TY - JOUR

T1 - Methanol feeding strategies for high-yield production of a collagen-based protein in Komagataella phaffii

AU - Ebbecke, Jan Peter

AU - Schlauch, Domenic

AU - Güler, Charlotte

AU - Pirmahboub, Hamidreza

AU - Kara, Selin

AU - Pepelanova, Iliyana

N1 - Publisher Copyright: © The Author(s) 2025.

PY - 2025/12/24

Y1 - 2025/12/24

N2 - 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 application of a three-phases fermentation strategy with 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 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. Key points: • Methanol-limiting feed enhances collagen expression in Komagataella phaffii bioprocesses.

AB - 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 application of a three-phases fermentation strategy with 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 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. Key points: • Methanol-limiting feed enhances collagen expression in Komagataella phaffii bioprocesses.

KW - Bioprocess optimization

KW - Collagen

KW - Komagataella phaffii

KW - Methanol feeding strategy

KW - Recombinant protein expression

KW - Stirred tank reactor

UR - http://www.scopus.com/inward/record.url?scp=105025889620&partnerID=8YFLogxK

U2 - 10.1007/s00253-025-13675-z

DO - 10.1007/s00253-025-13675-z

M3 - Article

C2 - 41436677

AN - SCOPUS:105025889620

VL - 109

JO - Applied Microbiology and Biotechnology

JF - Applied Microbiology and Biotechnology

SN - 0175-7598

IS - 1

M1 - 284

ER -