WP4_Identification and engineering of natural alleles that increase protein secretion in Saccharomyces cerevisiae and Komagataella phaffii

Abstract

WP4: Stacking superior alleles improves protein secretion across genetic backgrounds Combinatorial engineering of the alleles identified through the QTL study, consistently led to improved protein secretion (different stain backgrounds, target proteins and genetic elements controlling gene expression). Recombinant protein production in microbial cell factories like Saccharomyces cerevisiae or Komagataelle phaffii is a cornerstone of modern biotechnology. However, yields are often suboptimal and attempts to increase secretion have shown mixed and target-specific results. To identify broader engineering targets, we systematically evaluated the secreting capacity for β-glucosidase and a VHH-IgA nanobody across 230 genetically diverse natural S. cerevisiae strains. Notably, several strains outperform commonly used chassis strains like CEN.PK by up to fourfold. QTL analysis of high-performing strains revealed natural alleles affecting processes ranging from mRNA splicing (HSH155) to vesicle trafficking (SPO20) and vacuole targeting (ENV9). Stacking these alleles via gene editing increased secretion across strains and target proteins, including pre-insulin and a GLP1-like peptide. Furthermore, heterologous expression of these alleles in K. phaffii also improved secretion. Together, our findings highlight new engineering strategies and support development of more efficient yeast cell factories for production of a range of proteins for applications in medicine and industry.