Chun You

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

High-energy-density, green, safe batteries are highly desirable for meeting the rapidly growing needs of portable electronics. The incomplete oxidation of sugars mediated by one or a few enzymes in enzymatic fuel cells suffers from low energy densities and slow reaction rates. Here we show that nearly 24 electrons per glucose unit of maltodextrin can be produced through a synthetic catabolic pathway that comprises 13 enzymes in an air-breathing enzymatic fuel cell. This enzymatic fuel cell is based on non-immobilized enzymes that exhibit a maximum power output of 0.8 mW cm(-2) and a maximum current density of 6 mA cm(-2), which are far higher than the values for systems based on immobilized enzymes. Enzymatic fuel cells containing a 15% (wt/v) maltodextrin solution have an energy-storage density of 596 Ah kg(-1), which is one order of magnitude higher than that of lithium-ion batteries. Sugar-powered biobatteries could serve as next-generation green power sources, particularly for portable electronics.

Facilitated Substrate Channeling in a Self‐Assembled Trifunctional Enzyme Complex

Most cascade enzymes in metabolic pathways are spatially held together by noncovalent protein–protein interactions. The formation of a cascade enzyme complex often allows the product of one enzyme to be transferred to an adjacent enzyme where it acts as the substrate, thereby resulting in an enhanced reaction rate, because reaching equilibrium in the cytoplasm is not required; this mechanism is called substrate channeling. In nature, most intracellular enzyme complexes are dynamic so that they may be dissociated or associated, thereby resulting in forestallment of substrate competition among different pathways, regulation of metabolic fluxes, mitigation of metabolite inhibition, and circumvention of unfavorable equilibrium and kinetics. The simplest way to facilitate substrate channeling between cascade enzymes is the construction of fusion proteins, but substrate channeling in fusion proteins might not take place. The assembly of numerous enzymes and/or co-enzymes in vitro is called cascade enzyme biocatalysis and has been proposed for the implementation of complicated bioconversion that microbes and chemical catalysts cannot do, such as hydrogen production from cellulosic materials and water with high yield. Inspired by natural enzyme complexes (e.g., metabolons, which are complexes of sequential enzymes of a metabolic pathway), the construction of static rather than dynamic enzyme complexes could be an important approach to accelerating reaction rates among cascade enzymes and to avoiding the regulation of enzyme–enzyme interactions. For example, Wilner et al. linked glucose oxidase and horseradish peroxidase by DNA scaffolds of different lengths, resulting in reaction rates that were enhanced by 20–30-fold. However, DNA scaffolds may be too costly for scale-up as compared to protein scaffolds. Minteer and co-workers demonstrated that chemical cross-linking of proteins within the mitochondria of Saccharomyces cerevisiae resulted in significant increases of the power output in enzymatic fuel cells. But chemical covalent linking often impairs enzyme activity so that it may not be applied to most intracellular enzymes. Herein we demonstrate a general approach for constructing a static self-assembled enzyme complex by using the highaffinity interaction between cohesin and dockerin modules, which occur in natural extracellular complexed cellulase systems, called cellulosomes. Cohesin domains are part of the natural scaffoldin protein of the cellulosome, which is crucial to the construction of the cellulase complex by binding to enzymes carrying dockerin domains. Bayer et al. proposed to construct designed enzyme complexes by utilizing speciesspecific dockerins and cohesins, which can bind tightly in these complexes at a molar ratio of 1:1. Later, several synthetic mini-cellulosomes containing various extracellular glycoside hydrolases were constructed. However, no one attempted to construct an enzyme complex containing cascade enzymes from a metabolic pathway by using dockerins and cohesins and investigated its potential applications in cascade enzyme biocatalysis. Triosephosphate isomerase (TIM, EC 5.3.1.1), aldolase (ALD, EC 4.1.2.13), and fructose 1,6-bisphosphatase (FBP, EC3.1.3.11) are cascade enzymes in the glycolysis and gluconeogenesis pathways. TIM catalyzes the reversible conversion of glycer-aldehyde-3-phosphate (G3P) to dihydroxy-acetone phosphate (DHAP). ALD catalyzes the reversible aldol condensation of G3P and DHAP to fructose-1,6bisphosphate (F16P). FBP catalyzes the irreversible conversion of F16P to fructose-6-phosphate (F6P; Scheme 1). Previous studies reported that substrate channeling existed in dynamic metabolons of enzymes such as TIM, ALD, or FBP. Three dockerin-free proteins: Thermus thermophilus HB27 TIM (TTC0581) as well as the Thermotoga maritima ALD (TM0273) and FBP (TM1415) were expressed in E. coli and purified to homogeneity by using nickel–nitrilotriacetate (Ni–NTA) resin or a self-cleaving intein. However, a mixture of these three enzymes did not form a putative enzyme complex, as examined by affinity electrophoresis (data not shown). The synthetic static three-enzyme complex was assembled in vitro through a synthetic trifunctional scaffoldin containing a family 3 cellulose-binding module (CBM3) at the N terminus followed by three different types of cohesins from the Clostridium thermocellum ATCC 27405 CipA, Clostridium cellulovorans ATCC 35296 CbpA, and Ruminococcus flavefaciens ScaB (cohesins CTCoh, CCCoh, and RFCoh, [*] Dr. C. You, S. Myung, Y.-H. P. Zhang Biological Systems Engineering Department Virginia Tech, 304 Seitz Hall Blacksburg, VA 24061 (USA) E-mail: ypzhang@vt.edu Homepage: http://www.sugarcar.com

