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

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,...

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Published in:ChemSusChem p. e202401263
Main Authors: Li, Qiangzi, Su, Hao, Meng, Dongdong, Qin, Yanmei, Wu, Ranran, Zhu, Zhiguang, Sheng, Xiang, You, Chun, Zhang, Yi-Heng P Job
Format: Journal Article
Language:English
Published: Germany 09-10-2024
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Summary: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 (kcat/Km) 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 ([kcat/Km]NMN+/[kcat/Km]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.
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ISSN:1864-5631
1864-564X
1864-564X
DOI:10.1002/cssc.202401263