Journal
ACS CATALYSIS
Volume 12, Issue 14, Pages 8582-8592Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acscatal.2c01837
Keywords
Embden-Meyerhof-Parnas; glycolysis; nicotinamide mononucleotide; noncanonical redox cofactor; computational protein design; orthogonal pathway engineering glyceraldehyde-3-phosphate dehydrogenase
Categories
Funding
- University of California, Irvine
- National Science Foundation (NSF) [1847705]
- National Institutes of Health (NIH) [GM130367, DP2 GM137427]
- Advanced Research Projects Agency-Energy (ARPA-E) [DE-AR0001508]
- National Institute of Environmental Health Sciences [P42ES004699]
- National Institutes of Health [R01 GM 076324-11]
- National Science Foundation [1627539, 1805510, 1827246]
- NSF Graduate Research Fellowship Program [DGE1839285]
- Alfred Sloan Research Fellowship
- Directorate For Engineering
- Div Of Chem, Bioeng, Env, & Transp Sys [1847705] Funding Source: National Science Foundation
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This study redesigns the high-flux glycolytic pathway to generate reducing power using the noncanonical cofactor NMN+, and discovers variant enzymes with high NMN+-dependent activity and specific switch in cofactor specificity. The engineered enzymes enable orthogonal control of glucose utilization in Escherichia coli only in the presence of NMN+.
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 similar to 3.4 x 10(6)-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.
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