Stoichiometric Regeneration of Biomimetic Nicotinamide Coenzyme Powered by Biomass Sugars via In Vitro Synthetic...

Biomimetic nicotinamide coenzymes, including nicotinamide mononucleotide (NMN⁺), have been demonstrated as promising low-cost alternatives to nicotinamide adenine dinucleotide (phosphate) (NAD(P)⁺) in biocatalysis. Herein, to efficiently regenerate NMNH from NMN⁺ in vitro powered by biomass sugars, a thermophilic NADP⁺-dependent glucose 6-phosphate dehydrogenase from Thermotoga maritima (TmG6PDH) was engineered to increase the activity toward NMN⁺. The catalytic efficiency (k_cat/K_m) of optimal mutant (TmG6PDH-R7) toward NMN⁺ increased by 71.7-fold than TmG6PDH-WT. As a result, compared to the wild type, the coenzyme specificity ([k_cat/K_m]_NMN ⁺/[k_cat/K_m]_NADP ⁺) of TmG6PDH-R7 increased by ~2.0×10⁵-fold. The structural analysis revealed that the introduced hydrophobic and bulky residues lead to the formation of a smaller binding pocket, which resulting in a higher affinity for NMN⁺ with small size than NADP⁺. Then several in vitro synthetic enzymatic biosystems (ivSEBs) comprising this thermophilic TmG6PDH-R7 and a previously engineered thermophilic 6-phosphogluconate dehydrogenase were constructed. These ivSEBs harnessed the complete oxidation of renewable biomass sugars to facilitate the stoichiometric regeneration of 12 molecules of NMNH from 1 molecule of glucose, thereafter producing various products such as levodione, 2,3-butanediol or bioelectricity, over a wide temperature range. This study could pave the way for using stable and low-cost biomimetic coenzymes in ivSEBs for industrial biomanufacturing.