Enhancing co-translational folding of heterologous protein by deleting non-essential ribosomal proteins in Pichia pastoris

In this study, we screened 27 RP deletion strains of P. pastoris and expressed two heterologous proteins eGFP and phytase (Phy) in them.To verify the transcription level and translation initiation efficiency in RP deletion strains, the typical “enhancing” strains of rpl38∆, rpl9a∆, rps25∆ and a “non-enhancing” strain of rps7∆ expressing Phy are chosen as representative strains. While these strains accumulate heterologous proteins in a time-dependent manner, the mRNA level of the Phy remained constant over the time and across the strains, with a standard deviation of only 50ppm, determined by mRNA-seq quantified using rpkM. Meanwhile, the RNC-mRNA (mRNAs attached to the ribosomes) of Phy, representing the Phy mRNA which entered translation process, remained also constant over the time and across the strains, with a standard deviation of 153ppm, determined by RNC-seq. These results demonstrated that deletion of the non-essential RPs did not influence the transcription and translation engagement of the heterologous mRNA at all. mRNA and RNC-mRNA profiles of wild type and rpl38∆, rpl9a∆, rps25∆, rps7∆ at early and middle stage of fermentation by using next-generation sequencing, in duplicte.