Reestablishment Of Endoplasmic Reticulum Homeostasis And Regulation Of Wsc1p During Phytase Overproduction In Pichia Pastoris

Pichia pastoris is one of the most popular industrial host strains for recombinant protein production,which has been used to express more than 5000 kinds of recombinant proteins.Because of the insufficient protein processing capacity,overproduction of recombinant protein increases the load of unfolded proteins and causes ER stress,which has consequences for protein synthesis and cell growth.Co-expression of HAC1~i,encoding the transcription factor of unfolded protein response,can enhance the protein processing in the ER and increase recombinant protein production,which also sequentially increases the ER load.Therefore,cells need to maintain ER homeostasis and perform proper regulation to reduce the ER load.However,few reports have described this reestablishment of ER homeostasis.In this study,we used a dynamic proteomics to analyze the highly regulated pathways in response to phytase overproduction and HAC1~i co-expression,and investigate the mechanism and regulation of reestablishment of ER homeostasis during recombinant protein overproduction.(1)The construction of the phytase overproduction strainThe overproduction of phytase from Citrobacter amalonaticus CGMCC 1696 was achieved by optimizing the AOX1 promoter and?signal peptide,increasing the copy numbers of phytase gene cassettes to six copies and co-expressing HAC1~i in P.pastoris.The phytase activity reached 2119 U/mL after 96 h of methanol induction,which was 312%higher than that of the original strain.In 10 L fermenter,the expression of phytase of this strain reached35032 U/m L and the protein concentration reached 9.58 g/L after 120 h of methanol induction.(2)The analysis of protein synthesis network of the phytase overproduction strainWe analyzed the intracellular proteomics of the co-expressing HAC1~i strain and the control strain after induction for 24 h,72 h and 96 h by applying iTRAQ.We found that with increasing phytase expression,the amount of molecular chaperones and enzymes in the ER membrane and the level of protein processing in the ER increased while the ribosome levels down-regulated.The increasing ER membrane proteins and protein processing levels may require a larger ER,suggesting the ER may expand when the phytase expression increased.(3)The effect of phytase overproduction on ER expansionBy observing the appearance of ER and relative quantitative analysis of ER expansion,we found that ER expansion existed in the phytase overproduction strain.Moreover,the ER expansion increased with the increasing phytase expression.These results and the results from(2)suggest that the ER expansion can provide more space for the increasing ER membrane proteins and protein folding to promote protein glycosylation and folding,which can reduce the ER load and reestablish ER homeostasis.(4)The regulation of ribosome levels in the phytase overproduction strainThe down-regulation of ribosome levels can reduce ER load and reestablish ER homeostasis.To explore the regulatory mechanism of down-regulation of ribosome levels,we knocked out WSC1,encoding the signal protein Wsc1p.We found that the down-regulation of ribosome levels was restored in the phytase overproduction strain,which increased 31%phytase expression after 96 h of methanol induction.These results were confirmed in the xylanase overproduction strains.These results suggest that the down-regulation of ribosome levels is regulated through Wsc1p.This study elucidates the regulation of recombinant protein synthesis and reestablishment of ER homeostasis.The knockout of WSC1 relieves the suppression of ribosome synthesis caused by recombinant protein overproduction and further increases recombinant protein production,which provides more insights in fundamental understanding of pathway engineering to improve recombinant protein production in P.pastoris or other yeast.