Gate Fuels Inc.

Gate City, VA, United States

Gate Fuels Inc.

Gate City, VA, United States
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Zhang Y.-H.P.,Virginia Polytechnic Institute and State University | Zhang Y.-H.P.,Gate Fuels Inc. | Huang W.-D.,Virginia Polytechnic Institute and State University | Huang W.-D.,Hefei University of Technology
Trends in Biotechnology | Year: 2012

In this opinion, we suggest the electricity-carbohydrate-hydrogen (ECHo) cycle which bridges primary energies and secondary energies. Carbohydrates are sources of food, feed, liquid biofuels, and renewable materials and are a high-density hydrogen carrier and electricity storage compounds (e.g. >3000 Wh/kg). One element of this ECHo cycle can be converted to another reversibly and efficiently depending on resource availability, needs and costs. This cycle not only supplements current and future primary energy utilization systems for facilitating electricity and hydrogen storage and enhancing secondary energy conversion efficiencies, but also addresses such sustainability challenges as transportation fuel production, CO 2 utilization, fresh water conservation, and maintenance of a small closed ecosystem in emergency situations. © 2012 Elsevier Ltd.


You C.,Virginia Polytechnic Institute and State University | Myung S.,Virginia Polytechnic Institute and State University | Zhang Y.-H.P.,Virginia Polytechnic Institute and State University | Zhang Y.-H.P.,U.S. Department of Energy | Zhang Y.-H.P.,Gate Fuels Inc.
Angewandte Chemie - International Edition | Year: 2012

Three enzymes, triosephosphate isomerase (orange in picture), aldolase (cyan), and fructose 1,6-bisphosphatase (purple), which contained dockerins (red), self-assembled into a static trifunctional enzyme complex through interaction with a mini-scaffoldin protein consisting of three different cohesins (green). The synthetic enzyme complex exhibited an enhanced reaction rate compared to the noncomplexed three-enzyme mixture at the same enzyme concentration. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


Myung S.,Virginia Polytechnic Institute and State University | Zhang Y.-H.P.,Virginia Polytechnic Institute and State University | Zhang Y.-H.P.,Cell-Free Bioinnovations Inc. | Zhang Y.-H.P.,Gate Fuels Inc.
PLoS ONE | Year: 2013

Cell-free biosystems comprised of synthetic enzymatic pathways would be a promising biomanufacturing platform due to several advantages, such as high product yield, fast reaction rate, easy control and access, and so on. However, it was essential to produce (purified) enzymes at low costs and stabilize them for a long time so to decrease biocatalyst costs. We studied the stability of the four recombinant enzyme mixtures, all of which originated from thermophilic microorganisms: triosephosphate isomerase (TIM) from Thermus thermophiles, fructose bisphosphate aldolase (ALD) from Thermotoga maritima, fructose bisphosphatase (FBP) from T. maritima, and phosphoglucose isomerase (PGI) from Clostridium thermocellum. It was found that TIM and ALD were very stable at evaluated temperature so that they were purified by heat precipitation followed by gradient ammonia sulfate precipitation. In contrast, PGI was not stable enough for heat treatment. In addition, the stability of a low concentration PGI was enhanced by more than 25 times in the presence of 20 mg/L bovine serum albumin or the other three enzymes. At a practical enzyme loading of 1000 U/L for each enzyme, the half-life time of free PGI was prolong to 433 h in the presence of the other three enzymes, resulting in a great increase in the total turn-over number of PGI to 6.2×109 mole of product per mole of enzyme. This study clearly suggested that the presence of other proteins had a strong synergetic effect on the stabilization of the thermolabile enzyme PGI due to in vitro macromolecular crowding effect. Also, this result could be used to explain why not all enzymes isolated from thermophilic microorganisms are stable in vitro because of a lack of the macromolecular crowding environment. © 2013 Myung and Zhang.


