Xu, X.-J. | Search bioRxiv

bioRxiv2025

Knocking out two polyphenol oxidase genes significantly improves recombinant protein purification in Nicotiana benthamiana

Efficient purification remains one of the major bottlenecks in the development of plant-based systems for recombinant protein production. The complex metabolites, particularly polyphenols, which usually cause recombinant protein aggregation during purification. In this study, we identified two key polyphenol oxidase genes, PPOa and PPOb from N.benthamiana as responsible for these effects. Using CRISPR/Cas9, we generated two ppoa;ppob double knockout lines that significantly improved the purification of functional proteins like SARS-CoV-2 Spike trimer and influenza HA trimer. These lines showed reduced polyphenol-protein interactions, minimized aggregation, and higher purification yields. Our work establishes a clean, high-efficiency N. benthamiana chassis for scalable recombinant protein production.

synthetic biology↗

bioRxiv2022

Chlamydomonas mutant hpm91 lacking PGR5 is a scalable and valuable strain for algal hydrogen (H2) production

Clean and sustainable H2 production is essential toward a carbon-neutral world. H2 generation by Chlamydomonas reinhardtii is an attractive approach for solar-H2 from H2O. However, it is currently not scalable because of lacking ideal strains. Here, we explore hpm91, a previously reported PGR5-deletion mutant with remarkable H2 production, that possesses numerous valuable attributes towards large-scale application and in-depth study issues. We show that hpm91 is at least 100-fold scalable (upto 10 liter) with H2 collection sustained for averagely 26 days and 7287 ml H2/10L-HPBR. Also, hpm91 is robust and active over the period of sulfur-deprived H2 production, most likely due to decreased intracellular ROS relative to wild type. Moreover, quantitative proteomic analysis revealed its features in photosynthetic antenna, primary metabolic pathways and anti-ROS responses. Together with success of new high-H2-production strains derived from hpm91, we highlight that hpm91 is a potent strain toward basic and applied research of algal-H2 photoproduction.

biochemistry↗