Coenzyme Engineering of a Hyperthermophilic 6-Phosphogluconate Dehydrogenase from NADP+to NAD+with Its Application to Biobatteries.
Engineering the coenzyme specificity of redox enzymes plays an important role in metabolic engineering, synthetic biology, and biocatalysis, but it has rarely been applied to bioelectrochemistry. Here we develop a rational design strategy to change the coenzyme specificity of 6-phosphogluconate dehydrogenase (6PGDH) from a hyperthermophilic bacterium Thermotoga maritima from its natural coenzyme NADP+to NAD+. Through amino acid-sequence alignment of NADP+- and NAD+-preferred 6PGDH enzymes and computer-aided substrate-coenzyme docking, the key amino acid residues responsible for binding the phosphate group of NADP+were identified. Four mutants were obtained via site-directed mutagenesis. The best mutant N32E/R33I/T34I exhibited a ~6.4 × 104-fold reversal of the coenzyme selectivity from NADP+to NAD+. The maximum power density and current density of the biobattery catalyzed by the mutant were 0.135 mW cm-2and 0.255 mA cm-2, ~25% higher than those obtained from the wide-type 6PGDH-based biobattery at the room temperature. By using this 6PGDH mutant, the optimal temperature of running the biobattery was as high as 65 °C, leading to a high power density of 1.75 mW cm-2. This study demonstrates coenzyme engineering of a hyperthermophilic 6PGDH and its application to high-temperature biobatteries.
Biological Systems Engineering Department, Virginia Tech, 304 Seitz Hall, Blacksburg, Virginia 24061, USA.
Hui
H
Chen
Cell Free Bioinnovations Inc. 1800 Kraft Drive, Suite 222, Blacksburg, VA 24060, USA.|Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 West 7th Avenue, Tianjin Airport Economic Area, Tianjin 300308, China.
Zhiguang
Z
Zhu
Biological Systems Engineering Department, Virginia Tech, 304 Seitz Hall, Blacksburg, Virginia 24061, USA.
Rui
R
Huang
Biological Systems Engineering Department, Virginia Tech, 304 Seitz Hall, Blacksburg, Virginia 24061, USA.|Cell Free Bioinnovations Inc. 1800 Kraft Drive, Suite 222, Blacksburg, VA 24060, USA.|Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 West 7th Avenue, Tianjin Airport Economic Area, Tianjin 300308, China.
Yi-Heng Percival
YP
Zhang
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D001674:Bioelectric Energy Sources; D002384:Catalysis; D003067:Coenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005818:Genetic Engineering; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D010734:Phosphogluconate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D020124:Thermotoga maritima / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D003067:Coenzymes; D009243:NAD; D009249:NADP; D010734:Phosphogluconate Dehydrogenase