Directed evolution of a plant Rubisco chaperone with altered client recognition

• 成果类型: Article
• 署名作者: Li, Siyu; Lee, Byunguk; Lao, Yichong; Lertwiriyapiti, Sirawit; Huang, Xuhui; Wang, Tina
• 署名单位: University of Wisconsin System; University of Wisconsin Madison; University of Wisconsin System; University of Wisconsin Madison; University of Wisconsin System; University of Wisconsin Madison; University of Wisconsin System; University of Wisconsin Madison
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-10752
• DOI: 10.1073/pnas.2510701122
• 发表日期: 2025-09-16
• 关键词: particle mesh ewald; chloroplast chaperones; assembly chaperone; biogenesis; coli; protein

摘要

Improving the Calvin-Benson-Bassham cycle enzyme Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) has the potential to increase crop productivity. However, the selectivity of the chaperones mediating Rubisco assembly in vascular plants toward their cognate Rubisco presents a substantive roadblock to both Rubisco protein engineering and transgenic expression of heterologous Rubisco orthologs, both of which necessitate changes to the Rubisco sequence. Here, we ask whether a plant Rubisco chaperone can be reprogrammed by directed evolution to accommodate a nonnative client. We developed a selection strategy to assess Rubisco assembly factor activity in high-throughput and used this selection to identify mutants of the chaperone Raf1 from Arabidopsis thaliana (AtRaf1) that assemble Nicotiana tabacum Rubisco, for which wild-type AtRaf1 has minimal activity. We show that directed evolution can generate AtRaf1 variants that enable significantly increased N. tabacum Rubisco assembly compared to wild-type AtRaf1. Evaluation of evolved AtRaf1s indicates that they retain the ability to assemble their native client and can assemble other dicot Rubisco orthologs that they were not evolved to recognize. This work may provide a strategy for addressing the constraints chaperone selectivity impose upon Rubisco-centric efforts to improve plant photosynthesis.