High-yield hydrogen production from biomass by in vitro metabolic engineering: Mixed sugars coutilization and kinetic modeling

Significance Hydrogen (H2) has great potential to be used to power passenger vehicles. One solution to these problems is to distribute and store renewable carbohydrate instead, converting it to hydrogen as required. In this work more than 10 purified enzymes were combined into artificial enzymatic pathways and a high yield from both glucose and xylose to hydrogen was achieved. Also, gaseous hydrogen can be separated from aqueous substrates easily, greatly decreasing product separation costs, and avoid reconcentrating sugar solutions. This study describes high-yield enzymatic hydrogen production from biomass sugars and an engineered reaction rate increase achieved through the use of kinetic modeling. Distributed hydrogen production based on evenly distributed less-costly biomass could accelerate the implementation of the hydrogen economy. The use of hydrogen (H2) as a fuel offers enhanced energy conversion efficiency and tremendous potential to decrease greenhouse gas emissions, but producing it in a distributed, carbon-neutral, low-cost manner requires new technologies. Herein we demonstrate the complete conversion of glucose and xylose from plant biomass to H2 and CO2 based on an in vitro synthetic enzymatic pathway. Glucose and xylose were simultaneously converted to H2 with a yield of two H2 per carbon, the maximum possible yield. Parameters of a nonlinear kinetic model were fitted with experimental data using a genetic algorithm, and a global sensitivity analysis was used to identify the enzymes that have the greatest impact on reaction rate and yield. After optimizing enzyme loadings using this model, volumetric H2 productivity was increased 3-fold to 32 mmol H2⋅L−1⋅h−1. The productivity was further enhanced to 54 mmol H2⋅L−1⋅h−1 by increasing reaction temperature, substrate, and enzyme concentrations—an increase of 67-fold compared with the initial studies using this method. The production of hydrogen from locally produced biomass is a promising means to achieve global green energy production.

Enzymatic transformation of nonfood biomass to starch

The global demand for food could double in another 40 y owing to growth in the population and food consumption per capita. To meet the world’s future food and sustainability needs for biofuels and renewable materials, the production of starch-rich cereals and cellulose-rich bioenergy plants must grow substantially while minimizing agriculture’s environmental footprint and conserving biodiversity. Here we demonstrate one-pot enzymatic conversion of pretreated biomass to starch through a nonnatural synthetic enzymatic pathway composed of endoglucanase, cellobiohydrolyase, cellobiose phosphorylase, and alpha-glucan phosphorylase originating from bacterial, fungal, and plant sources. A special polypeptide cap in potato alpha-glucan phosphorylase was essential to push a partially hydrolyzed intermediate of cellulose forward to the synthesis of amylose. Up to 30% of the anhydroglucose units in cellulose were converted to starch; the remaining cellulose was hydrolyzed to glucose suitable for ethanol production by yeast in the same bioreactor. Next-generation biorefineries based on simultaneous enzymatic biotransformation and microbial fermentation could address the food, biofuels, and environment trilemma.

Simple Cloning via Direct Transformation of PCR Product (DNA Multimer) to Escherichia coli and Bacillus subtilis

ABSTRACT We developed a general restriction enzyme-free and ligase-free method for subcloning up to three DNA fragments into any location of a plasmid. The DNA multimer generated by prolonged overlap extension PCR was directly transformed in Escherichia coli [e.g., TOP10, DH5α, JM109, and BL21(DE3)] and Bacillus subtilis for obtaining chimeric plasmids.

