Engineering of Komagataella phaffii for efficient secretory expression of highly hydrophilic recombinant type III human-like collagen

Collagen, a key extracellular matrix protein, regulates cell adhesion, proliferation, and tissue integrity for skin health. In this study, a novel human-like type III collagen was engineered by substituting hydrophobic GXY triplets in the core fragment (Gly201-Asn498) of the human α1(III)type chain with hydrophilic motifs (GSP, GQP, GEP, and GSQ) and incorporating integrin-binding sequences (GER and RGD) at the C-terminus of the two consecutive sequence units. The codon-optimized gene was cloned into the pPIC9K vector, and transformed into Komagataella phaffii ( Pichia pastoris ) GS115. Multicopy transformants were selected by various G418 concentrations. Furthermore, a high yield of recombinant collagen at 19.4 g/L was achieved by fed-batch fermentation in a 5-L bioreactor. The purified protein exhibited excellent thermostability, remaining fully soluble after heating at 121℃ for 30 min. The collagen migrated on 10% SDS-PAGE with an apparent molecular weight approximately 2.18-fold greater than its theoretical mass, suggesting extensive hydration. Functionally, it significantly promoted the proliferation of human skin fibroblasts (HSF) and immortalized keratinocytes (HaCaT). In HSF cells, it upregulated the expression of COL1A1 , COL3A1 , and TIMP1 , while in HaCaT cells, it enhanced transcription of skin barrier-related genes, including KRT1 , KRT5 , KRT10 , KRT14 , IVL , LOR , and FLG . This designed collagen integrates high-yield production, thermostability, and dual bioactivity for dermal regeneration and epidermal barrier reinforcement, showing a promising application for skincare and tissue engineering.