PEX3 gene knockout influences recombinant xylanase expression by Komagataella phaffii

<i>Komagataella phaffii</i> is a methylotrophic yeast harboring a tightly regulated alcohol oxidase promoter (<i>P</i> _<i>AOX1</i> ), which is now widely used for recombinant protein production. During <i>P</i> _<i>AOX1</i> expression phase by methanol induction, a methanol metabolism organelle peroxisome enlarged and occupied 80 % of <i>K. phaffii</i> cell through peroxins functions of matrix protein import and organelle division. Using a <i>K. phaffii</i> expressing xylanase in this study, each of all 23 <i>PEX</i> genes of <i>K. phaffii</i>, encoding peroxin, was knockout to influence the peroxisome size, leading to changes of <i>K. phaffii</i> physiological status and recombinant xylanase expression. It was observed that <i>PEX3</i> knockout reduced peroxisome size by 54.3 %, increased xylanase expression by 29 %, decreased apoptosis ratio by 70.6 %. Transcriptome analysis revealed that <i>PEX3</i> gene knockout decreased 18 other <i>PEX</i> genes of all three steps of peroxisome propagation, biogenesis, matrix protein import, and peroxisome fission. <i>PEX3</i> gene knockout influenced expression of ribosomal subunit-related and protein transportation significantly based on gene function annotation and enrichment analysis. Additionally, Therefore, <i>PEX3</i> gene knockout promoted xylanase folding correctly via Sec63 complex, and PDI1 significantly. In a summary, <i>PEX3</i> gene knockout provided a novel strategy to enhance recombinant xylanase by <i>K. phaffii</i>.