Pooled CRISPR Screening Informs Rapid Remodeling of the Secretory Pathway in Non-Model Yeast

The yeast Komagataella phaffii (Pichia pastoris) is commonly used for the production of recombinant proteins due to its rapid growth to high cell densities and its ability to secrete proteins into the culture medium. Large-scale clinical manufacturers routinely achieve industrially relevant titers for protein therapeutics like insulin and subunit vaccines. There is growing interest in K. phaffii as an alternative host for the manufacturing of more complex, high-value protein therapeutics like monoclonal antibodies (mAbs), including several recent product approvals. Early engineering efforts yielded humanized yeast strains capable of secreting mAbs with specific quality attributes and post-translational modifications. Secreted titers must be increased, however, to realize cost-saving or scalability advantages over current hosts like CHO cells.

Yeasts devote a significant portion of cellular resources to protein secretion – including several hundred secreted endogenous host-cell proteins (HCPs). Many HCPs are not essential in laboratory or manufacturing conditions, and those resources could be rerouted to production of a recombinant protein. In non-model hosts like K. phaffii, however, it is not known which HCPs could be removed without cost to the cells, making large scale genome engineering a tedious effort. Here, we used an unbiased pooled CRISPR knockout library to identify non-essential genes in the yeast secretory pathway. We showed that genes predicted to be non-essential could be rapidly disrupted with little engineering effort. Finally, we evaluated the performance of engineered strains for secretion of several recombinant proteins, including a mAb. This study demonstrates a general strategy for facilitating genomic engineering in non-model organisms using unbiased knockout screens.