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

Inaccuracies in biochemically characterizing the amount and CO(2)-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...

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Autores principales: Zhou, Yu, Whitney, Spencer
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6834295/
https://www.ncbi.nlm.nih.gov/pubmed/31658746
http://dx.doi.org/10.3390/ijms20205019
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author Zhou, Yu
Whitney, Spencer
author_facet Zhou, Yu
Whitney, Spencer
author_sort Zhou, Yu
collection PubMed
description Inaccuracies in biochemically characterizing the amount and CO(2)-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 CO(2)-fixing properties. The RsRubisco contains four amino acid substitutions in the large subunit (RbcL) and an improved carboxylation rate (k(cat)(C), up 27%), carboxylation efficiency (k(cat)(C)/K(m) for CO(2), increased 17%), unchanged CO(2)/O(2) 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 (L(8)S(8)) 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).
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spelling pubmed-68342952019-11-25 Directed Evolution of an Improved Rubisco; In Vitro Analyses to Decipher Fact from Fiction Zhou, Yu Whitney, Spencer Int J Mol Sci Article Inaccuracies in biochemically characterizing the amount and CO(2)-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 CO(2)-fixing properties. The RsRubisco contains four amino acid substitutions in the large subunit (RbcL) and an improved carboxylation rate (k(cat)(C), up 27%), carboxylation efficiency (k(cat)(C)/K(m) for CO(2), increased 17%), unchanged CO(2)/O(2) 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 (L(8)S(8)) 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). MDPI 2019-10-10 /pmc/articles/PMC6834295/ /pubmed/31658746 http://dx.doi.org/10.3390/ijms20205019 Text en © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Zhou, Yu
Whitney, Spencer
Directed Evolution of an Improved Rubisco; In Vitro Analyses to Decipher Fact from Fiction
title Directed Evolution of an Improved Rubisco; In Vitro Analyses to Decipher Fact from Fiction
title_full Directed Evolution of an Improved Rubisco; In Vitro Analyses to Decipher Fact from Fiction
title_fullStr Directed Evolution of an Improved Rubisco; In Vitro Analyses to Decipher Fact from Fiction
title_full_unstemmed Directed Evolution of an Improved Rubisco; In Vitro Analyses to Decipher Fact from Fiction
title_short Directed Evolution of an Improved Rubisco; In Vitro Analyses to Decipher Fact from Fiction
title_sort directed evolution of an improved rubisco; in vitro analyses to decipher fact from fiction
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6834295/
https://www.ncbi.nlm.nih.gov/pubmed/31658746
http://dx.doi.org/10.3390/ijms20205019
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