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

本研究中，我们筛选了27株巴斯德毕赤酵母（P. pastoris）的核糖体蛋白（Ribosomal Protein，RP）缺失菌株，并在其中表达了两种异源蛋白：增强型绿色荧光蛋白（enhanced Green Fluorescent Protein，eGFP）与植酸酶（Phytase，Phy）。为验证核糖体蛋白缺失菌株的转录水平与翻译起始效率，我们选取了典型的“增强型”菌株rpl38Δ、rpl9aΔ、rps25Δ，以及一株表达植酸酶的“非增强型”菌株rps7Δ作为代表性菌株。尽管这些菌株的异源蛋白积累呈现时间依赖性，但经mRNA测序（mRNA-seq）并以每百万映射读段每千碱基转录本的读段数（reads per kilobase of transcript per million mapped reads，RPKM）定量后发现，植酸酶的mRNA水平在不同时间点及各菌株间均保持恒定，标准差仅为50ppm。与此同时，经核糖体结合mRNA测序（RNC-seq）测定，代表进入翻译过程的植酸酶mRNA的核糖体结合mRNA（RNC-mRNA，即附着于核糖体的mRNA）水平，在不同时间点及各菌株间同样保持恒定，标准差为153ppm。上述结果表明，非必需核糖体蛋白的缺失完全不会影响异源mRNA的转录与翻译参与度。整体实验设计：采用二代测序技术，对发酵早、中期的野生型巴斯德毕赤酵母及rpl38Δ、rpl9aΔ、rps25Δ、rps7Δ菌株的mRNA与核糖体结合mRNA谱进行重复实验。