发酵
重组DNA
甲醇
生物反应器
生物量(生态学)
食品科学
生物化学
化学
生物
蛋白质表达
细胞外
产量(工程)
限制
生物技术
生物过程
高蛋白
补料分批培养
营养物
毕赤酵母
酵母
效价
下游加工
细胞生物学
碳源
恒化器
细胞生长
蛋白质生物合成
化学定义介质
单细胞蛋白
拉伤
作者
Jan Peter Ebbecke,Domenic Schlauch,Cengiz Güler,Hamidreza Pirmahboub,Selin Kara,Iliyana Pepelanova
标识
DOI:10.1007/s00253-025-13675-z
摘要
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. • Exponential feeding favors biomass but lowers protein yield and process efficiency • Gradient feeding results in the highest collagen titers and sustained expression.
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