Zhang Y.-H.P.,Virginia Polytechnic Institute and State University | Zhang Y.-H.P.,U.S. Department of Energy | Zhang Y.-H.P.,Gate Fuels Inc.
Process Biochemistry | Year: 2011

Since biofuels is a hot topic, many researchers new to this field are eager to propose different solutions while they often seem not to have full understanding of the current status of technologies and numerous (hidden) constraints. As a result, the general public, policymakers, academic researchers, and industrial developers have been assaulted by a wave of biased, misinterpreted, or outright false information. In reality, only a small fraction of exploding biofuels R&D teams are addressing vital rather than trivial challenges associated with economically production of advanced biofuels. Biofuels R&D is not a completely basic science project; instead, it is a typical goal-oriented (engineering) project because so many constraints prevent economically competitive production of most advanced biofuels and are expected to do so in the future. In this opinion paper, I present some basic rules and facts in thermodynamics, physical chemistry, and special constraints in the transport sector, sort through and challenge some claimed breakthroughs or new directions, and identify vital topics to advance biofuels in the short and long terms. Simply speaking, energy efficiency is the most important long-term criterion whereas cost is the most important short-term criterion; eventually thermodynamics determines economics. For light-duty passenger vehicles, which consume ∼60% transportation fuels, cellulosic ethanol and butanol are the best short- and middle-term biofuels, whereas sugary hydrogen would be the ultimate biofuel in the long term. The top three priorities of biofuels R&D are (i) cost-effective release of sugars from lignocellulose, (ii) co-utilization of lignocellulose components for the production of value-added compounds that subsidize whole biorefineries, and (iii) enhancing the biomass-to-kinetic energy efficiency from conversions to prime movers through a potential evolutionary scenario from ethanol or butanol/internal combustion engines (ICE) to ethanol/hybrid diesel-like ICE to sugar hydrogen fuel cell vehicles. © 2011 Elsevier Ltd. All rights reserved.


Zhang Y.-H.P.,Virginia Polytechnic Institute and State University | Zhang Y.-H.P.,U.S. Department of Energy | Zhang Y.-H.P.,Gate Fuels Inc.
Biotechnology Advances | Year: 2011

Substrate channeling is a process of transferring the product of one enzyme to an adjacent cascade enzyme or cell without complete mixing with the bulk phase. Such phenomena can occur in vivo, in vitro, or ex vivo. Enzyme-enzyme or enzyme-cell complexes may be static or transient. In addition to enhanced reaction rates through substrate channeling in complexes, numerous potential benefits of such complexes are protection of unstable substrates, circumvention of unfavorable equilibrium and kinetics imposed, forestallment of substrate competition among different pathways, regulation of metabolic fluxes, mitigation of toxic metabolite inhibition, and so on. Here we review numerous examples of natural and synthetic complexes featuring substrate channeling. Constructing synthetic in vivo, in vitro or ex vivo complexes for substrate channeling would have great biotechnological potentials in metabolic engineering, multi-enzyme-mediated biocatalysis, and cell-free synthetic pathway biotransformation (SyPaB). © 2011 Elsevier Inc.


You C.,Virginia Polytechnic Institute and State University | Percival Zhang Y.-H.,Virginia Polytechnic Institute and State University | Percival Zhang Y.-H.,Gate Fuels Inc.
Advances in Biochemical Engineering/Biotechnology | Year: 2013

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.,H2, 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. © Springer-Verlag Berlin Heidelberg 2012.


Zhang Y.-H.P.,Virginia Polytechnic Institute and State University | Zhang Y.-H.P.,U.S. Department of Energy | Zhang Y.-H.P.,Gate Fuels Inc.
ACS Catalysis | Year: 2011

The production of biofuels from renewable sugars isolated from plants or produced through artificial photosynthesis would provide a sustainable transportation fuel alternative for decreasing reliance on crude oil, mitigating greenhouse gas emissions, creating new manufacturing jobs, and enhancing national energy security. Since sugar costs usually account for at least 50% of biofuels selling prices, it is vital to produce desired biofuels with high product yields and at low production costs. Here I suggest high-product yield and potentially low-cost biofuels production through cell-free synthetic enzymatic pathway biotransformation (SyPaB) by in vitro assembly of stable enzymes and (biomimetic) coenzymes. SyPaB can achieve high product yields or high energy efficiencies that living entities cannot achieve. Great potentials of SyPaB, from chiral compounds, biodegradable sugar batteries, sulfur-free jet fuel, hydrogen, sugar hydrogen fuel cell vehicles, high-density electricity storage, to synthetic starch, are motivation to solve the remaining obstacles by using available technologies, such as protein engineering, enzyme immobilization, unit operations, and technology integration. The biotransformation through in vitro assembly of numerous enhanced-performance and stable enzymes in one bioreactor that can last a very long reaction time (e.g., several months or even years) would be an out-of-the-box solution for high-yield and low-cost biofuels production. © 2011 American Chemical Society.


