Tailoring the stress response of Komagataella phaffii for recombinant protein production on sustainable carbon sources

Date

2026-6-16

Author

Raja, Wajeeha Aslam

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Komagataella phaffii is an established eukaryotic expression host for recombinant protein production, conventionally cultured using methanol as both a carbon source and inducer of the strong AOX1 promoter. The metabolic stress imposed by alternative sustainable carbon sources (SCSs) like methanol, ethanol, acetate, and formate motivate the development of stress-tolerant engineered strains capable of meaningful growth and production under these stress-inducing conditions. This study’s first objective was to characterize the induction power of the glyoxylate shunt enzyme isocitrate lyase’s promoter (pICL1) across methanol, ethanol, acetate, formate, and glycerol, and benchmark it against two of the most widely used promoters in this host, pAOX1 and pADH2, using enhanced green fluorescence protein as a reporter protein. The second objective was to investigate whether overexpression of HOG1, the central effector kinase of the high osmolarity glycerol (HOG) MAPK stress-response cascade, driven by the carbon-source-responsive promoters pICL1 and pADH2, could improve cell generation and recombinant protein production on SCSs. pICL1 showed weak but consistent induction on these carbon sources, with increases in fluorescence ranging from 1.2-fold on glycerol and 1.3-fold on both methanol and formate, to 1.4-fold on ethanol and 1.5-fold on acetate at the 30th hour. Subsequently, HOG1 overexpression under pICL1 produced the most substantial phenotype on methanol and formate, with cell generation improvements of up to 4.55-fold over wild-type, compared to 1.25-fold for HOG1 overexpression under pADH2, signifying that a slightly-more-than-basal level of HOG1 expression, rather than a significantly higher level of expression, is sufficient and appropriate to confer meaningful stress response benefits. HOG1 overexpression also resulted in enhanced oxidative stress tolerance across carbon sources and under H2O2 conditions. These findings establish stress-response engineering via HOG1 as a viable strategy for improving K. phaffii performance on sustainable carbon sources.

Subject Keywords

Komagataella phaffii, High osmolarity glycerol pathway, Sustainable carbon sources, Stress-response engineering, Promoter characterization

URI

https://hdl.handle.net/11511/119724

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Graduate School of Natural and Applied Sciences, Thesis