Supplementary file 1_Engineering small peptide secretion in Komagataella phaffii.docx

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.</p>Methods<p>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.</p>Results<p>Secretion efficiency exhibited a strong dependency on peptide chain length and signal sequence selection, likely reflecting limitations during endoplasmic reticulum translocation.