Effects Of Codon Usage Bias On Regulating Protein Expression,Secretion And Structure In Pichia Pastoris

Protein synthesis in biological cells is regulated by many factors,among which codon usage is a well known factor to determine translation rate.With the development of related studies recently,codon usage bias has been proven to regulate many other processes besides translation,such as transcription and co-translationol folding.The Pichia pastoris system has absolute advantages and has been widely used to express recombiant proteins.Gene codon optimization of heterologous genes is always the firt step before expression.Therefore,the purpose of this research is to expore the special codon usage bias of P.pasotris genome,as well as its biological significance.Firstly,we calculated parameters such as tRNA adaptation index(tAI),codon usage index(cui),relative synonymous codon usage(RSCU)and codon adaptation index(CAI)for P.pastoris genome,based on genomic sequence and RNA-seq data.Then,the relationships between tAI and cui in different organisms were analyzed and we found P.pastoris has a stronger positive correlation.After that,we focused on the signal sequence MATa and designed codon optimization and de-optimization sequences.Reporter gene amylase(aF-AmyA)was fused to compare secretive efficiency.We found that codon de-optimization at the N terminus strongly inhibited protein expression,while the first 20 optimal codons did not contribute much.The analysis of 5' end rare codon patch lenth in P.pastoris and Saccharomyces cerevisiae genomes suggested that it was shorter in P.pastoris.Besides signal sequence,we also analyzed the codon bias of other regions towards gene expression and protein function.According to the genomic CAI distribution,we picked three candidates with low overall CAI and high structural disorder:PAS_chr2-2₀376(EAF7),PAS_chr2-1₀233(RRM)and PAS_chr2-2₀432(ATG13).Then we disnged codon optimizd sequences(opt-EAF7,opt-ATG13,optC-ATG13,opt-RRM,optC-RRM)and expressed them under the constitutive promoter PGAP.Our results suggested that codon bias not only affects mRNA and protein levels,but also regulates protein structure.As a fast developing expression system and chassis cell,P.pastoris now is frequently used for recombinant protein expression and gene clusters assembling.Codon optimization is always the first step for sucessful protein expression.In this study,we found that P.pastoris genome has a stronger preference for codons with high translation rate,and at the same time,rare codons play a significant role in regulating expression and protein structure in certain genes.These results would be helpful towards codon engineering based P.pastoris system optimization.