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Understanding exopolysaccharide byproduct formation in Komagataella phaffii fermentation processes for recombinant protein production

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Title: Understanding exopolysaccharide byproduct formation in Komagataella phaffii fermentation processes for recombinant protein production
Authors: Thomas Steimann, Zoe Heite, Andrea Germer, Lars Mathias Blank, Jochen Büchs, Marcel Mann, Jørgen Barsett Magnus
Source: Microbial Cell Factories, Vol 23, Iss 1, Pp 1-10 (2024)
Publisher Information: BMC, 2024.
Publication Year: 2024
Collection: LCC:Microbiology
Subject Terms: Exopolysaccharide, Pichia pastoris, Komagataella phaffii, Stirred tank reactor, Recombinant protein production, Cell wall composition, Microbiology, QR1-502
Description: Abstract Background Komagataella phaffii (Pichia pastoris) has emerged as a common and robust biotechnological platform organism, to produce recombinant proteins and other bioproducts of commercial interest. Key advantage of K. phaffii is the secretion of recombinant proteins, coupled with a low host protein secretion. This facilitates downstream processing, resulting in high purity of the target protein. However, a significant but often overlooked aspect is the presence of an unknown polysaccharide impurity in the supernatant. Surprisingly, this impurity has received limited attention in the literature, and its presence and quantification are rarely addressed. Results This study aims to quantify this exopolysaccharide in high cell density recombinant protein production processes and identify its origin. In stirred tank fed-batch fermentations with a maximal cell dry weight of 155 g/L, the polysaccharide concentration in the supernatant can reach up to 8.7 g/L. This level is similar to the achievable target protein concentration. Importantly, the results demonstrate that exopolysaccharide production is independent of the substrate and the protein production process itself. Instead, it is directly correlated with biomass formation and proportional to cell dry weight. Cell lysis can confidently be ruled out as the source of this exopolysaccharide in the culture medium. Furthermore, the polysaccharide secretion can be linked to a mutation in the HOC1 gene, featured by all derivatives of strain NRRL Y-11430, leading to a characteristic thinner cell wall. Conclusions This research sheds light on a previously disregarded aspect of K. phaffii fermentations, emphasizing the importance of monitoring and addressing the exopolysaccharide impurity in biotechnological applications, independent of the recombinant protein produced.
Document Type: article
File Description: electronic resource
Language: English
ISSN: 1475-2859
Relation: https://doaj.org/toc/1475-2859
DOI: 10.1186/s12934-024-02403-3
Access URL: https://doaj.org/article/5646e2e0617842c9be89981d7069f326
Accession Number: edsdoj.5646e2e0617842c9be89981d7069f326
Database: Directory of Open Access Journals
Description
Abstract:Abstract Background Komagataella phaffii (Pichia pastoris) has emerged as a common and robust biotechnological platform organism, to produce recombinant proteins and other bioproducts of commercial interest. Key advantage of K. phaffii is the secretion of recombinant proteins, coupled with a low host protein secretion. This facilitates downstream processing, resulting in high purity of the target protein. However, a significant but often overlooked aspect is the presence of an unknown polysaccharide impurity in the supernatant. Surprisingly, this impurity has received limited attention in the literature, and its presence and quantification are rarely addressed. Results This study aims to quantify this exopolysaccharide in high cell density recombinant protein production processes and identify its origin. In stirred tank fed-batch fermentations with a maximal cell dry weight of 155 g/L, the polysaccharide concentration in the supernatant can reach up to 8.7 g/L. This level is similar to the achievable target protein concentration. Importantly, the results demonstrate that exopolysaccharide production is independent of the substrate and the protein production process itself. Instead, it is directly correlated with biomass formation and proportional to cell dry weight. Cell lysis can confidently be ruled out as the source of this exopolysaccharide in the culture medium. Furthermore, the polysaccharide secretion can be linked to a mutation in the HOC1 gene, featured by all derivatives of strain NRRL Y-11430, leading to a characteristic thinner cell wall. Conclusions This research sheds light on a previously disregarded aspect of K. phaffii fermentations, emphasizing the importance of monitoring and addressing the exopolysaccharide impurity in biotechnological applications, independent of the recombinant protein produced.
ISSN:14752859
DOI:10.1186/s12934-024-02403-3