Abstract
In photosynthesis Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyses the often rate limiting CO2-fixation step in the Calvin cycle. This makes Rubisco both the gatekeeper for carbon entry into the biosphere and a target for functional improvement to enhance photosynthesis and plant growth. Encumbering the catalytic performance of Rubisco is its highly conserved, complex catalytic chemistry. Accordingly, traditional efforts to enhance Rubisco catalysis using protracted “trial and error” protein engineering approaches have met with limited success. Here we demonstrate the versatility of high throughput directed (laboratory) protein evolution for improving the carboxylation properties of a non-photosynthetic Rubisco from the archaea Methanococcoides burtonii. Using chloroplast transformation in the model plant Nicotiana tabacum (tobacco) we confirm the improved forms of M. burtonii Rubisco increased photosynthesis and growth relative to tobacco controls producing wild-type M. burtonii Rubisco. Our findings indicate continued directed evolution of archaeal Rubisco offers new potential for enhancing leaf photosynthesis and plant growth.
Highlights
In photosynthesis Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyses the often rate limiting CO2-fixation step in the Calvin cycle
The initial selection was performed under high-Rubisco inducing (0.5 mM IPTG) and low-PRK inducing conditions (0.05% (w/v) arabinose) in air supplemented with 2.5% (v/v) CO2
Colony growth was scored relative to MM1-prk cells expressing wild-type Methanococcoides burtonii L10 Rubisco (MbR) that could not grow on media containing 0.1% (w/v) arabinose (Fig. 1b)
Summary
In photosynthesis Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyses the often rate limiting CO2-fixation step in the Calvin cycle. Improving the performance of the CO2-fixing enzyme Rubisco has the potential to significantly enhance photosynthetic efficiency and yield[1] Strategies to achieve this goal involve either modifying the biochemistry and ultrastructure of leaf chloroplasts to concentrate CO2 around Rubisco, or directly improving Rubisco catalysis itself by genetic crossing or transgenic modification[2]. Despite five decades of research, a dramatic amplification in computational power and more than 25 X-ray structures for different Rubisco isoforms[10] we remain unable to improve Rubisco catalysis by rational design[11,12,13] This limitation has led to the development of directed (in vitro or laboratory) protein evolution approaches tailored to select for Rubisco mutants with improved function[12].
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