UC Riverside

The production of recombinant products, including enzymes, biomaterials, and therapeutics, is a driving force in biotechnology addressing various global challenges. Non-conventional microbes are particularly appealing for metabolic engineering due to their uniquely evolved characteristics that can simplify the engineering process compared to more traditional model organisms. The methylotrophic yeast Komagataella phaffii stands out for its ability to grow to high cell densities, perform post-translational modifications, and secrete high titers of recombinant proteins with minimal endogenous host protein secretion. Although significant progress has been made in developing K. phaffii to produce biopharmaceuticals and other value-added products, there remains a need for advanced synthetic biology tools facilitating genome engineering, functional genomic screening, and rapid strain optimization to fully harness its potential. We have sought to overcome these limitations by providing a detailed protocol for designing a highly active genome-wide sgRNA knockout library. We measured the cutting efficiency of the library using an experimental workflow involved with transforming the library into cells with a deficient dominant DNA repair pathway and performing growth screens. Our results demonstrated that over 98% of the sgRNAs in the library were active. The activity validation ensures accurate and precise screening outcomes. We then performed growth screens using glucose as the sole carbon source and defined a set of consensus essential genes for K. phaffii. Comparative analysis of these genes with essential genes from other known yeast species revealed a core set of essential genes in K. phaffii, many of which are linked to vital cell processes. This analysis also revealed essential genes exclusive to K. phaffii related to key metabolic engineering targets, such as protein production, secretion, and glycosylation. Additionally, we employed ribosome profiling and next-generation sequencing to examine the global and early secretory demands of K. phaffii, focusing on host protein synthesis and endoplasmic reticulum trafficking before and after methanol induction. This analysis was conducted using an industrial strain of K. phaffii engineered to produce human serum albumin (HSA) under methanol conditions. By identifying key host proteins that impose the greatest constraints on the biogenetic machinery and subsequently targeting these genes using the CRISPR-Cas9 system, we achieved a 35% increase in HSA secretion. The highly active, genome-wide CRISPR library as well as the generated Ribo-req protocol and data developed in this study facilitates functional genomic screening in K. phaffii, provides insights into this cell’s biology, and holds potential for enabling a wide range of engineering into this host cell.