Abstract
The specificity of enzymes for nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) as redox carriers can pose a significant hurdle for metabolic engineering and synthetic biology applications, where switching the specificity might be beneficial. We have developed an easy-to-use computational tool (CSR-SALAD) for the design of mutant libraries to simplify the process of reversing the cofactor specificity of an enzyme. Here, we describe the optimal use of this tool and present methods for its application in a laboratory setting.
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Acknowledgements
This work was supported by the Gordon and Betty Moore Foundation through grant number GBMF2809 to the Caltech Programmable Molecular Technology Initiative and by American Recovery and Reinvestment Act (ARRA) funds through the National Institutes of Health Shared Instrumentation Grant Program, grant number S10RR027203 to F.H.A. J.K.B.C acknowledges the support of the Resnick Sustainability Institute (Caltech). The cloning method described in Subheading 3.3 was developed by Martin Engqvist.
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Cahn, J.K.B., Brinkmann-Chen, S., Arnold, F.H. (2018). Enzyme Nicotinamide Cofactor Specificity Reversal Guided by Automated Structural Analysis and Library Design. In: Jensen, M.K., Keasling, J.D. (eds) Synthetic Metabolic Pathways. Methods in Molecular Biology, vol 1671. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7295-1_2
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DOI: https://doi.org/10.1007/978-1-4939-7295-1_2
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