(498a) Engineering Embden-Meyerhof-Parnas (EMP) Glycolysis to Generate Noncanonical Reducing Power | AIChE

(498a) Engineering Embden-Meyerhof-Parnas (EMP) Glycolysis to Generate Noncanonical Reducing Power

Authors 

King, E., UC Irvine
Cui, Y., University of California, Davis
Maxel, S., University of California Irvine
Siegel, J., University of California Davis
Nicklen, F., University of California, Irvine
Zhang, L., University of California, Irvine
Li, H., University of California, Irvine
Aitchison, E., University of California, Irvine
Luo, R., University of California, Irvine
Noncanonical cofactors such as nicotinamide mononucleotide (NMN+) supplant the electron transfer function of natural cofactors nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate (NAD(P)+) at 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 this noncanonical cofactor. Through iterative rounds of rational design, we discover the variant GapN Penta with high NMN+-dependent activity, and GapN Ortho with ~3.4 × 106-fold switch in cofactor specificity from its native cofactor NADP+ to NMN+. GapN Ortho is further demonstrated to only function in Escherichia coli with 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 residue. These results provide a strategy to guide future designs of NMN+-dependent enzymes and the first steps toward an orthogonal EMP pathway with biomanufacturing potential.