Improving heterologous protein production in yeast with massively parallel CRISPR genome editing

Heterologous protein production is an indispensable tool in biotechnology and biopharma for manufacturing enzymes, protein therapeutics, and more. Generating robust production strains involves strategies that target the protein itself as well as the host genome. Despite advances in strain engineering, optimization of protein production is still limited by the ability to access the broad sequence space, constrained by traditional, low-throughput, and laborious methods like promoter substitutions or random mutagenesis. Here we use a genome-wide, multiplexed, targeted editing approach that can generate diverse edit types in both the heterologous gene and across the host genome to optimize production of cellobiohydrolase I enzyme (CHB1) in S. cerevisiae. The edits conferring improved CBH1 expression span cellular processes important for protein production, including transcription, translation, secretion, and protein degradation pathways.