Stabilizing heterologous protein production in Phaeodactylum tricornutum through cystatin overexpression

Fantino, E., Mérindol, N., Gajón Robles, C. G., Awwad, F., Gonçalves dos Santos, K. C., Rhéaume, K.-L., Goulet, M.-C., Michaud, D. et Desgagné-Penix, I. (2026). Stabilizing heterologous protein production in Phaeodactylum tricornutum through cystatin overexpression. Algal Research, 95 . Article 104670. ISSN 2211-9264 DOI 10.1016/j.algal.2026.104670

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Résumé

Abstract

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 (PtCYS), could stabilize heterologous protein production in P. tricornutum. We generated extrachromosomal expression strains co-expressing the yellow fluorescent protein (yfp) gene with PtCYS. The anti-protease activity of PtCYS was verified in vitro. Co-expression of YFP with the full-length PtCYS coding sequence resulted in higher YFP accumulation (~1.6-fold). Increased heterologous protein accumulation was impaired by fusion with PtCYS and dependent on the presence of an endogenous signal peptide. The reintroduction of PtCYS 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.

Type de document:	Article
Mots-clés libres:	Marine diatoms Cystatin Biofactories Metabolic engineering Mean fluorescence intensity
Date de dépôt:	01 juin 2026 12:59
Dernière modification:	01 juin 2026 12:59
Version du document déposé:	Version officielle de l'éditeur
URI:	https://depot-e.uqtr.ca/id/eprint/12918

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