Translational landscape and protein biogenesis demands of the early secretory pathway in Komagataella phaffii

Background: Eukaryotes 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. Results: By 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. Conclusion: A 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.

Automated summary

By analyzing the translatomes of Komagataella phaffii, it was found that cell-wall components represent the greatest number of nascent chains entering the ER during exponential growth in rich media. Transcripts encoding the transmembrane protein PMA1 sequester more ribosomes at the ER membrane than any others. Given the bottlenecks in heterologous protein production, certain host proteins may be potential targets for pathway improvement.