Expression screening and functional verification of recombinant hirudin from novel chassis cell Chlamydomonas reinhardtii

Hirudin is a potent anticoagulant peptide whose clinical application is limited by scarce natural supplies and the suboptimal activity of recombinant versions from conventional microbial hosts lacking key post-translational modifications. Here, for the first time, we establish the “Generally Regarded as Safe” (GRAS) microalga, Chlamydomonas reinhardtii , as a novel biological chassis for producing fully functional hirudin. Using high-density heterotrophic fermentation, we generated a bioactive lyophilized algal powder suitable for oral delivery. Crucially, the algal-derived hirudin underwent proper tyrosine sulfation—a key modification absent in prokaryotic hosts—conferring exceptionally high thrombin-inhibitory activity (up to 20,000 ATU/mg). When orally administered to a murine thrombosis model, this hirudin-loaded alga demonstrated potent antithrombotic efficacy. It also exhibited a superior safety profile, showing no signs of the thrombocytopenia or bleeding associated with traditional anticoagulants. This study establishes a green, scalable biomanufacturing and oral delivery platform for therapeutics, highlighting transformative potential at the nexus of agricultural bioengineering, functional foods, and biomedicine.