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Biochemical analysis of Komagataella phaffii oxidative folding proposes novel regulatory mechanisms of disulfide bond formation in yeast.

ABSTRACT: Oxidative protein folding in the endoplasmic reticulum (ER) is driven mainly by protein disulfide isomerase PDI and oxidoreductin Ero1. Their activity is tightly regulated and interconnected with the unfolded protein response (UPR). The mechanisms of disulfide bond formation have mainly been studied in human or in the yeast Saccharomyces cerevisiae. Here we analyze the kinetics of disulfide bond formation in the non-conventional yeast Komagataella phaffii, a common host for the production of recombinant secretory proteins. Surprisingly, we found significant differences with both the human and S. cerevisiae systems. Specifically, we report an inactive disulfide linked complex formed by K. phaffii Ero1 and Pdi1, similarly to the human orthologs, but not described in yeast before. Furthermore, we show how the interaction between K. phaffii Pdi1 and Ero1 is unaffected by the introduction of unfolded substrate into the system. This is drastically opposed to the previously observed behavior of the human pathway, suggesting a different regulation of the UPR and/or possibly different interaction mechanics between K. phaffii Pdi1 and Ero1.

SUBMITTER: Palma A

PROVIDER: S-EPMC10471769 | biostudies-literature | 2023 Aug

REPOSITORIES: biostudies-literature

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Biochemical analysis of Komagataella phaffii oxidative folding proposes novel regulatory mechanisms of disulfide bond formation in yeast.

Palma Arianna A Rettenbacher Lukas A LA Moilanen Antti A Saaranen Mirva M Pacheco-Martinez Christian C Gasser Brigitte B Ruddock Lloyd L

Scientific reports 20230831 1

Oxidative protein folding in the endoplasmic reticulum (ER) is driven mainly by protein disulfide isomerase PDI and oxidoreductin Ero1. Their activity is tightly regulated and interconnected with the unfolded protein response (UPR). The mechanisms of disulfide bond formation have mainly been studied in human or in the yeast Saccharomyces cerevisiae. Here we analyze the kinetics of disulfide bond formation in the non-conventional yeast Komagataella phaffii, a common host for the production of rec ...[more]

PMID: 37652992

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Project description: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 summaryOverexpression of recombinant protein stimulates the hyperproduction of high-molecular-weight, mannose-based, exopolysaccharides by the industrial yeast Komagataella phaffii.

Project description:The protein secretory pathway must maintain homoeostasis while producing a wide assortment of proteins in different conditions. It is also used extensively to produce many useful proteins in biotechnology. As such, secretory pathway dysfunction can be highly detrimental to the cell, resulting in the molecular basis for many human diseases, and can drastically inhibit product titers in biochemical production. Because the secretory pathway is a highly-integrated, multi-organelle system, dysfunction can happen at many levels and dissecting the root cause can be challenging. To better understand some of these dysfunctions, we measured multiple systems-level states of the cell (physiology, transcriptome, metabolism) while secreting a small protein (insulin precursor) or a large protein (?-amylase). This was carried out in the presence and absence of HAC1, a key transcription factor in maintaining secretory homeostasis. Clear trends in cellular stress were apparent across multiple data resulting from our perturbations. In particular, processes involving (1) degradation of protein / recycling amino acids, (2) overall transcription/translation repression, and (3) oxidative stress. Apparent runaway oxidative radical production was explained by a thermodynamic model that we put forward for disulfide formation in the endoplasmic reticulum. This model predicts that balancing the relative rates of protein folding and disulfide bond formation are key to easing oxidative stress. These predictions have direct implications in how to engineer a broad range of recombinant proteins for secretion and provide potential hypotheses for the root causes of several secretory-associated diseases. Yeast strains were constructed that produce and secrete (a) IP or (b) ?-amylase and were compared to yeast strains containing (c) an empty vector in both wild-type and HAC1 deletion backgrounds. These strains are named WN (WT with empty vector), WI (WT secreting IP), WA (WT secreting ?-amylase), dN (?hac1 with empty vector), dI (?hac1 secreting IP), and dA (?hac1 secreting ?-amylase). Strains were characterized in batch fermentation and samples were taken in mid-exponential phase. Triplicate fermentations were carried out for each strain.

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Biochemical analysis of Komagataella phaffii oxidative folding proposes novel regulatory mechanisms of disulfide bond formation in yeast.

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Biochemical analysis of Komagataella phaffii oxidative folding proposes novel regulatory mechanisms of disulfide bond formation in yeast.

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