Engineering a nicotinamide mononucleotide redox cofactor system for biocatalysis

• Published in: Nature chemical biology Vol. 16; no. 1; pp. 87 - 94
• Main Authors: Black, William B., Zhang, Linyue, Mak, Wai Shun, Maxel, Sarah, Cui, Youtian, King, Edward, Fong, Bonnie, Sanchez Martinez, Alicia, Siegel, Justin B., Li, Han
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01.01.2020; Nature Publishing Group
• Subjects: 631/553/318; 631/92/469; 631/92/603; Biocatalysis; Biochemical Engineering; Biochemistry; Bioorganic Chemistry; Carbon - chemistry; Catalysis; Cell Biology; Chemistry; Chemistry and Materials Science; Chemistry/Food Science; Chromatography, Gas; Cyclohexanones - chemistry; E coli; Enzymatic activity; Escherichia coli - metabolism; Glucose dehydrogenase; Kinetics; Metabolism; NAD; NAD - chemistry; NADP - chemistry; NADPH; Nicotinamide; Nicotinamide Mononucleotide - chemistry; Nicotinamide Mononucleotide - genetics; Organic chemistry; Oxidation-Reduction; Protein Conformation; Protein Engineering; Pseudomonas putida - metabolism; Ralstonia - metabolism; Redesign; Software
• ISSN: 1552-4450; 1552-4469
• DOI: 10.1038/s41589-019-0402-7

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 (~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. Redesign of a glucose dehydrogenase to use nicotinamide mononucleotide (NMN + ) instead of NAD(P) + enables the development of a noncanonical cofactor system that can be used to support redox chemistries both in vitro and in Escherichia coli .