UC Davis

UNLABELLED: The yeast Komagataella phaffii has become a popular host strain among biotechnology start-up companies for producing recombinant proteins for food and adult nutrition applications. Komagataella phaffii is a host of choice due to its long history of safe use, open access to protocols and strains, a secretome free of host proteins and proteases, and contract manufacturing organizations with deep knowledge in bioprocess scale-up. However, a recent publication highlighted the abundance of an unknown polysaccharide that accumulates in the supernatant during fermentation. This poses a significant challenge in using K. phaffii as a production host. This polysaccharide leads to difficulties in achieving high purity products and requires specialized and costly downstream processing steps for removal. In this study, we describe the use of the common K. phaffii host strain YB-4290 for production of the bioactive milk protein lactoferrin. Upon purification of lactoferrin using membrane-based separation methods, significant amounts of carbohydrate were copurified with the protein. It was determined that the carbohydrate is mostly composed of mannose residues with minor amounts of glucose and glucosamine. The polysaccharide fraction has an average molecular weight of 50 kDa and consists mainly of mannan, galactomannan, and amylose. In addition, a large fraction of the carbohydrate has an unknown structure likely composed of oligosaccharides. Additional strains were tested in fermentation to further understand the source of the carbohydrates. The commonly used industrial hosts, BG10 and YB-4290, produce a basal level of exopolysaccharide; YB-4290 producing slightly more than BG10. Overexpression of recombinant protein stimulates exopolysaccharide production well above levels produced by the host strains alone. Overall, this study aims to provide a foundation for developing methods to improve the economics of recombinant protein production using K. phaffii as a production host. ONE-SENTENCE SUMMARY: Overexpression of recombinant protein stimulates the hyperproduction of high-molecular-weight, mannose-based, exopolysaccharides by the industrial yeast Komagataella phaffii.