UC Irvine

Biological production of chemicals often requires the use of cellular cofactors, such as nicotinamide adenine dinucleotide phosphate (NADP⁺). These cofactors are expensive to use in vitro and difficult to control in vivo. We demonstrate the development of a noncanonical redox cofactor system based on nicotinamide mononucleotide (NMN⁺). The key enzyme in the system is a computationally designed glucose dehydrogenase with a 10⁷-fold cofactor specificity switch toward NMN⁺ over NADP⁺ based on apparent enzymatic activity. We demonstrate that this system can be used to support diverse redox chemistries in vitro with high total turnover number (~39,000), to channel reducing power in Escherichia coli whole cells specifically from glucose to a pharmaceutical intermediate, levodione, and to sustain the high metabolic flux required for the central carbon metabolism to support growth. Overall, this work demonstrates efficient use of a noncanonical cofactor in biocatalysis and metabolic pathway design.