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Title: Rewinding evolution in planta: A Rubisco-null platform validates high-performance ancestral enzymes
Improving the photosynthetic enzyme Rubisco is a key target for enhancing C3crop productivity, but progress has been hampered by the difficulty of evaluating engineered variants in planta without interference from the native enzyme. Here, we report the creation of a Rubisco-nullNicotiana tabacumplatform by using CRISPR-Cas9 to knock out all 11 nuclear-encoded small subunit (rbcS) genes. Knockout was achieved in a line expressing cyanobacterial Rubisco from the plastid genome, allowing the recovery of viable plants. We then developed a chloroplast expression system for coexpressing both large and small subunits from the plastid genome. We expressed two resurrected ancestral Rubiscos from the Solanaceae family. The resulting transgenic plants were phenotypically normal and accumulated Rubisco to wild-type levels. Importantly, kinetic analyses of the purified ancestral enzymes revealed they possessed a 16 to 20% higher catalytic efficiency (kcat,air/Kc,air) under ambient conditions, driven by a significantly faster turnover rate (kcat,air). We have demonstrated that our system allows robust in vivo assessment of novel Rubiscos and that ancestral reconstruction is a powerful strategy for identifying superior enzymes to improve photosynthesis in C3crops.  more » « less
Award ID(s):
2131582
PAR ID:
10661022
Author(s) / Creator(s):
; ; ;
Publisher / Repository:
National Academy of Sciences
Date Published:
Journal Name:
Proceedings of the National Academy of Sciences
Volume:
123
Issue:
3
ISSN:
0027-8424
Subject(s) / Keyword(s):
Rubisco CRISPR chloroplast transformation ancestral transgenic plant
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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