title: High-Throughput Screening of Coenzyme Preference Change of Thermophilic 6-Phosphogluconate Dehydrogenase from NADP(+) to NAD(.).
abstract: 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(+). 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.
authors: Biological Systems Engineering Department, Virginia Tech, 304 Seitz Hall, Blacksburg, Virginia 24061, USA.
authors: Rui
authors: R
authors: Huang
authors: Biological Systems Engineering Department, Virginia Tech, 304 Seitz Hall, Blacksburg, Virginia 24061, USA.
authors: Hui
authors: H
authors: Chen
authors: Biological Systems Engineering Department, Virginia Tech, 304 Seitz Hall, Blacksburg, Virginia 24061, USA.
authors: Chao
authors: C
authors: Zhong
authors: Biological Systems Engineering Department, Virginia Tech, 304 Seitz Hall, Blacksburg, Virginia 24061, USA.
authors: Jae Eung
authors: JE
authors: Kim
authors: Biological Systems Engineering Department, Virginia Tech, 304 Seitz Hall, Blacksburg, Virginia 24061, USA.|Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 West 7th Avenue, Tianjin Airport Economic Area, Tianjin 300308, China.
authors: Yi-Heng Percival
authors: YH
authors: Zhang
mesh_terms: D001483:Base Sequence; D003067:Coenzymes / Q000378:metabolism*; D003116:Color; D003124:Colorimetry; D057166:High-Throughput Screening Assays / Q000379:methods*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D009243:NAD / Q000737:chemistry / Q000378:metabolism*; D009249:NADP / Q000737:chemistry / Q000378:metabolism*; D010734:Phosphogluconate Dehydrogenase / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D015203:Reproducibility of Results; D013696:Temperature
chemical_list: D003067:Coenzymes; D009243:NAD; D009249:NADP; D010734:Phosphogluconate Dehydrogenase