(607b) Developing Formate Dehydrogenase into a Modular Cofactor Regeneration and Sequestration Tool

Nicotinamide-dependent oxidoreductases can be used as biocatalysts for industrial production, offering a greener alternative to conventional chemical pathways. High stereoselectivity and ambient, aqueous catalysis make these enzymes especially attractive for the production of pharmaceuticals, where they can reduce energy costs and environmental risks associated with the use of organic solvents. However, their stoichiometric NAD(P)(H) requirements impose prohibitive costs for any large scale synthesis. Cofactor regeneration techniques reduce the amount of cofactor required for synthesis, but fail to ameliorate the costs associated with downstream separation of cofactor from product. We have recently demonstrated the creation of a novel cofactor-independent oxidoreductase through the introduction of NAD(H)-swing arms to two covalently fused dehydrogenase enzymes (1). The maleimide functionalized swing arms were covalently attached to cysteines on the surface of a Formate Dehydrogenase-Spycatcher (FDH-Spycatcher) fusion protein, which was then linked to a cystolic Malate Dehydrogenase-Spytag (cMDH-Spytag) fusion protein. The resulting complex was able to reduce oxaloacetate to malate, using the covalently tethered NADH continuously regenerated by the FDH. The costly NADH requirement of any Spy-tagged enzyme can therefore be eliminated by use of FDH-Spycatcher and inexpensive formate. We have further developed FDH-Spycatcher as a modular cofactor regeneration and sequestration tool. We have created FDH-Spycatcher mutants with additional cysteines strategically located on the surface of the fused Spycatcher. We demonstrate that the additional cysteines result in increased swing arm attachment, increasing the amount of cofactor that can be loaded onto FDH-Spycatcher. Use of the improved FDH-Spycatcher in the FDH-cMDH complex results in a higher catalytic turnover while maintaining predictable kinetic behavior. This demonstrates that the performance of the mutants can be selectively tuned through swing arm attachment.

(1) Ozbakir, H.F., Garcia, K.E., and Banta, S. (2018). Creation of a formate: malate oxidoreductase by fusion of dehydrogenase enzymes with PEGylated cofactor swing arms. Protein Eng Des Sel 31, 103–108