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Open Access Publications from the University of California

Scholarly Works (11 results)

  • Thesis
  • Peer Reviewed
UC Irvine Electronic Theses and Dissertations (2021)

Noncanonical redox cofactors, or cofactor mimics, which operate in an orthogonal manner to the natural counterpart nicotinamide adenine dinucleotide (phosphate), NAD(P)+, in the cells, are considered promising tools to increase the specificity and decrease the cost of biomanufacturing. While the reversing of enzymatic nicotinamide-based coenzyme specificity of all NAD(P)-utilizing enzymes has become a mature technology, shifting enzymes’ cofactor preferences towards noncanonical redox cofactors remains a challenging task. In this work, we demonstrated the development and application of growth-based, high-throughput selection platforms based on redox balance. These selection platforms enable generic and efficient engineering of NMN(H)-dependent enzymes through directed evolution.First, we developed a selection platform based on the NADPH-dependent redox balance to elucidate the relationship between the redox balance principle and the growth of Escherichia coli (E. coli). In this part, we switched the cofactor preference of glycolysis by introducing a heterologous NADPH-generating glyceraldehyde-3-phosphate dehydrogenase (GapDH) into a gapA deficient E. coli strain. The growth of the resulting strain specifically depended on the heterologously-introduced NADPH-oxidizing reactions under anaerobic condition, and automatically eliminated nonfunctional variants. This part of work served as a proof of concept, and we applied this principle for having extensive application in the directed evolution of NMN(H)-dependent redox enzymes. Next, we attempted to engineer redox enzymes to favor NMN+ under the guidance of computational simulation. After three rounds of rational design, we engineered glucose dehydrogenase (GDH) from Bacillus megaterium to specifically reduce nicotinamide mononucleotide (NMN+) using glucose. The resultant mutant (GDH Ortho) had an overall specificity switch of ~2 × 107- and ~1 × 107-fold toward NMN+ from NAD+ and NADP+, respectively, based on apparent kinetic parameters. The activity of GDH Ortho can be linked to the growth rate of cells by introducing the GDH Ortho along with the Entner–Doudoroff pathway (ED pathway) into strain lacking the Embden–Meyerhof–Parnas (EMP) pathway and pentose phosphate pathway (PPP). The resulting strain allowed us to develop a high-throughput selection method for obtaining NMN+-dependent enzymes through directed evolution. Moreover, as many redox enzymes share key structural features, the engineering strategy in GDH Ortho may be adopted to devise additional NMN(H)-dependent redox enzymes. Finally, we utilized our experience in NMN-dependent protein engineering and redox balance-based selection construction to develop an efficient selection method to obtain NMN(H)-dependent enzymes. In this selection platform, a life-essential enzyme, glutathione reductase (Gor) was engineered to specifically require the noncanonical reducing power through NMNH. This enzyme links thiol-disulfide balance-dependent cell growth and the intracellular NMNH level, which enables the selection of NMN+ reducing enzymes in directed evolution. After optimization and characterization, the selection platform was applied to engineer an efficient NMNH-generation phosphite dehydrogenase (TS-PTDH), which is a widely used enzyme for reducing power generation in industrial bioprocesses. Compared with wild type TS-PTDH, the best mutant A155N-E175A-A176F obtained from selection exhibited about 146-fold higher apparent catalytic efficiency (kcat / Km) when using NMN+ as the cofactor. Combined with computational library design, this selection platform will allow the broader bioengineering community to make their own NMN(H)-utilizing enzymes in a streamlined fashion.

