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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.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Key points<\/jats:title>\n                    <jats:p>\n                      \u2022\n                      <jats:italic>Methanol-limiting feed enhances collagen expression in Komagataella phaffii bioprocesses.<\/jats:italic>\n                    <\/jats:p>\n                    <jats:p>\n                      \u2022\n                      <jats:italic>Exponential feeding favors biomass but lowers protein yield and process efficiency<\/jats:italic>\n                    <\/jats:p>\n                    <jats:p>\n                      \u2022\n                      <jats:italic>Gradient feeding results in the highest collagen titers and sustained expression<\/jats:italic>\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Graphical abstract<\/jats:title>\n                  <\/jats:sec>","DOI":"10.1007\/s00253-025-13675-z","type":"journal-article","created":{"date-parts":[[2025,12,24]],"date-time":"2025-12-24T01:05:52Z","timestamp":1766538352000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Methanol feeding strategies for high-yield production of a collagen-based protein in Komagataella phaffii"],"prefix":"10.1007","volume":"109","author":[{"given":"Jan Peter","family":"Ebbecke","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Domenic","family":"Schlauch","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Charlotte","family":"G\u00fcler","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hamidreza","family":"Pirmahboub","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Selin","family":"Kara","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Iliyana","family":"Pepelanova","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,12,24]]},"reference":[{"key":"13675_CR1","doi-asserted-by":"publisher","first-page":"1344","DOI":"10.1111\/1751-7915.14264","volume":"16","author":"MA Boojari","year":"2023","unstructured":"Boojari MA, Rajabi Ghaledari F, Motamedian E, Soleimani M, Shojaosadati SA (2023) Developing a metabolic model-based fed-batch feeding strategy for Pichia pastoris fermentation through fine-tuning of the methanol utilization pathway. 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