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Title: High-throughput screening of coenzyme preference change of thermophilic 6-phosphogluconate dehydrogenase from NADP + to NAD +

Coenzyme 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 +. Furthermore, this screening method could be widely used to detect numerous redox enzymes, particularly for thermophilic ones, which canmore » generate NAD(P)H reacted with the redox dye TNBT.« less
Authors:
 [1] ;  [1] ;  [1] ;  [1] ;  [2]
  1. Virginia Polytechnic Inst. and State Univ. (Virginia Tech), Blacksburg, VA (United States)
  2. Virginia Polytechnic Inst. and State Univ. (Virginia Tech), Blacksburg, VA (United States); Chinese Academy of Sciences (CAS), Tianjin (China)
Publication Date:
Grant/Contract Number:
EE0006968
Type:
Accepted Manuscript
Journal Name:
Scientific Reports
Additional Journal Information:
Journal Volume: 6; Journal Issue: 1; Journal ID: ISSN 2045-2322
Publisher:
Nature Publishing Group
Research Org:
Virginia Polytechnic Inst. and State Univ. (Virginia Tech), Blacksburg, VA (United States)
Sponsoring Org:
USDOE Office of Energy Efficiency and Renewable Energy (EERE), Fuel Cell Technologies Office (EE-3F)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; biocatalysis; oxidoreductases
OSTI Identifier:
1363900

Huang, Rui, Chen, Hui, Zhong, Chao, Kim, Jae Eung, and Zhang, Yi-Heng Percival. High-throughput screening of coenzyme preference change of thermophilic 6-phosphogluconate dehydrogenase from NADP+ to NAD+. United States: N. p., Web. doi:10.1038/srep32644.
Huang, Rui, Chen, Hui, Zhong, Chao, Kim, Jae Eung, & Zhang, Yi-Heng Percival. High-throughput screening of coenzyme preference change of thermophilic 6-phosphogluconate dehydrogenase from NADP+ to NAD+. United States. doi:10.1038/srep32644.
Huang, Rui, Chen, Hui, Zhong, Chao, Kim, Jae Eung, and Zhang, Yi-Heng Percival. 2016. "High-throughput screening of coenzyme preference change of thermophilic 6-phosphogluconate dehydrogenase from NADP+ to NAD+". United States. doi:10.1038/srep32644. https://www.osti.gov/servlets/purl/1363900.
@article{osti_1363900,
title = {High-throughput screening of coenzyme preference change of thermophilic 6-phosphogluconate dehydrogenase from NADP+ to NAD+},
author = {Huang, Rui and Chen, Hui and Zhong, Chao and Kim, Jae Eung and Zhang, Yi-Heng Percival},
abstractNote = {Coenzyme 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+. Furthermore, 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.},
doi = {10.1038/srep32644},
journal = {Scientific Reports},
number = 1,
volume = 6,
place = {United States},
year = {2016},
month = {9}
}