UC Riverside

The production of biologics such as enzymes, biomaterials, and therapeutics play a leading role in biotechnology. As a microbial cell factory, Komagataella phaffii stands out for its high secretion capacity, ability to metabolize methanol as its primary carbon source, safety record as a source of biologics, and extensive literature compared to other non-model yeasts. Large scale production of biologics is simplified if the host secretes the proteins into its medium. However, this is difficult to achieve because an organism''s protein biogenesis machinery have evolved under the demands of its proteome and cells struggle to express and secrete non-native proteins. Heterologous protein production requires the harmonization of biogenetic machinery like ribosomes for synthesis, signal recognition particles and their cognate receptor for intracellular targeting, and protein folding chaperones for post-translational modification. These biogenetic components represent limited pools of resources that are distributed between heterologous proteins and the host proteome. While in use, these components are sequestered and unavailable for other tasks. Accurate accounting of these resources allows strains to be engineered in ways that relieve bottlenecks specific to producing particular heterologous proteins.

We survey the translational landscape of Komagataella phaffii using Ribo-seq under different conditions to elucidate which host proteins sequester the most biogenetic resources. Ribo-seq is a high throughput sequencing technique used to monitor protein synthesis by measuring ribosome abundancies at each codon of each transcript. Herein, a novel Ribo-seq pipeline was used to prepare mRNA footprint libraries for Illumina sequencing, create new annotations for protein-encoding genes, and address biases that are inherent to the technique and complicate quantification of protein synthesis. Using this pipeline, Ribo-seq was used with subcellular fractionation techniques to measure translation in the cytosol and on the endoplasmic reticulum membrane to uncover how and what proteins traffic through the early secretory pathway of yeasts. Finally, this developed protocol was used to identify which host proteins sequester the most biogenetic resources during heterologous expression. Genes encoding these proteins represent targets for rational strain engineering.