PEX3 gene knockout influences recombinant xylanase expression by Komagataella phaffii

Abstract

Komagataella phaffii is a methylotrophic yeast harboring a tightly regulated alcohol oxidase promoter (P_AOX1), which is now widely used for recombinant protein production. During P_AOX1 expression phase by methanol induction, a methanol metabolism organelle peroxisome enlarged and occupied 80 % of K. phaffii cell through peroxins functions of matrix protein import and organelle division. Using a K. phaffii expressing xylanase in this study, each of all 23 PEX genes of K. phaffii, encoding peroxin, was knockout to influence the peroxisome size, leading to changes of K. phaffii physiological status and recombinant xylanase expression. It was observed that PEX3 knockout reduced peroxisome size by 54.3 %, increased xylanase expression by 29 %, decreased apoptosis ratio by 70.6 %. Transcriptome analysis revealed that PEX3 gene knockout decreased 18 other PEX genes of all three steps of peroxisome propagation, biogenesis, matrix protein import, and peroxisome fission. PEX3 gene knockout influenced expression of ribosomal subunit-related and protein transportation significantly based on gene function annotation and enrichment analysis. Additionally, Therefore, PEX3 gene knockout promoted xylanase folding correctly via Sec63 complex, and PDI1 significantly. In a summary, PEX3 gene knockout provided a novel strategy to enhance recombinant xylanase by K. phaffii.