Abstract

Recombinant expression of proteins is widely required in the biopharmaceutical and biomanufacturing fields. Komagataella phaffii has served as a dominant eukaryotic host for protein expression, yet its methanol-dependent induction mode limits its versatility for safe production of bulk edible and therapeutic proteins. In this study, we report a glucose-responsive expression system that enables efficient methanol-free production of heterologous proteins. Glucose-responsive promoters were mined from transcriptome data and validated to show strong repression at high glucose levels and activation under glucose limitation. Incorporation of 28 promoters with an improved transcriptional signal amplification device resulted in glucose-responsive expression of various proteins. For example, the nanobody drug caplacizumab achieved efficient secretory expression using this synthetic system. Further transcriptomic analysis of engineered strains with variable gene dosages of caplacizumab guided the identification of 43 production-related candidate targets. Among these, overexpression of the functionally uncharacterized PAS_chr2-2_0003 and the phospholipid methyltransferase PAS_chr3_0859 further improved nanobody expression capacity. The engineered strains achieved caplacizumab titers of 1270.5 mg/L (glucose induction) and 1424.8 mg/L (glycerol induction) in well plates, and 26.2 g/L (glucose induction) and 26.6 g/L (glycerol induction) in 3-L bioreactors. These production titers far exceeded previously reported levels of caplacizumab in various hosts, including methanol-dependent expression in K. phaffii . Our study also reports the highest production levels among reported nanobodies. This glucose-responsive expression system provides a safe, economical, and sustainable platform for large-scale production of nanobodies and other proteins, and offers insights for the design of next-generation methanol-decoupled K. phaffii cell factories.