Engineering a nicotinamide mononucleotide redox cofactor system for biocatalysis

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Abstract

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.

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Fig. 1: Engineering Bs GDH to use NMN+ as redox cofactor efficiently in biotransformation in vitro.
Fig. 2: Engineering Bs GDH to exclude NADP+.
Fig. 3: In vivo NMN+ cycling supports E. coli growth.
Fig. 4: Bs GDH Ortho selectively provides reducing power for levodione production in E. coli whole cells.

Data availability

The authors declare that all relevant data supporting the findings of this study are available within the manuscript and its Supplementary Information.

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Acknowledgements

H.L. acknowledges support from University of California, Irvine, the National Science Foundation (NSF) (award no. 1847705), and the National Institutes of Health (NIH) (award no. DP2 GM137427). S.M. acknowledges support from the NSF Graduate Research Fellowship Program (grant no. DGE-1839285). W.B.B. acknowledges support from Graduate Assistance in Areas of National Need fellowship funded by the U.S. Department of Education. J.B.S., W.S.M. and Y.C. acknowledge support from the University of California, Davis, by the NSF (award nos. 1827246, 1805510, 1627539), the National Institute of Environmental Health Sciences of the NIH (award no. P42ES004699) and the NIH (award no. R01 GM 076324-11). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or the NSF. We thank the University of California, Irvine Mass Spectrometry Facility and F. Grun for help with LC–MS analysis.

Author information

H.L. and J.B.S. conceived the research. L.Z., W.B.B., and E.K. performed mutant enzyme kinetics characterization. W.B.B., L.Z., S.M., E.K., and B.F. performed the in vitro biotransformation. W.B.B., S.M., B.F., and A.S.M. performed the whole-cell biotransformation. W.B.B. performed the intracelluar NMN+ and NAD+ level analysis. S.M. performed the NMN+-dependent growth experiments. W.S.M. and Y.C. performed the computational modeling. All authors analyzed the data and wrote the manuscript.

Correspondence to Justin B. Siegel or Han Li.

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Black, W.B., Zhang, L., Mak, W.S. et al. Engineering a nicotinamide mononucleotide redox cofactor system for biocatalysis. Nat Chem Biol 16, 87–94 (2020) doi:10.1038/s41589-019-0402-7

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