Using Host Cell Guidance to Produce Human-Like Sugar Patterns in Komagataella phaffii

A humanized glycosylation pathway was reconstructed in Komagataella phaffii to produce the biantennary G0 glycan structure.

• Overexpression of C. elegans α1-2 mannosidase and deletion of the native OCH1 gene resulted in previously undescribed synthetic lethality.
• Using weaker native promoters for modified genes in the glycosylation pathway could mitigate the synthetic lethality observed.
• Engineered strains showed reduced growth rates compared to unmodified strains.
• RNA sequencing indicated upregulation of stress response and cell cycle pathways in glycoengineered strains.
• Transcriptomics-guided gene knockouts in the MAPK signaling cascade partially restored growth in a G0-engineered strain.

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