Engineering small peptide secretion in Komagataella phaffii

Small peptides have emerged as an important class of biologics with broad therapeutic applications. Limitations of conventional solid-phase synthesis have intensified the interest in microbial production systems. Komagataella phaffii, a widely used host for secretory protein production, represents a promising alternative platform; however, the efficient secretion of very small peptides (<3.5 kDa) remains a major biotechnological challenge due to constraints in detection, proteolytic processing, and overall secretion efficiency.

To systematically investigate bottlenecks in small peptide secretion, we established the 3×FLAG peptide (23 amino acids) as a tractable, immunologically detectable model system. We evaluated the impact of peptide chain length and signal sequence selection on secretory performance. Furthermore, we assessed the effect of overexpressing the translocon components and conducted a random mutagenesis screen to identify novel genetic determinants influencing secretion. Finally, we tested the transferability of our engineering strategies on the disulfide-rich peptide dQ-brazzein and the therapeutic fusion peptides α-melanocyte-stimulating hormone and salmon calcitonin.

Secretion efficiency exhibited a strong dependency on peptide chain length and signal sequence selection, likely reflecting limitations during endoplasmic reticulum translocation. Overexpression of Sec61 partially alleviated this bottleneck, enhancing secretion approximately 2-fold. Through the random mutagenesis screen, we identified AQR1, FMS1-2, SKG3, and CDC48 as novel targets, which increased secretion up to 9-fold. These engineering strategies were tested on dQ-brazzein, yielding up to ∼100% improvement, though the magnitude of transfer was cargo-dependent. Furthermore, the pronounced secretion limitations observed for α-melanocyte-stimulating hormone and salmon calcitonin based fusion peptides were partially mitigated utilizing these strategies, resulting in a ∼2-fold improvement.

This study provides a systematic evaluation of engineering strategies targeting the unique constraints of very small (<3.5 kDa) peptide secretion in K. phaffii. The identified genetic targets and mechanistic insights serve as a starting point for cargo-specific optimization efforts and highlight the unique challenges that short peptides pose to the native secretory machinery.