(622d) Shifting Redox Reaction Equilibrium on Demand Using an Orthogonal Cofactor

In Natural metabolism redox cofactors NAD(H) and NADP(H) are insulated from each other to enable individual control of catabolism and anabolism, two processes operating in opposite directions. However, in whole-cell biomanufacturing, dependence on NAD(P)(H) often limits the control of reaction direction, as the desired redox reaction becomes tied to all native reactions in the host that rely on the same cofactor. In cell-free biomanufacturing, NADP(H) is avoided due to its formidable cost and low stability. This leaves NAD(H) as the only electron carrier which cannot efficiently support both oxidation and reduction simultaneously without forming a futile cycle. To overcome these limitations, we sought to leverage an artificial cofactor, nicotinamide mononucleotide (NMN), to enable universal control of reaction direction supported by orthogonal enzymatic parts. NMN is much less expensive than NAD(P) and we have demonstrated its capability to serve as a redox cofactor in vivo. Here, we present the application of NMN in stereo-upgrading of meso-(2,3)-butanediol to (2S,3S)-butanediol, which requires first oxidizing the R-chiral center and then reducing it to install an S-chiral center. To drive these two opposing redox reactions both to completion, we used NAD(P) to mediate the oxidation and NMN the reduction, enabled by an engineered NMN-specific (2S,3S)-butanediol dehydrogenase. We demonstrated that NMN(H) is maintained at a dramatically different reduction potential than NAD(P)(H) in a single reaction mixture in vitro and in Escherichia coli whole cells. Our current NMN system achieves 85.7% conversion to >93% pure (2S,3S)-butanediol, compared to 21.0% conversion and a roughly equal mixture of all three butanediol isomers obtained with NAD(P) systems. Altogether, we envision that artificial cofactors will provide a route to controlled electron transfer reactions in vivo and a low-cost alternative to natural cofactors in vitro.