Abstract
The methylotrophic yeast Komagataella phaffii (Pichia pastoris) has been developed into a highly successful system for heterologous protein expression in both academia and industry. However, overexpression of recombinant protein often leads to severe burden on the physiology of K. phaffii and triggers cellular stress. To elucidate the global effect of protein overexpression, we set out to analyze the differential transcriptome of recombinant strains with 12 copies and a single copy of phospholipase A2 gene (PLA2) from Streptomyces violaceoruber. Through GO, KEGG and heat map analysis of significantly differentially expressed genes, the results indicated that the 12-copy strain suffered heavy cellular stress. The genes involved in protein processing and stress response were significantly upregulated due to the burden of protein folding and secretion, while the genes in ribosome and DNA replication were significantly downregulated possibly contributing to the reduced cell growth rate under protein overexpression stress. Three most upregulated heat shock response genes (CPR6, FES1, and STI1) were co-overexpressed in K. phaffii and proved their positive effect on the secretion of reporter enzymes (PLA2 and prolyl endopeptidase) by increasing the production up to 1.41-fold, providing novel helper factors for rational engineering of K. phaffii.
Highlights
Over the last few decades, Komagataella phaffii expression system has been used successfully for production of various recombinant heterologous proteins[1,2]
These studies demonstrate that genetic engineering of K. phaffii through helper factors has been served as valuable tools for maximizing protein production
In order to select suitable multi-copy strains for RNA-seq analysis we evaluated the effect of gene dosage on the production of phospholipase A2 gene (PLA2) by K. phaffii
Summary
The methylotrophic yeast Komagataella phaffii (Pichia pastoris) has been developed into a highly successful system for heterologous protein expression in both academia and industry. Coexpression of helper genes or molecular chaperones has been used to improve the heterologous expression in K. phaffii by overcoming the burden of protein folding and secretion. After screening ten helper genes involved in protein folding and stress response, the yield of a glucose oxidase was improved by 5.11-fold in K. phaffii when coexpressed with three chaperones, Sec. 53, Cne[1] and Gcn[413]. For the first time, we performed global transcriptomic analyses of the recombinant K. phaffii strains with 12 copies and a single copy of phospholipase A2 gene (PLA2) from Streptomyces violaceoruber, which produced two levels of PLA2 in titer over 1g/L, and evaluated the effect of the potential helper genes on recombinant protein secretion by gene coexpression
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
