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Open AccessArticle

Directed Evolution of an Improved Rubisco; In Vitro Analyses to Decipher Fact from Fiction

Australian Research Council Center of Excellence for Translational Photosynthesis, Research School of Biology, The Australian National University, 134 Linnaeus Way, Acton, ACT 0200, Australia
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2019, 20(20), 5019;
Received: 30 August 2019 / Revised: 2 October 2019 / Accepted: 4 October 2019 / Published: 10 October 2019
Inaccuracies in biochemically characterizing the amount and CO2-fixing properties of the photosynthetic enzyme Ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase continue to hamper an accurate evaluation of Rubisco mutants selected by directed evolution. Here, we outline an analytical pipeline for accurately quantifying Rubisco content and kinetics that averts the misinterpretation of directed evolution outcomes. Our study utilizes a new T7-promoter regulated Rubisco Dependent Escherichia coli (RDE3) screen to successfully select for the first Rhodobacter sphaeroides Rubisco (RsRubisco) mutant with improved CO2-fixing properties. The RsRubisco contains four amino acid substitutions in the large subunit (RbcL) and an improved carboxylation rate (kcatC, up 27%), carboxylation efficiency (kcatC/Km for CO2, increased 17%), unchanged CO2/O2 specificity and a 40% lower holoenzyme biogenesis capacity. Biochemical analysis of RsRubisco chimers coding one to three of the altered amino acids showed Lys-83-Gln and Arg-252-Leu substitutions (plant RbcL numbering) together, but not independently, impaired holoenzyme (L8S8) assembly. An N-terminal Val-11-Ile substitution did not affect RsRubisco catalysis or assembly, while a Tyr-345-Phe mutation alone conferred the improved kinetics without an effect on RsRubisco production. This study confirms the feasibility of improving Rubisco by directed evolution using an analytical pipeline that can identify false positives and reliably discriminate carboxylation enhancing amino acids changes from those influencing Rubisco biogenesis (solubility). View Full-Text
Keywords: photosynthesis; carbon fixation; synthetic biology; metabolic engineering photosynthesis; carbon fixation; synthetic biology; metabolic engineering
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MDPI and ACS Style

Zhou, Y.; Whitney, S. Directed Evolution of an Improved Rubisco; In Vitro Analyses to Decipher Fact from Fiction. Int. J. Mol. Sci. 2019, 20, 5019.

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