Stabilizing heterologous protein production in Phaeodactylum tricornutum through cystatin overexpression

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.