    • Article
    • Peer Reviewed
    UC Irvine Previously Published Works (2018)
    Bacteria undergoing anaerobic fermentation must maintain redox balance. In vivo metabolic evolution schemes based on this principle have been limited to targeting NADH-dependent reactions. Here, we developed a facile, specific, and high-throughput growth-based selection platform for NADPH-consuming reactions in vivo, based on an engineered NADPH-producing glycolytic pathway in Escherichia coli. We used the selection system in the directed evolution of a NADH-dependent d-lactate dehydrogenase from Lactobacillus delbrueckii toward utilization of NADPH. Through one round of selection, we obtained multiple enzyme variants with superior NADPH-dependent activities and protein expression levels; these mutants may serve as important tools in biomanufacturing d-lactate as a renewable polymer building block. Importantly, sequence analysis and computational protein modeling revealed that diverging evolutionary paths during the selection resulted in two distinct cofactor binding modes, which suggests that the high throughput of our selection system allowed deep searching of protein sequence space to discover diverse candidates en masse.
      Cover page: Development of a High-Throughput, In Vivo Selection Platform for NADPH-Dependent Reactions Based on Redox Balance Principles
      • Article
      • Peer Reviewed
      UC Irvine Previously Published Works (2018)
      Bacteria undergoing anaerobic fermentation must maintain redox balance. In vivo metabolic evolution schemes based on this principle have been limited to targeting NADH-dependent reactions. Here, we developed a facile, specific, and high-throughput growth-based selection platform for NADPH-consuming reactions in vivo, based on an engineered NADPH-producing glycolytic pathway in Escherichia coli. We used the selection system in the directed evolution of a NADH-dependent d-lactate dehydrogenase from Lactobacillus delbrueckii toward utilization of NADPH. Through one round of selection, we obtained multiple enzyme variants with superior NADPH-dependent activities and protein expression levels; these mutants may serve as important tools in biomanufacturing d-lactate as a renewable polymer building block. Importantly, sequence analysis and computational protein modeling revealed that diverging evolutionary paths during the selection resulted in two distinct cofactor binding modes, which suggests that the high throughput of our selection system allowed deep searching of protein sequence space to discover diverse candidates en masse.
        Cover page: Development of a High-Throughput, In Vivo Selection Platform for NADPH-Dependent Reactions Based on Redox Balance PrinciplesCreative Commons 'BY' 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 cofactor
              • Article
              • Peer Reviewed
              UC Irvine Previously Published Works (2020)
              Cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIMB 9871 is characterized as having wide substrate versatility for the biooxidation of (cyclic) ketones into esters and lactones with high stereospecificity. Despite industrial potential, CHMO usage is restricted by poor thermostability. Limited high-throughput screening tools and challenges in rationally engineering thermostability have impeded CHMO engineering efforts. We demonstrate the application of an aerobic, high-throughput growth selection platform in Escherichia coli (strain MX203) for the discovery of thermostability enhancing mutations for CHMO. The selection employs growth for the easy readout of CHMO activity in vivo, by requiring nicotinamide adenine dinucleotide phosphate (NADPH)-consuming enzymes to restore cellular redox balance. In the presence of the native substrate cyclohexanone, variant CHMO GV (A245G-A288V) was discovered from a random mutagenesis library screened at 42 °C. This variant retained native activity, exhibited ~4.4-fold improvement in residual activity after 30 °C incubation, and demonstrated ~5-fold higher cyclohexanone conversion at 37 °C compared to the wild type. Molecular modeling indicates that CHMO GV experiences more favorable residue packing and supports additional backbone hydrogen bonding. Further rational design resulted in CHMO A245G-A288V-T415C with improved thermostability at 45 °C. Our platform for oxygenase evolution enabled the rapid engineering of protein stability critical for industrial scalability.
                Cover page: In Vivo, High-Throughput Selection of Thermostable Cyclohexanone Monooxygenase (CHMO).
                • Article
                • Peer Reviewed
                UC Irvine Previously Published Works (2020)
                Biological production of chemicals often requires the use of cellular cofactors, such as nicotinamide adenine dinucleotide phosphate (NADP+). These cofactors are expensive to use in vitro and difficult to control in vivo. We demonstrate the development of a noncanonical redox cofactor system based on nicotinamide mononucleotide (NMN+). The key enzyme in the system is a computationally designed glucose dehydrogenase with a 107-fold cofactor specificity switch toward NMN+ over NADP+ based on apparent enzymatic activity. We demonstrate that this system can be used to support diverse redox chemistries in vitro with high total turnover number (~39,000), to channel reducing power in Escherichia coli whole cells specifically from glucose to a pharmaceutical intermediate, levodione, and to sustain the high metabolic flux required for the central carbon metabolism to support growth. Overall, this work demonstrates efficient use of a noncanonical cofactor in biocatalysis and metabolic pathway design.
                  Cover page: Engineering a nicotinamide mononucleotide redox cofactor system for biocatalysis
                  • Article
                  • Peer Reviewed
                  UC Irvine Previously Published Works (2022)
                  Noncanonical redox cofactors are attractive low-cost alternatives to nicotinamide adenine dinucleotide (phosphate) (NAD(P)+) in biotransformation. However, engineering enzymes to utilize them is challenging. Here, we present a high-throughput directed evolution platform which couples cell growth to the in vivo cycling of a noncanonical cofactor, nicotinamide mononucleotide (NMN+). We achieve this by engineering the life-essential glutathione reductase in Escherichia coli to exclusively rely on the reduced NMN+ (NMNH). Using this system, we develop a phosphite dehydrogenase (PTDH) to cycle NMN+ with ~147-fold improved catalytic efficiency, which translates to an industrially viable total turnover number of ~45,000 in cell-free biotransformation without requiring high cofactor concentrations. Moreover, the PTDH variants also exhibit improved activity with another structurally deviant noncanonical cofactor, 1-benzylnicotinamide (BNA+), showcasing their broad applications. Structural modeling prediction reveals a general design principle where the mutations and the smaller, noncanonical cofactors together mimic the steric interactions of the larger, natural cofactors NAD(P)+.
                    Cover page: Directed evolution of phosphite dehydrogenase to cycle noncanonical redox cofactors via universal growth selection platformCreative Commons 'BY' version 4.0 license
                    • Article
                    • Peer Reviewed
                    UC Irvine Previously Published Works (2020)
                    Biological production of chemicals often requires the use of cellular cofactors, such as nicotinamide adenine dinucleotide phosphate (NADP+). These cofactors are expensive to use in vitro and difficult to control in vivo. We demonstrate the development of a noncanonical redox cofactor system based on nicotinamide mononucleotide (NMN+). The key enzyme in the system is a computationally designed glucose dehydrogenase with a 107-fold cofactor specificity switch toward NMN+ over NADP+ based on apparent enzymatic activity. We demonstrate that this system can be used to support diverse redox chemistries in vitro with high total turnover number (~39,000), to channel reducing power in Escherichia coli whole cells specifically from glucose to a pharmaceutical intermediate, levodione, and to sustain the high metabolic flux required for the central carbon metabolism to support growth. Overall, this work demonstrates efficient use of a noncanonical cofactor in biocatalysis and metabolic pathway design.
                      Cover page: Engineering a nicotinamide mononucleotide redox cofactor system for biocatalysis.Creative Commons 'BY' version 4.0 license
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