Functional Characterization Of Ribosomal Protein From Pichia Pastoris And Regulation On Heterologous Protein Synthesis

Ribosome protein(RP)is the most abundant structural protein in the cell.It is the basic component for maintaining the structure and function of ribosomes,and has potential regulatory relationship with protein synthesis and endoplasmic reticulum(ER)stress.In order to charaterize the function of Pichia pastoris ribosomal protein and its regulation on heterologous protein synthesis,a large-scale library of RP deletion was constructed to screen for mutation strains that promote the expression of heterologous proteins,and study the molecular basis of these regulations.At the same time,overexpressing signal factors of RP synthesis found that Bcy1 significantly increased heterologous protein synthesis.In addition,deletion of Rpl26 is highly resistant to ER stress independen of Hac1.The uncommon mechanism of this ER stress response was analyzed from the assembly and function of ribosome.Details as follows:(1)A single gene knockout of 76 RPs in P.pastoris was performed,and 27 deletants were successfully obtained.Green fluorescent protein(eGFP)and phytase(Phy)were used as reporter proteins to analyze the expression efficiency of heterologous proteins,and the expression efficiency of 17 RP deletion strains was improved.The "enhancing" strains rpl38?,rpl9a?,rps25? and the "non-enhancing" strain rps7? expressing Phy were studied.The results showed that RP deletion impaired the assembly of the 60 S subunit and reduced the translation elongation speed,which increased the co-translational folding efficiency,reduced protein aggregates,and thereby increased the yield of heterologous protein without changing its transcriptional level and translation initiation efficiency.(2)Five RP synthesis signal factors were overexpressed in Pichia pastoris,respectively.We found that overexpression of the protein kinase A(PKA)regulatory subunit Bcy1 significantly increased the accumulation of cell biomass and increased the expression of eGFP and Phy during fermentation.Transcriptome analysis revealed that overexpression of Bcy1 reduces PKA activity,which significantly down-regulated RP expression and increased the expression of stress response element genes and post-diauxic shift genes.These results demonstrate a novel insight for maintaining normal cell growth during heterologous protein synthesis.(3)Rpl26 deletion was highly resisted to ER stress,which was independent of Hacl-mediated unfolded protein response pathway.Transcriptome data found that ER stress was greatly alleviated and the ribosome biosynthesis pathway was distinctly blocked in rpl26? strain.Combined with cell growth,ribosome assembly and rRNA maturation analysis,it was found that the maturation of 25 S rRNA and function of ribosome were affected in Rpl26 deletion strain,which reduced the flux of newly synthesized proteins into ER and thereby counteracted the ER stress.This study reveals the regulation of ribosomal protein on cell growth,heterologous protein synthesis and ER stress.By knocking out or down-regulating RPs,it affects the synthesis and function of ribosomes,improves cell growth and counteracts ER stress and improves the expression of heterologous proteins.This study provides a theoretical basis for the regulation of heterologous protein synthesis by ribosome regulation in Pichia pastoris or other hosts.