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High-Throughput Screening of Coenzyme Preference Change of Thermophilic 6-Phosphogluconate Dehydrogenase from NADP⁺ to NAD⁺

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dc.contributorVirginia Techen
dc.contributor.authorHuang, Ruien
dc.contributor.authorChen, Huien
dc.contributor.authorZhong, Chaoen
dc.contributor.authorKim, Jae Eungen
dc.contributor.authorZhang, Yi-Heng Percivalen
dc.contributor.departmentBiological Sciencesen
dc.date.accessioned2017-03-28T18:12:25Zen
dc.date.available2017-03-28T18:12:25Zen
dc.date.issued2016-09-02en
dc.description.abstractCoenzyme engineering that changes NAD(P) selectivity of redox enzymes is an important tool in metabolic engineering, synthetic biology, and biocatalysis. Here we developed a high throughput screening method to identify mutants of 6-phosphogluconate dehydrogenase (6PGDH) from a thermophilic bacterium Moorella thermoacetica with reversed coenzyme selectivity from NADP⁺ to NAD⁺. Colonies of a 6PGDH mutant library growing on the agar plates were treated by heat to minimize the background noise, that is, the deactivation of intracellular dehydrogenases, degradation of inherent NAD(P)H, and disruption of cell membrane. The melted agarose solution containing a redox dye tetranitroblue tetrazolium (TNBT), phenazine methosulfate (PMS), NAD⁺, and 6-phosphogluconate was carefully poured on colonies, forming a second semi-solid layer. More active 6PGDH mutants were examined via an enzyme-linked TNBT-PMS colorimetric assay. Positive mutants were recovered by direct extraction of plasmid from dead cell colonies followed by plasmid transformation into E. coli TOP10. By utilizing this double-layer screening method, six positive mutants were obtained from two-round saturation mutagenesis. The best mutant 6PGDH A30D/R31I/T32I exhibited a 4,278-fold reversal of coenzyme selectivity from NADP⁺ to NAD⁺. This screening method could be widely used to detect numerous redox enzymes, particularly for thermophilic ones, which can generate NAD(P)H reacted with the redox dye TNBT.en
dc.description.sponsorshipBiological System Engineering Department, Virginia Polytechnic Institute and State University, Virginia, USA; Department of Energy, Office of Energy Efficiency and Renewable Energy, Fuel Cell Technologies Office [DE-EE0006968]; Virginia Agricultural Experiment Station; Hatch Program of the National Institute of Food and Agriculture, U.S. Department of Agricultureen
dc.format.extent10en
dc.format.mimetypeapplication/pdfen
dc.identifier.doihttps://doi.org/10.1038/srep32644en
dc.identifier.issn2045-2322en
dc.identifier.pmid27587230en
dc.identifier.urihttp://hdl.handle.net/10919/76718en
dc.identifier.volume6en
dc.language.isoen_USen
dc.publisherNatureen
dc.rightsCreative Commons Attribution 4.0 Internationalen
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/en
dc.subject2,5-diketo-d-gluconic acid reductaseen
dc.subjectcofactor-binding pocketen
dc.subjectescherichia-colien
dc.titleHigh-Throughput Screening of Coenzyme Preference Change of Thermophilic 6-Phosphogluconate Dehydrogenase from NADP⁺ to NAD⁺en
dc.title.serialScientific Reportsen
dc.typeArticle - Refereeden
dc.type.dcmitypeTexten

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