Shifting Redox Reaction Equilibria on Demand Using an Orthogonal Redox Cofactor.

Natural metabolism relies on chemical compartmentalization of two redox cofactors, NAD+ and NADP+, to orchestrate life-essential redox reaction directions. However, in whole cells the reliance on these canonical cofactors limits flexible control of redox reaction direction as these reactions are permanently tied to catabolism or anabolism. In cell-free systems, NADP+ is too expensive in large scale. We have previously reported the use of nicotinamide mononucleotide, (NMN+) as a low-cost, noncanonical redox cofactor capable of specific electron delivery to diverse chemistries. Here, we present Nox Ortho, an NMNH-specific water-forming oxidase, that completes the toolkit to modulate NMNH/NMN+ ratio. This work uncovers an enzyme design principle that succeeds in parallel engineering of six butanediol dehydrogenases as NMN(H)-orthogonal biocatalysts consistently with a 103 - 106 -fold cofactor specificity switch from NAD(P)+ to NMN+. We combine these to produce chiral-pure 2,3-butanediol (Bdo) isomers without interference from NAD(H) or NADP(H) in vitro and in E. coli cells. We establish that NMN(H) can be held at a distinct redox ratio on demand, decoupled from both NAD(H) and NADP(H) redox ratios in vitro and in vivo.

Minimal synthetic enhancers reveal control of the probability of transcriptional engagement and its timing by a morphogen gradient.

How enhancers interpret morphogen gradients to generate gene expression patterns is a central question in developmental biology. Recent studies have proposed that enhancers can dictate whether, when, and at what rate promoters engage in transcription, but the complexity of endogenous enhancers calls for theoretical models with too many free parameters to quantitatively dissect these regulatory strategies. To overcome this limitation, we established a minimal promoter-proximal synthetic enhancer in embryos of Drosophila melanogaster. Here, a gradient of the Dorsal activator is read by a single Dorsal DNA binding site. Using live imaging to quantify transcriptional activity, we found that a single binding site can regulate whether promoters engage in transcription in a concentration-dependent manner. By modulating the binding-site affinity, we determined that a gene's decision to transcribe and its transcriptional onset time can be explained by a simple model where the promoter traverses multiple kinetic barriers before transcription can ensue.

Orthogonal glycolytic pathway enables directed evolution of noncanonical cofactor oxidase.

Noncanonical cofactor biomimetics (NCBs) such as nicotinamide mononucleotide (NMN+) provide enhanced scalability for biomanufacturing. However, engineering enzymes to accept NCBs is difficult. Here, we establish a growth selection platform to evolve enzymes to utilize NMN+-based reducing power. This is based on an orthogonal, NMN+-dependent glycolytic pathway in Escherichia coli which can be coupled to any reciprocal enzyme to recycle the ensuing reduced NMN+. With a throughput of >106 variants per iteration, the growth selection discovers a Lactobacillus pentosus NADH oxidase variant with ~10-fold increase in NMNH catalytic efficiency and enhanced activity for other NCBs. Molecular modeling and experimental validation suggest that instead of directly contacting NCBs, the mutations optimize the enzyme's global conformational dynamics to resemble the WT with the native cofactor bound. Restoring the enzyme's access to catalytically competent conformation states via deep navigation of protein sequence space with high-throughput evolution provides a universal route to engineer NCB-dependent enzymes.