Complete Oxidation of Xylose for Bioelectricity Generation by Reconstructing a Bacterial Xylose Utilization Pathway in vitro

Stoichiometric Conversion of Cellulosic Biomass by in Vitro Synthetic Enzymatic Biosystems for Biomanufacturing

Self-Powered Enzymatic Electrosynthesis of l-3,4-Dihydroxyphenylalanine in a Hybrid Bioelectrochemical System

Construction of Enzyme-Cofactor/Mediator Conjugates for Enhanced in Vitro Bioelectricity Generation

Coevolution of both Thermostability and Activity of Polyphosphate Glucokinase from Thermobifida fusca YX

ABSTRACT Thermostability and specific activity of enzymes are two of the most important properties for industrial biocatalysts. Here, we developed a petri dish-based double-layer high-throughput screening (HTS) strategy for rapid identification of desired mutants of polyphosphate glucokinase (PPGK) from a thermophilic actinobacterium, Thermobifida fusca YX, with both enhanced thermostability and activity. Escherichia coli colonies representing a PPGK mutant library were grown on the first-layer Phytagel-based plates, which can remain solid for 1 h, even at heat treatment temperatures of more than 100°C. The second layer that was poured on the first layer contained agarose, substrates, glucose 6-phosphate dehydrogenase (G6PDH), the redox dye tetranitroblue tetrazolium (TNBT), and phenazine methosulfate. G6PDH was able to oxidize the product from the PPGK-catalyzed reaction and generate NADH, which can be easily examined by a TNBT-based colorimetric assay. The best mutant obtained after four rounds of directed evolution had a 7,200-fold longer half-life at 55°C, 19.8°C higher midpoint of unfolding temperature (Tm), and a nearly 3-fold enhancement in specific activities compared to those of the wild-type PPGK. The best mutant was used to produce 9.98 g/liter myo-inositol from 10 g/liter glucose, with a theoretical yield of 99.8%, along with two other hyperthermophilic enzymes at 70°C. This PPGK mutant featuring both great thermostability and high activity would be useful for ATP-free production of glucose 6-phosphate or its derived products.IMPORTANCE Polyphosphate glucokinase (PPGK) is an enzyme that transfers a terminal phosphate group from polyphosphate to glucose, producing glucose 6-phosphate. A petri dish-based double-layer high-throughput screening strategy was developed by using ultrathermostable Phytagel as the first layer instead of agar or agarose, followed by a redox dye-based assay for rapid identification of ultrathermostable PPGK mutants. The best mutant featuring both great thermostability and high activity could produce glucose 6-phosphate from glucose and polyphosphate without in vitro ATP regeneration.

In vitro synthetic enzymatic biosystems at the interface of the food-energy-water nexus: A conceptual framework and recent advances

Co-utilization of mixed sugars in an enzymatic fuel cell based on an in vitro enzymatic pathway

Protein engineering of oxidoreductases utilizing nicotinamide-based coenzymes, with applications in synthetic biology

In vitro metabolic engineering of bioelectricity generation by the complete oxidation of glucose

Water Splitting for High-Yield Hydrogen Production Energized by Biomass Xylooligosaccharides Catalyzed by an Enzyme Cocktail

Coenzyme Engineering of a Hyperthermophilic 6-Phosphogluconate Dehydrogenase from NADP+ to NAD+ with Its Application to Biobatteries

Use of nonimmobilized enzymes and mediators achieved high power densities in closed biobatteries

A high-energy-density sugar biobattery based on a synthetic enzymatic pathway

Cell-free Biosystems in the Production of Electricity and Bioenergy

Cellulose solvent-based pretreatment for corn stover and avicel: concentrated phosphoric acid versus ionic liquid [BMIM]Cl

Deep oxidation of glucose in enzymatic fuel cells through a synthetic enzymatic pathway containing a cascade of two thermostable dehydrogenases

Cellulose solvent- and organic solvent-based lignocellulose fractionation enabled efficient sugar release from a variety of lignocellulosic feedstocks

One-step production of lactate from cellulose as the sole carbon source without any other organic nutrient by recombinant cellulolytic Bacillus subtilis

Biohydrogenation from Biomass Sugar Mediated by In Vitro Synthetic Enzymatic Pathways

Maltodextrin-powered enzymatic fuel cell through a non-natural enzymatic pathway

Fusion of a family 9 cellulose-binding module improves catalytic potential of Clostridium thermocellum cellodextrin phosphorylase on insoluble cellulose

Toward low-cost biomanufacturing through in vitro synthetic biology: bottom-up design

Bamboo saccharification through cellulose solvent-based biomass pretreatment followed by enzymatic hydrolysis at ultra-low cellulase loadings

Cellulose solvent-based biomass pretreatment breaks highly ordered hydrogen bonds in cellulose fibers of switchgrass

Increasing cellulose accessibility is more important than removing lignin: A comparison of cellulose solvent-based lignocellulose fractionation and soaking in aqueous ammonia

Comparative study of corn stover pretreated by dilute acid and cellulose solvent-based lignocellulose fractionation: Enzymatic hydrolysis, supramolecular structure, and substrate accessibility

Direct quantitative determination of adsorbed cellulase on lignocellulosic biomass with its application to study cellulase desorption for potential recycling

Efficient Sugar Release by the Cellulose Solvent-Based Lignocellulose Fractionation Technology and Enzymatic Cellulose Hydrolysis