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

Edward King; Youtian Cui; Derek Aspacio; Frances Nicklen; Linyue Zhang; Sarah Maxel; Ray Luo; Justin B Siegel; Erick Aitchison; Han Li

doi:10.1021/acscatal.2c01837

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

ACS Catal. 2022 Jul 15;12(14):8582-8592. doi: 10.1021/acscatal.2c01837. Epub 2022 Jul 5.

Authors

Edward King¹, Youtian Cui², Derek Aspacio³, Frances Nicklen⁴, Linyue Zhang³, Sarah Maxel³, Ray Luo⁵, Justin B Siegel⁶, Erick Aitchison¹, Han Li⁷

Affiliations

¹ Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, California 92697-3900, United States.
² Genome Center, University of California, Davis, Davis, California 95616, United States.
³ Department of Chemical and Biomolecular Engineering, University of California, Irvine, Irvine, California 92697-3900, United States.
⁴ Department of Biomedical Engineering, University of California, Irvine, Irvine, California 92697-3900, United States.
⁵ Department of Molecular Biology and Biochemistry, Department of Chemical and Biomolecular Engineering, and Department of Biomedical Engineering, University of California, Irvine, Irvine, California 92697-3900, United States.
⁶ Department of Chemistry, Genome Center, and Department of Biochemistry and Molecular Medicine, University of California, Davis, Davis, California 95616, United States.
⁷ Department of Chemical and Biomolecular Engineering and Department of Biomedical Engineering, University of California, Irvine, Irvine, California 92697-3900, United States.

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.

Keywords: Embden–Meyerhof–Parnas; Glycolysis; computational protein design; glyceraldehyde-3-phosphate dehydrogenase; nicotinamide mononucleotide; noncanonical redox cofactor; orthogonal pathway engineering.

Abstract

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