A chimeric rather than native secretion signal elicits stress-related transcriptional adaption during 75k γ-secalin production in Komagataella phaffii

Komagataella phaffii (formerly Pichia pastoris) is a popular host for protein production, enabling their targeted application and in-depth study. Among relevant targets are gluten proteins, which are essential for cereal-based food properties, but can also trigger immune reactions such as coeliac disease. Previously, production of rye (Secale cereale) prolamin 75k γ-secalin in K. phaffii was shown to yield the highest titres with the native secretion signal compared to co- and post-translational signals commonly used for enhanced production. Here, we compared the host's transcriptomic response to 75k γ-secalin expressed with the native signal (gSec) or a chimeric OST1-pre-MATα-pro leader (S4gSec) against a non-producing null mutant. Production of the native 75k γ-secalin showed minimal impact; whereas stress-, amino acid transport-, and flocculation-related pathways were upregulated in K. phaffii S4gSec. Transcriptome investigation yielded GCN4 as potential marker for transcriptomic stress monitoring during heterologous protein production. Despite K. phaffii's significance as protein production host, many of its genes are only putatively annotated. Of the 31 (gSec) and 349 (S4gSec) differentially expressed genes, 29% and 59.3% are putative, respectively. This study presents a tentative in silico characterization of these genes, highlighting the importance of continued functional characterization of the K. phaffii genome.