Abstract
BackgroundEukaryotes use distinct networks of biogenesis factors to synthesize, fold, monitor, traffic, and secrete proteins. During heterologous expression, saturation of any of these networks may bottleneck titer and yield. To understand the flux through various routes into the early secretory pathway, we quantified the global and membrane-associated translatomes of Komagataella phaffii.ResultsBy coupling Ribo-seq with long-read mRNA sequencing, we generated a new annotation of protein-encoding genes. By using Ribo-seq with subcellular fractionation, we quantified demands on co- and posttranslational translocation pathways. During exponential growth in rich media, protein components of the cell-wall represent the greatest number of nascent chains entering the ER. Transcripts encoding the transmembrane protein PMA1 sequester more ribosomes at the ER membrane than any others. Comparison to Saccharomyces cerevisiae reveals conservation in the resources allocated by gene ontology, but variation in the diversity of gene products entering the secretory pathway.ConclusionA subset of host proteins, particularly cell-wall components, impose the greatest biosynthetic demands in the early secretory pathway. These proteins are potential targets in strain engineering aimed at alleviating bottlenecks during heterologous protein production.
Highlights
Eukaryotes use distinct networks of biogenesis factors to synthesize, fold, monitor, traffic, and secrete proteins
We find that the majority of nascent chains synthesized in K. phaffii are from genes involved in translation, ribosomal structure and biogenesis, as expected for log-phase growth
Biogenesis demands in the early secretory pathway We investigated the global demands for machinery needed for translocation into the endoplasmic reticulum (ER)
Summary
Our goal was to quantify the costs of protein synthesis, and so we argue that Ribo-seq is a more appropriate tool than mass spectrometry
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