A streamlined strain engineering workflow with genome-wide screening detects enhanced protein secretion in Komagataella phaffii

Expression of secreted recombinant proteins burdens the protein secretion machinery, limiting production. Here, we describe an approach to improving protein production by the non-conventional yeast Komagataella phaffii comprised of genome-wide screening for effective gene disruptions, combining them in a single strain, and recovering growth reduction by adaptive evolution. For the screen, we designed a multiwell-formatted, streamlined workflow to high-throughput assay of secretion of a single-chain small antibody, which is cumbersome to detect but serves as a good model of proteins that are difficult to secrete. Using the consolidated screening system, we evaluated >19,000 mutant strains from a mutant library prepared by a modified random gene-disruption method, and identified six factors for which disruption led to increased antibody production. We then combined the disruptions, up to quadruple gene knockouts, which appeared to contribute independently, in a single strain and observed an additive effect. Target protein and promoter were basically interchangeable for the effects of knockout genes screened. We finally used adaptive evolution to recover reduced cell growth by multiple gene knockouts and examine the possibility for further enhancing protein secretion. Our successful, three-part approach holds promise as a method for improving protein production by non-conventional microorganisms. A high-throughput workflow involving a streamlined genome-wide screen improves protein production in the non-conventional yeast, Komagataella phaffii, following adaptive laboratory evolution.

Automated summary

This study presents a method to improve protein production in the non-conventional yeast Komagataella phaffii. The method involves gene knockout screening, combining gene disruptions, and adaptive evolution to recover growth reduction. The researchers used a multiwell-formatted screening system to evaluate a large number of mutant strains and identified multiple gene disruptions that increased antibody production. They also found that the target protein and promoter could be interchangeable for the effects of gene knockouts. Adaptive evolution was used to recover reduced cell growth caused by multiple gene knockouts and explore the possibility of further enhancing protein secretion. The overall approach holds promise for improving protein production in non-conventional microorganisms.