A Chimeric Rather Than Native Secretion Signal Elicits Stress-Related Transcriptional Adaption during 75k γ-Secalin Production in 𝘒𝘰𝘮𝘢𝘨𝘢𝘵𝘢𝘦𝘭𝘭𝘢 𝘱𝘩𝘢𝘧𝘧𝘪𝘪

The prolamin 75k γ-secalin of rye (Secale cereale) cultivar Elias was produced in Komagataella phaffii MutS strain BSYBG11. In total, three different mutants were investigated: i) the producer of the native 75k γ-secalin K. phaffii gSec, ii) the strain K. phaffii S4gSec, where the native γ-secalin secretion signal was replaced by the chimeric OST1-pre-MATα-pro leader, and iii) the null-mutant K. phaffii ΔgSec, where the target gene is replaced by a truncated version without ATG start codons. Cultures were grown in baffled shaker flasks (500 mL volume filled with 1/10 of the total capacity) in buffered minimal medium at pH 6, 28°C and 180 rpm. Four samples per strain were harvested 96 h post-induction (0.5% vol. MeOH every 12 h), total RNA was extracted with the Roboklon Universal RNA Kit according to the manufacturer's instructions (Roboklon GmbH, Germany), and sequencing libraries were constructed using the Illumina stranded mRNA Prep kit (Illumina, USA). mRNA libraries were sequenced on an Illumina platform NextSeq 1000 with P2 XLEAP-SBS Reagents (Illumina, USA) as dark cycle sequencing, generating paired-end reads of 99 bp length.