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Scholarly Works (5 results)

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UC Irvine Electronic Theses and Dissertations (2023)

Biomanufacturing is an approach to production that repurposes cellular metabolism to transform inexpensive, renewable resources into value-added chemicals. While recent efforts haverapidly expanded product scope and improved product yields, many products developed at lab scale fail to reach markets. This problem exists largely due to the staggering complexity of natural metabolism that distributes crucial resources, such as electrons, across the vast metabolic network. Natural metabolism relies on chemical compartmentalization of two redox cofactors, NAD+ and NADP+, to orchestrate the directions of life-essential redox reactions. These redox cofactors are insulated from each other to permit thermodynamically incompatible reactions to occur simultaneously and in the same space.

Here, we investigate the recreation of this indispensable design in cellular metabolism,chemical compartmentalization by specific coenzymes, using a new-to-Nature redox cofactor, nicotinamide mononucleotide (NMN+). In Chapter 1, I introduce a framework to evaluate control of redox reaction equilbria and review relevant literature. In Chapter 2, we engineer the enzymes necessary to test our hypotheses on the control of redox reaction equilibria that can be achieved with noncanonical redox cofactors. In Chapter 3, we evaluate application of a complete toolkit for the modulation of NMNH/NMN+ ratio in vitro and in vivo, both in the presence of dramatically opposed NADH/NAD+ or NADPH/NADP+ ratios. In Chapter 4, I discuss efforts to engineer Escherichia coli ’s pyruvate dehydrogenase complex to specifically interface NMN+ in an effort to develop growth-coupled NMN+ reduction. Finally, in Chapter 5, I explore engineering the pyruvate dehydrogenase complex as a test-bed for extremely far-from-equilibrium redox biochemistry.

This work presents the first generation of a full infrastructure of using NMN+ as anorthogonal redox cofactor with its designated electron source and electron sink. The work presented here focuses on applications that directly address limitations in biomanufacturing. However, looking far forward, access to genetically encoded tools for precise control of reduction potential in specific metabolic processes could also be essential for applications that address challenges in human health by precise maintenance of the delicate redox patterns that support life. This work aims to be a foundation upon which future orthogonal metabolic systems are established.

    Creative Commons 'BY-NC-ND' version 4.0 license
    • Article
    • Peer Reviewed
    UC Irvine Previously Published Works (2020)
    We report an aerobic, growth-based selection platform founded on NADP(H) redox balance restoration in Escherichia coli, and we demonstrate its application in the high-throughput evolution of an oxygenase. A single round of selection followed by a facile growth assay enabled Pseudomonas aeruginosa 4-hydroxybenzoate hydroxylase (PobA) to efficiently hydroxylate both 4-hydroxybenzoic acid (4-HBA) and 3,4-dihydroxybenzoic acid (3,4-DHBA), two consecutive steps in gallic acid biosynthesis. Structural modeling suggests precise reorganization of active site hydrogen bond network, which is difficult to obtain without deep navigation of combinatorial sequence space. We envision universal application of this selection platform in engineering NADPH-dependent oxidoreductases.
      Cover page: A Growth-Based, High-Throughput Selection Platform Enables Remodeling of 4‑Hydroxybenzoate Hydroxylase Active SiteCreative Commons 'BY' version 4.0 license
      • Article
      • Peer Reviewed
      UC Irvine Previously Published Works (2021)
      Cyclohexanone monooxygenases (CHMO) consume molecular oxygen and NADPH to catalyze the valuable oxidation of cyclic ketones. However, CHMO usage is restricted by poor stability and stringent specificity for NADPH. Efforts to engineer CHMO have been limited by the sensitivity of the enzyme to perturbations in conformational dynamics and long-range interactions that cannot be predicted. We demonstrate an aerobic, high-throughput growth selection platform in Escherichia coli for oxygenase evolution based on NADH redox balance. We applied this NADH-dependent selection to alter the cofactor specificity of CHMO to accept NADH, a less expensive cofactor than NADPH. We first identified the variant CHMO DTNP (S208D-K326T-K349N-L143P) with a ∼1200-fold relative cofactor specificity switch from NADPH to NADH compared to the wild type through semirational design. Molecular modeling suggests CHMO DTNP activity is driven by cooperative fine-tuning of cofactor contacts. Additional evolution of CHMO DTNP through random mutagenesis yielded the variant CHMO DTNPY with a ∼2900-fold relative specificity switch compared to the wild type afforded by an additional distal mutation, H163Y. These results highlight the difficulty in engineering functionally innovative variants from static models and rational designs, and the need for high throughput selection methods. Our introduced tools for oxygenase engineering accelerate the advancements of characteristics essential for industrial feasibility.
        Cover page: Growth-Based, High-Throughput Selection for NADH Preference in an Oxygen-Dependent Biocatalyst.
        • Article
        • Peer Reviewed
        UC Irvine Previously Published Works (2020)

        Background

        Noncanonical redox cofactors are emerging as important tools in cell-free biosynthesis to increase the economic viability, to enable exquisite control, and to expand the range of chemistries accessible. However, these noncanonical redox cofactors need to be biologically synthesized to achieve full integration with renewable biomanufacturing processes.

        Results

        In this work, we engineered Escherichia coli cells to biosynthesize the noncanonical cofactor nicotinamide mononucleotide (NMN+), which has been efficiently used in cell-free biosynthesis. First, we developed a growth-based screening platform to identify effective NMN+ biosynthetic pathways in E. coli. Second, we explored various pathway combinations and host gene disruption to achieve an intracellular level of ~ 1.5 mM NMN+, a 130-fold increase over the cell's basal level, in the best strain, which features a previously uncharacterized nicotinamide phosphoribosyltransferase (NadV) from Ralstonia solanacearum. Last, we revealed mechanisms through which NMN+ accumulation impacts E. coli cell fitness, which sheds light on future work aiming to improve the production of this noncanonical redox cofactor.

        Conclusion

        These results further the understanding of effective production and integration of NMN+ into E. coli. This may enable the implementation of NMN+-directed biocatalysis without the need for exogenous cofactor supply.
          Cover page: Metabolic engineering of Escherichia coli for optimized biosynthesis of nicotinamide mononucleotide, a noncanonical redox cofactorCreative Commons 'BY' version 4.0 license
          • Article
          • Peer Reviewed
          UC Irvine Previously Published Works (2022)
          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.
            Cover page: Engineering Embden–Meyerhof–Parnas Glycolysis to Generate Noncanonical Reducing Power
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