Systematic engineering of COPII machinery and cell wall remodeling enables gram-level secretion of recombinant human serum albumin with native-like structural features in Kluyveromyces marxianus

Abstract

Recombinant human serum albumin (rHSA) is an important alternative to plasma-derived HSA, but its microbial production is constrained by the secretion burden associated with its large size and 17 intramolecular disulfide bonds. Here, we developed a systematic secretory-network engineering strategy in the food-safe yeast Kluyveromyces marxianus. The α-factor leader enabled efficient secretion and correct N-terminal processing of rHSA. A system-wide screen of 35 SEC genes implicated coat protein complex II (COPII)-mediated endoplasmic reticulum export as a major intracellular constraint. Combinatorial engineering identified SEC23/SEC13/SEC31 as the optimal COPII module, increasing rHSA secretion by 60.7% although with a reduction in growth. Screening of 13 cell wall-related genes revealed the cell wall as an engineerable interface. Combining COPII and cell wall engineering increased extracellular rHSA by 287.3% (COPII+gas1Δkre1Δ) and 316.0% (COPII+kre6Δyps1Δ) relative to TOP1-rHSA. RT-qPCR revealed strain-specific transcriptional changes in selected COPII-, ER folding-, and cell wall-related genes, while stress phenotyping, transmission electron microscopy, and cell wall staining supported strain-specific cell wall remodeling rather than overt cell wall damage. In 5 L fed-batch fermentation with defined nutrient supplementation, the engineered strains achieved high-cell-density growth and extracellular rHSA accumulation of 2.32–2.37 g/L. Far-UV circular dichroism, Native PAGE, and LC–HRMS/MS demonstrated native-like secondary structure, non-denaturing electrophoretic mobility comparable to serum-derived HSA, and all 17 expected intramolecular disulfide linkages consistent with native HSA. This work establishes a food-safe yeast chassis through multi-layer secretion engineering for the production of rHSA with native-like structural features and provides a generalizable strategy for secreting complex disulfide-rich proteins.