Engineering a nicotinamide mononucleotide redox cofactor system for biocatalysis

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 10(7)-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 (similar to 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.