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Development of a High-Throughput, In Vivo Selection Platform for NADPH-Dependent Reactions Based on Redox Balance Principles.

ABSTRACT: Bacteria undergoing anaerobic fermentation must maintain redox balance. In vivo metabolic evolution schemes based on this principle have been limited to targeting NADH-dependent reactions. Here, we developed a facile, specific, and high-throughput growth-based selection platform for NADPH-consuming reactions in vivo, based on an engineered NADPH-producing glycolytic pathway in Escherichia coli. We used the selection system in the directed evolution of a NADH-dependent d-lactate dehydrogenase from Lactobacillus delbrueckii toward utilization of NADPH. Through one round of selection, we obtained multiple enzyme variants with superior NADPH-dependent activities and protein expression levels; these mutants may serve as important tools in biomanufacturing d-lactate as a renewable polymer building block. Importantly, sequence analysis and computational protein modeling revealed that diverging evolutionary paths during the selection resulted in two distinct cofactor binding modes, which suggests that the high throughput of our selection system allowed deep searching of protein sequence space to discover diverse candidates en masse.

SUBMITTER: Zhang L

PROVIDER: S-EPMC7195872 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Development of a High-Throughput, In Vivo Selection Platform for NADPH-Dependent Reactions Based on Redox Balance Principles.

Zhang Linyue L King Edward E Luo Ray R Li Han H

ACS synthetic biology 20180629 7

Bacteria undergoing anaerobic fermentation must maintain redox balance. In vivo metabolic evolution schemes based on this principle have been limited to targeting NADH-dependent reactions. Here, we developed a facile, specific, and high-throughput growth-based selection platform for NADPH-consuming reactions in vivo, based on an engineered NADPH-producing glycolytic pathway in Escherichia coli. We used the selection system in the directed evolution of a NADH-dependent d-lactate dehydrogenase fro ...[more]

PMID: 29939709

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Dataset Information

Development of a High-Throughput, In Vivo Selection Platform for NADPH-Dependent Reactions Based on Redox Balance Principles.

Publications

Development of a High-Throughput, In Vivo Selection Platform for NADPH-Dependent Reactions Based on Redox Balance Principles.

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