(629g) Engineering an Orthogonal Redox Cofactor System for Cell-Free and Whole-Cell Biocatalysis (Invited Speaker)

Biological production of chemicals often requires the use of cellular cofactors, such as nicotinamide adenine dinucleotide (phosphate) (NAD(P)⁺). These cofactors are expensive to use in vitro and difficult to control in vivo. We demonstrate the development of a non-canonical redox cofactor system based on nicotinamide mononucleotide (NMN⁺). The key enzyme in the system is a computationally designed glucose dehydrogenase (GDH) with a 10⁷-fold cofactor specificity switch towards NMN⁺ over NAD(P)⁺ based on apparent enzymatic activity. We demonstrate that this system can be used (1) to support diverse redox chemistries in vitro with high robustness, (2) to eliminate side-reactions in crude lysate-based cell-free biotransformation, (3) to specifically channel reducing power in Escherichia coli whole cells from glucose to a pharmaceutical intermediate, levodione, and (4) to sustain the high metabolic flux required for the central carbon metabolism to support growth. In addition, we also established high-throughput selection platforms which allow NMN⁺-dependent enzymes to be obtained on demand through directed evolution. Overall, this work demonstrates efficient utilization of a non-canonical cofactor in biocatalysis and metabolic pathway design, and points out a path for rapidly expanding its applications.