Abstract
<title>Abstract</title> <p> <italic>Komagataella phaffii</italic> is a common alternative host to mammalian cell lines for heterologous production of recombinant proteins due to its cost-effectiveness and accelerated development. Its native N-linked glycosylation, however, yields high-mannose structures that differ from those commonly found on secreted recombinant proteins used in biopharmaceuticals. The feasibility of humanizing <italic>K. phaffii</italic> ’s glycosylation pathway has been demonstrated, but routine engineering of this attribute remains underdeveloped. In this study, we reconstructed a humanized glycosylation pathway capable of producing the biantennary GlcNAc2Man3GlcNAc2 (G0) glycan structure. We report a previously undescribed synthetic lethality related to the overexpression of <italic>C. elegans</italic> α1–2 mannosidase (MNS1) and the deletion of the native OCH1 gene that can be mitigated by using weaker native promoters for genes modified in the glycosylation pathway. Engineered strains exhibited reduced growth rates compared to unmodified strains, and RNA sequencing revealed upregulated stress response and cell cycle pathways in glycoengineered strains. Through transcriptomics-guided gene knockouts, particularly in the MAPK signaling cascade, we partially restored growth in a G0-engineered strain. This study demonstrates a targeted, host biology-informed strategy for improving glycoengineering in <italic>K. phaffii</italic> by combining a CRISPR-Cas9 genome-editing system and transcriptomic analysis. </p>