Methanol Feeding Strategies for High-Yield Production of a Collagen-Based Protein in Komagataella phaffii

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Abstract

The recombinant production of extracellular matrix proteins is a promising approach for replacing animal-derived materials in biomedical applications. K. phaffii represents a favorable expression host because it combines the ability of higher eukaryotes for secreted protein production with the ability to grow to high cell densities on simple, low-cost media. Additionally, this well-studied host allows for tight control of recombinant protein expression using the methanol-inducible AOX1 promoter. In this study, different methanol feeding strategies were evaluated to optimize the expression of a collagen-mimetic protein (ColMP-His). A methanol feed approach with carbon as a limiting nutrient resulted in the highest target protein production, whereas exponential feeding resulted in fast biomass accumulation with reduced protein expression. Moreover, the limited feeding strategy resulted in 25% lower oxygen consumption, despite the longer fermentation time, which has a positive impact on process cost efficiency. The addition of a preceding glycerol-fed batch phase to increase biomass did not improve product titers and was associated with reduced expression efficiency. A variation in the methanol feeding rate was also investigated for induction. A gradient-based methanol feed, which increased incrementally over time, achieved the highest final product concentration (83.9 g L⁻¹) and sustained expression over extended fermentation periods. Compared with the initial process, the yield was increased by a factor of 11. Despite statistical limitations due to high variability, the results highlight the importance of adaptive process control in balancing cell growth and recombinant protein production. The presented gradient-based strategy provides a foundation for animal-free, scalable production of recombinant collagen materials.

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