Production of prolyl-hydroxylated human type III collagen in Physcomitrella photobioreactors

Collagens are important structural proteins of the extracellular matrix that are essential for skin elasticity and integrity and are widely used in dietary supplements and cosmetics. Conventional collagens of animal origin raise concerns regarding ethics, safety, and sustainability. As a vegan alternative, we report on the production of a 30 kDa prolyl-hydroxylated human collagen polypeptide from Physcomitrella moss plants. For secretion-based production and formulation compatibility, a hydrophilic region encompassing 334 amino acids from human type III collagen was selected, which includes four protein domains involved in cell adhesion, collagen binding, integrin recognition and wound healing. Transgenic moss lines were generated via PEG-mediated protoplast transformation. Immunodetection identified collagen polypeptide-producing lines, and mass spectrometric analysis validated the product and confirmed its prolyl-hydroxylation. The presence of this important post-translational modification underscores the high biomimetic quality of the product derived from moss. To enable industrial-scale production, the transformants were quantitatively analysed at the genomic, transcript, and protein levels. The most productive lines were forwarded to process development, where culture conditions including CO ₂ supplementation, pH, and light intensity were optimized. Upscaling to 5 L photobioreactors established a robust, light- and biomass-dependent production regime that yielded nearly 1 mg/L of secreted collagen polypeptide after 11 days of cultivation. Taken together, this study presents the first scalable moss-based production of a post-translationally modified human collagen and offers a sustainable and vegan alternative to conventional collagens. This highlights the versatility of Physcomitrella as a production host for high-quality proteins with industrial applicability that also meet consumer requirements.