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.

Coenzyme Engineering of a Hyperthermophilic 6-Phosphogluconate Dehydrogenase from NADP+ to NAD+ with Its Application to Biobatteries

“…2) indicates that the formation of a new hydrogen bond with NAD + through the replacement of alkaline amino acid by acidic one is favorable to increase the enzyme activity on NAD + , and even reverse the coenzyme preference from NADP + to NAD + . Moreover, our previous research on the directed evolution of Moorella thermoacetica 6PGDH also demonstrated that the introduction of an acidic amino acid, aspartate, in the loop of the Rossmann fold to form a new hydrogen bond with 2′-hydroxyl group of NAD + resulted in a 4,300-fold reversal of coenzyme selectivity from NADP + to NAD +  30. Reports on many other dehydrogenases also confirmed the positive effect of the formation of new hydrogen bond in reversing the coenzyme preference21243031323334.…”

Coevolution of both Thermostability and Activity of Polyphosphate Glucokinase from Thermobifida fusca YX

“…Previously, Zhang and his coworkers developed a simple petri dish-based double-layer screening for the identification of mutants of thermophilic NADP-dependent 6-phosphogluconate dehydrogenase with enhanced catalytic efficiencies on NAD ϩ . In this screening, the reduced NADH can react with the redox dye TNBT, generating black TNBT formazan (27). This screening method cannot be applicable to ultrathermophilic enzymes due to low melting temperatures of agar or agarose.…”

“…The E. coli TOP10 is a good strain for mutant library construction because of the high transformation efficiencies (e.g., 10 8 to 10 9 CFU/g plasmid DNA) ( Table 1). However, its ability for recombinant protein expression is much lower than that for E. coli BL21(DE3) based on the pET expression system, which suffers from low transformation efficiency (e.g., 10 6 CFU/g plasmid DNA) (27). To fulfill high transformation efficiency for directed evolution in E. coli TOP10 and high protein expression in E. coli BL21(DE3), an inducible tac promoter was inserted into pET28a-ppgk ( Fig.…”

“…The second layer was composed of the substrates for the following cascade enzyme reaction coupled with a colorimetric assay. This strategy was an extension of the previous method for detecting dehydrogenase that generates NAD(P)H (27). This Phytagel-based HTS method could be widely used for screening other ultrathermostable enzymes.…”

“…However, this method is labor-intensive and consumes many reagents and/or requires costly automated machines. In contrast, petri dishbased HTS approaches have the following benefits: higher screening capacity; less time and labor intensity used for colony picking, liquid cell culture, cell lysis, and enzyme activity assays; less reagent consumption; and no need for costly automated machine (26,27). The key point of petri dish-based HTS techniques is the development of a visual signal of colonies growing on solid plates, such as halo (28), color change (22), and fluorescence (29).…”