Ribo-seq guided design of enhanced protein secretion in  Komagataella phaffii
The production of recombinant proteins requires the precise coordination of various biological processes, including protein synthesis, folding, trafficking, and secretion. The overproduction of a heterologous protein can impose various bottlenecks on these networks. Identifying and alleviating these bottlenecks can guide strain engineering efforts to enhance protein production. The methylotrophic yeast Komagataella phaffii is used for its high capacity to produce recombinant proteins. Here, we use ribosome profiling to identify bottlenecks in protein secretion during heterologous expression of human serum albumin (HSA). Validation of this analysis showed that the knockout of non-essential genes whose gene products target the ER, through co- and post-translational mechanisms, and have high ribosome utilization can increase production of a heterologous protein, HSA. A triple knockout in co-translationally translocated carbohydrate and acetate transporter Gal2p, cell wall maintenance protein Ydr134cp, and the post-translationally translocated cell wall protein Aoa65896.1 increased HSA production by 35%. This data-driven strain engineering approach uses cell-level information to identify gene targets for phenotype improvement. This specific case identifies hits and creates strains with improved HSA production, with Ribo-seq and bioinformatic analysis to identify non-essential ER targeted proteins that are high ribosome utilizers.