Engineering Embden–Meyerhof–Parnas Glycolysis to Generate Noncanonical Reducing Power

Abstract

Noncanonical cofactors such as nicotinamide mononucleotide (NMN+) supplant the electron-transfer functionality of the natural cofactors, NAD­(P)+, at a lower cost in cell-free biomanufacturing and enable orthogonal electron delivery in whole-cell metabolic engineering. Here, we redesign the high-flux Embden–Meyerhof–Parnas (EMP) glycolytic pathway to generate NMN+-based reducing power, by engineering Streptococcus mutans glyceraldehyde-3-phosphate dehydrogenase (Sm GapN) to utilize NMN+. Through iterative rounds of rational design, we discover the variant GapN Penta (P179K-F153S-S330R-I234E-G210Q) with high NMN+-dependent activity and GapN Ortho (P179K-F153S-S330R-I234E-G214E) with ∼3.4 × 106-fold switch in cofactor specificity from its native cofactor NADP+ to NMN+. GapN Ortho is further demonstrated to function in Escherichia coli only in the presence of NMN+, enabling orthogonal control of glucose utilization. Molecular dynamics simulation and residue network connectivity analysis indicate that mutations altering cofactor specificity must be coordinated to maintain the appropriate degree of backbone flexibility to position the catalytic cysteine. These results provide a strategy to guide future designs of NMN+-dependent enzymes and establish the initial steps toward an orthogonal EMP pathway with biomanufacturing potential.

Computer-Aided Engineering of a Non-Phosphorylating Glyceraldehyde-3-Phosphate Dehydrogenase to Enable Cell-Free Biocatalysis

“…Other targets of interest in cofactor engineering include mimetics of NAD­(P) for application in cell-free biocatalysis, as demonstrated by the Li group . The interest in these (semi-)­synthetic molecules derives from three weaknesses of natural cofactorstheir instability, their cost, and the difficulty in balancing oxidation and reduction in a cell-free reaction.…”

“…Other targets of interest in cofactor engineering include mimetics of NAD(P) for application in cell-free biocatalysis, as demonstrated by the Li group. 36 The interest in these (semi-)synthetic molecules derives from three weaknesses of natural cofactors�their instability, their cost, and the difficulty in balancing oxidation and reduction in a cell-free reaction. Synthetic NAD(P) cofactors offer the possibility of cheaper and more robust cofactors that could be reduced in situ, allowing long-lasting industrial biocatalytic reactions in which reducing equivalents could be supplied via a biomimetic cofactor from electricity through an anode.…”

Orthogonal glycolytic pathway enables directed evolution of noncanonical cofactor oxidase

“…A similar binding mode is expected to be necessary for catalysis since MNAH is a truncated version of the native cofactor. Modeling preparations and docking of NMNH were done using Rosetta 57 . Models for the variant Lp Nox were produced with Rosetta.…”

In Vitro Inhibitory Activity and Molecular Docking Study of Selected Natural Phenolic Compounds as AR and SDH Inhibitors**

“…This case significantly increases the formation of advanced glycation end products, which causes ROS formation at the last of the polyol pathway [33] . In addition, in the glycolytic pathway, NAD + is a cofactor essential for converting glyceraldehyde 3‐phosphate to 1,3‐bisphosphoglycerate [34] . Decreased amount of NAD + causes increment flux into the pentose phosphate pathway in glucose metabolism [35] .…”

“…[33] In addition, in the glycolytic pathway, NAD + is a cofactor essential for converting glyceraldehyde 3phosphate to 1,3-bisphosphoglycerate. [34] Decreased amount of NAD + causes increment flux into the pentose phosphate pathway in glucose metabolism. [35] Since approximately 30 % of blood glucose may flux from the polyol pathway during hyperglycemia, this metabolic pathway is known to be the primary pathway regulating redox balance metabolizable between NADH and NAD + .…”