Knocking Out Two Polyphenol Oxidase Genes Significantly Improves Recombinant Protein Purification in Nicotiana benthamiana.

Efficient purification remains a key technical challenge affecting the recovery efficiency of recombinant proteins in plant-based systems. Complex metabolites, particularly polyphenols, often cause recombinant protein aggregation during purification. In this study, we identified two key polyphenol oxidase genes, PPOa and PPOb from Nicotiana benthamiana, as responsible for these effects. Using CRISPR-Cas9, we generated two ppoa;ppob double knockout lines that significantly improved the purification of virus surface proteins like SARS-CoV-2 Spike trimer and influenza HA trimer. These lines showed reduced polyphenol-protein interactions, minimised aggregation, and higher purification yields. Our work establishes a clean, high-efficiency N. benthamiana chassis for scalable recombinant protein production.