Development of a noncanonical redox cofactor platform in Saccharomyces cerevisiae

Saccharomyces cerevisiae is a keystone host for biomanufacturing, yet its metabolic engineering is often complicated by the crosstalk between heterologous pathways and native metabolism. In particular, allocating and balancing the universal reducing equivalents nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) typically requires laborious genetic modifications. To address this challenge, we established an orthogonal redox cofactor infrastructure in S. cerevisiae based on nicotinamide mononucleotide (NMN(H)), which is decoupled from the host's NAD(P)H-based metabolism. Because NMN(H)-dependent pathway networks comprise only a small number of user-defined reactions, their optimization is substantially simplified. First, by rewiring glycolysis to eliminate NAD(P)H generation from glucose, we repurposed glucose as a dedicated electron source for NMNH reducing power. We then demonstrated that NMN(H) selectively drives the reduction of citral to citronellal, both unstable aldehydes, while suppressing the rapid over-reduction to alcohols observed with native cofactors without identifying or disrupting the numerous endogenous alcohol dehydrogenases in resting S. cerevisiae cells. Finally, we engineer S. cerevisiae to accumulate an intracellular NMN⁺ pool of ∼2.9 mM, comparable to NAD⁺ intracellular levels, enabling the first self-sustained, new-to-nature redox cofactor system in a eukaryotic organism. This work establishes NMN(H) as a functional third nicotinamide-based redox cofactor in yeast and provides a generalizable eukaryotic platform for orthogonal redox biocatalysis.