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. Author manuscript; available in PMC: 2021 Dec 1.
Published in final edited form as: Curr Opin Biotechnol. 2020 Sep 18;66:217–226. doi: 10.1016/j.copbio.2020.08.005

Figure 3. Outline of protocol to engineer enzymes for mNAD activity.

Figure 3

An initial screen is performed with the mNAD of interest and wild type enzyme to determine the baseline performance. Positions surrounding the cofactor binding pocket and those that contribute to cofactor specificity found through sequence alignment are chosen for mutagenesis. Variants are screened through colorimetric assay measuring activity through color development reflecting production of reduced cofactor. Future tool developments to improve throughput will include growth-based selection assays where the ability of the cell to regenerate mNAD is linked to survival.