Engineering microalgal cell wall-anchored proteins using GP1 PPSPX motifs and releasing with intein-mediated fusion

Harnessing and controlling the localization of recombinant proteins is essential for advancing synthetic biology, industrial biotechnology, and drug delivery. This study presents a dual system for protein anchoring and controlled release in Chlamydomonas reinhardtii . Using truncated variants of the GP1 glycoprotein fused to the plastic-degrading enzyme PHL7, we identified the PPSPX motif as critical for anchoring proteins to the cell wall. Constructs with increased PPSPX content exhibited reduced secretion but enhanced anchoring, revealing key anchor-signal sites within GP1. To enable controlled release, we incorporated a pH-sensitive intein from derived from Mycobacterium tuberculosis RecA. Under acidic conditions (pH 5.5), this intein efficiently cleaved and released mCherry and PHL7 from the GP1 anchor. Fluorescence kinetics demonstrated significant mCherry release from the mCherry-intein-GP1 construct within 4 hours at pH 5.5, while release was minimal at pH 8.0 and negligible for the mCherry-GP1 control. Western blot analysis confirmed efficient cleavage, showing a lower band corresponding to free mCherry at pH 5.5 and no release at pH 8.0. This anchor-release strategy integrates glycomodules with pH-sensitive inteins to enable precise spatial and temporal protein control. The system offers broad utility for targeted drug delivery, environmental biosensing, and biocatalyst deployment. Overall, we establish a versatile framework for optimizing protein localization and environmentally triggered release in C. reinhardtii , with broad implications for proteomics, biofilm engineering, and scalable therapeutic delivery systems.