Efficient Secretory Expression of Soybean Peroxidase in Saccharomyces cerevisiae Through Systematic Engineering

Soybean peroxidase (SBP), a thermostable and pH-robust enzyme, holds promise as a biopolymer cross-linking agent. This study pioneers the secretory expression of SBP in Saccharomyces cerevisiae, a GRAS (generally recognized as safe) microbial host. To address expression bottlenecks, a synthetic hybrid promoter was designed through modular assembly, enabling stringent glucose-dependent repression and high induction ratios. Transcriptional efficiency was further optimized via systematic terminator screening to enhance mRNA stability. Furthermore, the signal peptide was systematically redesigned by coordinating the functions of the prepeptide and pro-peptide to enhance endoplasmic reticulum transport and secretion flux. Finally, the engineered strain achieved a maximal extracellular SBP activity of 18358.03 U/L in fed-batch bioreactor cultivation. Functional validation demonstrated the ability of recombinant enzyme to mediate covalent cross-linking between α-lactalbumin and wheat arabinoxylan. This systematic engineering framework establishes a paradigm for overcoming secretion bottlenecks in heterologous protein production for food and biomanufacturing systems.