFP with the full-length Pt CYS coding sequence resulted in higher YFP accumulation (~1.6-fold). Increased heterologous protein accumulation was impaired by fusion with Pt CYS and dependent on the presence of an endogenous signal peptide. The reintroduction of Pt CYS into three independent YFP-only lines with low initial fluorescence led to a marked increase, by ~2-fold, in YFP signal in one genetic background, highlighting the potential of this strategy as well as strain-dependent effects. Importantly, changes in YFP accumulation were not associated with increased yfp transgene copy number. Together, these results demonstrate that cystatin-mediated modulation of proteolytic activity can influence heterologous protein stability in P. tricornutum , while also revealing strong context- and genetic background–dependent limitations. This work provides critical design considerations for improving recombinant protein production in diatom-based biofactories. • Identification & characterization of a cysteine protease inhibitor, cystatin ( Pt CYS) in the diatom Phaeodactylum tricornutum • Pt CYS modulates heterologous protein accumulation in a context- and genetic background-dependent manner. • Pt CYS co-expression alters YFP stability & fluorescence depending on fusion design & signal peptide usage. • Pt CYS enhances P. tricornutum 's potential as a sustainable recombinant protein biofactory.
Fantino et al. (Sun,) studied this question.