In vivo directed evolution of an ultrafast Rubisco from a semianaerobic environment imparts oxygen resistance

• 成果类型: Article
• 署名作者: McDonald, Julie L.; Shapiro, Nathan P.; Mengiste, Amanuella A.; Kaines, Sarah; Whitney, Spencer M.; Wilson, Robert H.; Shoulders, Matthew D.
• 署名单位: Massachusetts Institute of Technology (MIT); Massachusetts Institute of Technology (MIT); Australian National University; Harvard University; Massachusetts Institute of Technology (MIT); Broad Institute; Massachusetts Institute of Technology (MIT)
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-9415
• DOI: 10.1073/pnas.2505083122
• 发表日期: 2025-07-08
• 关键词: form-i; selection; specificity; improvements; prospects; food

摘要

Carbon dioxide (CO2) assimilation by the enzyme Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) underpins biomass accumulation in photosynthetic bacteria and eukaryotes. Despite its pivotal role, Rubisco has a slow carboxylation rate (k(cat)(CO2)) and is competitively inhibited by oxygen (O-2). These traits impose limitations on photosynthetic efficiency, making Rubisco a compelling target for improvement. Interest in Form II Rubisco from Gallionellaceae bacteria, which comprise a dimer or hexamer of large subunits, arises from their nearly fivefold higher k(cat)(CO2) than the average Rubisco enzyme. As well as having a fast k(cat)(CO2) (25.8 s(-1) at 25 degrees C), we show that Gallionellaceae Rubisco (GWS1B) is extremely sensitive to O-2 inhibition, consistent with its evolution under semianaerobic environments. We therefore used an in vivo mutagenesis-mediated screening pipeline to evolve GWS1B over six rounds under oxygenic selection, identifying three catalytic point mutants with improved ambient carboxylation efficiency: Thr-29-Ala (T29A), Glu-40-Lys (E40K), and Arg-337-Cys (R337C). Full kinetic characterization showed that each substitution enhanced the CO2 affinity of GWS1B under oxygenic conditions by subduing oxygen affinity, leading to 25% (E40K), 11% (T29A), and 8% (R337C) enhancements in carboxylation efficiency under ambient O-2 at 25 degrees C. By contrast, under the near anaerobic natural environment of Gallionellaceae, the carboxylation efficiency of each mutant was impaired similar to 16%. These findings demonstrate the efficacy of artificial directed evolution to access distinctive regions of catalytic space in Rubisco.