Enhanced Microbial Utilization of Recalcitrant Cellulose by an Ex Vivo Cellulosome-Microbe Complex

ABSTRACT A cellulosome-microbe complex was assembled ex vivo on the surface of Bacillus subtilis displaying a miniscaffoldin that can bind with three dockerin-containing cellulase components: the endoglucanase Cel5, the processive endoglucanase Cel9, and the cellobiohydrolase Cel48. The hydrolysis performances of the synthetic cellulosome bound to living cells, the synthetic cellulosome, a noncomplexed cellulase mixture with the same catalytic components, and a commercial fungal enzyme mixture were investigated on low-accessibility recalcitrant Avicel and high-accessibility regenerated amorphous cellulose (RAC). The cell-bound cellulosome exhibited 4.5- and 2.3-fold-higher hydrolysis ability than cell-free cellulosome on Avicel and RAC, respectively. The cellulosome-microbe synergy was not completely explained by the removal of hydrolysis products from the bulk fermentation broth by free-living cells and appeared to be due to substrate channeling of long-chain hydrolysis products assimilated by the adjacent cells located in the boundary layer. Our results implied that long-chain hydrolysis products in the boundary layer may inhibit cellulosome activity to a greater extent than the short-chain products in bulk phase. The findings that cell-bound cellulosome expedited the microbial cellulose utilization rate by 2.3- to 4.5-fold would help in the development of better consolidated bioprocessing microorganisms (e.g., B. subtilis) that can hydrolyze recalcitrant cellulose rapidly at low secretory cellulase levels.

In vitro metabolic engineering of hydrogen production at theoretical yield from sucrose

Hydrogen is one of the most important industrial chemicals and will be arguably the best fuel in the future. Hydrogen production from less costly renewable sugars can provide affordable hydrogen, decrease reliance on fossil fuels, and achieve nearly zero net greenhouse gas emissions, but current chemical and biological means suffer from low hydrogen yields and/or severe reaction conditions. An in vitro synthetic enzymatic pathway comprised of 15 enzymes was designed to split water powered by sucrose to hydrogen. Hydrogen and carbon dioxide were spontaneously generated from sucrose or glucose and water mediated by enzyme cocktails containing up to 15 enzymes under mild reaction conditions (i.e. 37°C and atm). In a batch reaction, the hydrogen yield was 23.2mol of dihydrogen per mole of sucrose, i.e., 96.7% of the theoretical yield (i.e., 12 dihydrogen per hexose). In a fed-batch reaction, increasing substrate concentration led to 3.3-fold enhancement in reaction rate to 9.74mmol of H2/L/h. These proof-of-concept results suggest that catabolic water splitting powered by sugars catalyzed by enzyme cocktails could be an appealing green hydrogen production approach.

Potential hydrophobic interaction between two cysteines in interior hydrophobic region improves thermostability of a family 11 xylanase from Neocallimastix patriciarum.

In this study, we employed directed evolution and site‐directed mutagenesis to screen thermostable mutants of a family 11 xylanase from Neocallimastix patriciarum, and found that the thermostability and specific activity are both enhanced when mutations (G201C and C60A) take place in the interior hydrophobic region of the enzyme. Far‐ultraviolet circular dichroism analysis showed that the melting temperatures (Tm) of the G201C and C60A–G201C mutants are higher than that of the wild type by about 10 and 12°C, respectively. At 72°C, their specific activities are about 4 and 6 times as that of the wild type, respectively. Homology modeling and site‐directed mutagenesis demonstrated that the enhanced thermostability of the G201C and C60A–G201C mutants may be mainly attributed to a potential stronger hydrophobic interaction between the two well‐packed cysteines at sites 50 and 201, rather than the disulfide bond formation which was ruled out by thiol titration with dithionitrobenzoic acid (DTNB). And the strength of such interaction depends on the packing of the side‐chain and hydrophobicity of residues at these two sites. This suggests that cysteine could stabilize a protein not only by forming a disulfide bond, but also by the strong hydrophobicity itself. Biotechnol. Bioeng. 2010;105: 861–870.

Beta‐xylosidase activity of a GH3 glucosidase/xylosidase from yak rumen metagenome promotes the enzymatic degradation of hemicellulosic xylans

Aims:  To characterize the duel activities of a glycosyl hydrolase family 3 β‐glucosidase/xylosidase from rumen bacterial metagenome and to investigate the capabilities of its β‐d‐xylosidase activities for saccharification of hemicellulosic xylans.

Cell-Free Biosystems for Biomanufacturing

Although cell-free biosystems have been used as a tool for investigating fundamental aspects of biological systems for more than 100 years, they are becoming an emerging biomanufacturing platform in the production of low-value biocommodities (e.g., H(2), ethanol, and isobutanol), fine chemicals, and high-value protein and carbohydrate drugs and their precursors. Here we would like to define the cell-free biosystems containing more than three catalytic components in a single reaction vessel, which although different from one-, two-, or three-enzyme biocatalysis can be regarded as a straightforward extension of multienzymatic biocatalysis. In this chapter, we compare the advantages and disadvantages of cell-free biosystems versus living organisms, briefly review the history of cell-free biosystems, highlight a few examples, analyze any remaining obstacles to the scale-up of cell-free biosystems, and suggest potential solutions. Cell-free biosystems could become a disruptive technology to microbial fermentation, especially in the production of high-impact low-value biocommodities mainly due to the very high product yields and potentially low production costs.