ARTP Mutagenesis and Omics‐Guided Target Validation for Improved Human Serum Albumin Fusion Protein Production in Yeast

Efficient production of human serum albumin (HSA) fusion proteins in yeast remains constrained by the combined demands of folding, secretion, and cellular resource allocation. Rational optimization of endoplasmic reticulum folding and unfolded protein response pathways can improve production, but additional cellular constraints may persist in engineered strains. Here, we combined iterative atmospheric and room-temperature plasma (ARTP) mutagenesis, activity-coupled screening, and genome- and transcriptome-guided validation to improve production of an amylase-HSA fusion protein (AGSH) in Saccharomyces cerevisiae. Using extracellular α-amylase activity as the screening indicator, five rounds of ARTP mutagenesis in the folding-optimized strain AH28 generated mutants with enhanced AGSH production. The final mutant N5S14 reached 2.12-fold the AH28 level in AGSH-associated α-amylase activity. Physiological profiling showed that enhanced production was associated with altered growth-production balance and changes in glucose, ethanol, and glycerol profiles. Whole-genome sequencing identified 33 candidate mutation sites, and reverse engineering validated genes associated with improved AGSH production, including BUD27, NPR3, NPR2, and IML1. RNA-seq revealed distinct transcriptional states involving central carbon metabolism, protein folding, and stress-related processes. Transcriptome-guided validation identified PFY1 deletion and SSA3 overexpression as additional beneficial interventions. Overall, this strategy improved AGSH production and identified engineering targets beyond previously optimized folding-related modules.