Knocking Out Two Polyphenol Oxidase Genes Significantly Improves Recombinant Protein Purification in Nicotiana benthamiana
Hai-Ping Diao, Hui-Xin Meng, Xue-Jiao Xu, Zeng-Lin Zhang, Jian-Feng Zhang, Yong-Feng Guo, Inhwan Hwang, Shi-Jian Song
Journal：PLANT BIOTECHNOLOGY JOURNAL
IF：12.8
DOI：10.1111/pbi.70575
PMID：
Published：2026-02-08
research field：分子生物学重组蛋白生产基因工程植物生物技术
Abstract
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.
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