Browsing by Author "Kim, Jae Eung"

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    High-Throughput Screening of Coenzyme Preference Change of Thermophilic 6-Phosphogluconate Dehydrogenase from NADP⁺ to NAD⁺
    Huang, Rui; Chen, Hui; Zhong, Chao; Kim, Jae Eung; Zhang, Yi-Heng Percival (Nature, 2016-09-02)
    Coenzyme engineering that changes NAD(P) selectivity of redox enzymes is an important tool in metabolic engineering, synthetic biology, and biocatalysis. Here we developed a high throughput screening method to identify mutants of 6-phosphogluconate dehydrogenase (6PGDH) from a thermophilic bacterium Moorella thermoacetica with reversed coenzyme selectivity from NADP⁺ to NAD⁺. Colonies of a 6PGDH mutant library growing on the agar plates were treated by heat to minimize the background noise, that is, the deactivation of intracellular dehydrogenases, degradation of inherent NAD(P)H, and disruption of cell membrane. The melted agarose solution containing a redox dye tetranitroblue tetrazolium (TNBT), phenazine methosulfate (PMS), NAD⁺, and 6-phosphogluconate was carefully poured on colonies, forming a second semi-solid layer. More active 6PGDH mutants were examined via an enzyme-linked TNBT-PMS colorimetric assay. Positive mutants were recovered by direct extraction of plasmid from dead cell colonies followed by plasmid transformation into E. coli TOP10. By utilizing this double-layer screening method, six positive mutants were obtained from two-round saturation mutagenesis. The best mutant 6PGDH A30D/R31I/T32I exhibited a 4,278-fold reversal of coenzyme selectivity from NADP⁺ to NAD⁺. This screening method could be widely used to detect numerous redox enzymes, particularly for thermophilic ones, which can generate NAD(P)H reacted with the redox dye TNBT.
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    In Vitro Synthetic Biology Platform and Protein Engineering for Biorefinery
    Kim, Jae Eung (Virginia Tech, 2017-07-17)
    In order to decrease our dependence on non-renewable petrochemical resources, it is urgently required to establish sustainable biomass-based biorefineries. Replacing fossil fuels with renewable biomass as a raw feedstock for the production of chemicals and biofuels is a main driving force of biorefinering. Almost all kinds of biomass can be converted to biochemicals, biomaterials and biofuels via continuing advances on conversion technologies. In vitro synthetic biology is an emergent biomanufacturing platform that circumvents cellular constraints so that it can implement some biotransformations better than whole-cell fermentation, which spends a fraction of energy and carbon sources for cellular duplication and side-product formation. In this work, the in vitro synthetic (enzymatic) biosystem is used to produce a future carbon-neutral transportation fuel, hydrogen, and two high-value chemicals, a sugar phosphate and a highly marketable sweetener, representing a new portfolio for new biorefineries. Hydrogen gas is a promising future energy carrier as a transportation fuel, offering a high energy conversion efficiency via fuel cells, nearly zero pollutants produced to end users, and high mass-specific and volumetric energy densities compared to rechargeable batteries. Distributed production of cost-competitive green hydrogen from renewable biomass will be vital to the hydrogen economy. Substrate costs contribute to a major portion of the production cost for low-value bulk biocommodities, such as hydrogen. The reconstitution of 17 thermophilic enzymes enabled to construct an artificial enzymatic pathway converting all glucose units of starch, regardless of the branched and linear contents, to hydrogen gas at a theoretic yield (i.e., 12 H2 per glucose), three times of the theoretical yield from dark microbial fermentation. Using a biomimetic electron transport chain, a maximum volumetric productivity was increased by more than 200-fold to 90.2 mmol of H2/L/h at a high starch concentration from the original study in 2007. In order to promote economics of biorefineries, the production of a sugar phosphate and a fourth-generation sweetener is under development. D-xylulose 5-phosphate (Xu5P), which cannot be prepared efficiently by regular fermentation due to the negatively charged and hydrophilic phosphate groups, was synthesized from D-xylose and polyphosphate via a minimized two-enzyme system using a promiscuous activity of xylulose kinase. Under the optimized condition, 32 mM Xu5P was produced from 50 mM xylose and polyphosphate, achieving a 64% conversion yield, after 36 h at 45 °C. L-arabinose, a FDA-approved zero-calorie sweetener, was produced from D-xylose via a novel enzymatic pathway consisting of xylose isomerase, L-arabinose isomerase and xylulose 4-epimerase (Xu4E). Promiscuous activity of Xu4E, a monosaccharide C4-epimerase, was discovered for the first time. Directed evolution of Xu4E enabled to increase the catalytic function of C4-epimerization on D-xylulose as a substrate by more than 29-fold from the wild-type enzyme. Together, these results demonstrate that the in vitro synthetic biosystem as a feasible biomanufacturing platform has great engineering, and can be used to convert renewable biomass resources to a spectrum of marketable products and renewable energy. As future efforts are addressed to overcome remaining challenges, for example, decreasing enzyme production costs, prolonging enzyme lifetime, engineering biomimetic coenzymes to replace natural coenzymes, and so on. This in vitro synthetic biology platform would become a cornerstone technology for biorefinery industries and advanced biomanufacturing (Biomanufacturing 4.0).