You C.,Virginia Polytechnic Institute and State University | Zhang Y.-H.P.,Virginia Polytechnic Institute and State University | Zhang Y.-H.P.,U.S. Department of Energy | Zhang Y.-H.P.,Gate Fuels Inc.
ACS Synthetic Biology | Year: 2013

One-step purification of a multi-enzyme complex was developed based on a mixture of cell extracts containing three dockerin-containing enzymes and one family 3 cellulose-binding module (CBM3)-containing scaffoldin through high-affinity adsorption on low-cost solid regenerated amorphous cellulose (RAC). The three-enzyme complex, called synthetic metabolon, was self-assembled through the high-affinity interaction between the dockerin in each enzyme and three cohesins in the synthetic scaffoldin. The metabolons were either immobilized on the external surface of RAC or free when the scaffoldin contained an intein between the CBM3 and three cohesins. The immobilized and free metabolons containing triosephosphate isomerase, aldolase, and fructose 1,6-biphosphatase exhibited initial reaction rates 48 and 38 times, respectively, that of the non-complexed three-enzyme mixture at the same enzyme loading. Such reaction rate enhancements indicated strong substrate channeling among synthetic metabolons due to the close spatial organization among cascade enzymes. These results suggested that the construction of synthetic metabolons by using cohesins, dockerins, and cellulose-binding modules from cellulosomes not only decreased protein purification labor and cost for in vitro synthetic biology projects but also accelerated reaction rates by 1 order of magnitude compared to non-complexed enzymes. Synthetic metabolons would be an important biocatalytic module for in vitro and in vivo synthetic biology projects. © 2012 American Chemical Society.


Grant
Agency: Department of Energy | Branch: | Program: STTR | Phase: Phase I | Award Amount: 150.00K | Year: 2013

Formic acid (FA, CH2O2) is the simplest carboxylic acid. It is mainly used as a preservative and antibacterial agent in livestock feed. A significant fraction of FA is used in the leather-processing, textile and rubber industries and a small fraction of formic acid is used as a cleaning agent replacing mineral acids. Aqueous FA is a promising liquid hydrogen-storage carrier with a hydrogen storage density of 4.3% H2 weight. On industrial scales, most formic acid is produced through carbonylation of methanol, which is produced from fossil fuels. Since carbohydrate (CH2O), the most abundant renewable chemical energy, has low costs (e.g., $~0.30/kg), we propose to fix CO2 to formic acid powered by sugars through a novel synthetic enzyme pathway comprising 13 enzymes. The overall stoichiometric reaction is 6 CO2 + 7 H2O + C6H10O5 (starch, Phase I; cellulose, Phase II) -7 12 CH2O2. Cell-free biosystems are in vitro assembly of numerous enzymes and/or cofactors for implementing complicated biological reactions that microbes and chemical catalysts cannot do, for example, 12 mol of dihydrogen generated from per glucose, enzymatic conversion of cellulose to starch. In this project, we (Gate Fuels Inc. and Virginia Tech) will validate the technological feasibility of enzymatic conversion of 6 CO2 and starch to 12 formic acid by putting 13 enzymes together under modest reaction conditions (e.g., ~30-40 oC and ~1 atm) and will use a biomimetic cofactor replacing a costly and unstable cofactor NAD. The production of formic acid from high-concentration CO2 released by power stations and renewable sugars would bring numerous benefits: (i) utilize CO2 for the production of a value-added chemical, which is produced from fossil fuels, (ii) decrease net CO2 emissions, (iii) create high-paying biomanufacturing jobs and promote rural economy, (iv) utilize abundant domestic renewable resources, (v) enhance national energy security, and (vi) enhance technology export in the future. Cell-free systems would become a disruptive biomanufacturing platform, compared to living entities.


Grant
Agency: Department of Energy | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 142.19K | Year: 2012

This project will provide a low-cost route for high-yield fumarate production from pretreated non-food cellulosic biomass mediated by recombinant cellulolytic Bacillus subtilis. The cellulose fraction of biomass feedstock contains more than one half oxygen by weight, making it a good starting material for the production of oxygen-containing polymeric monomers. Fumaric acid or fumarate, among the DOEs top building block chemicals is a precursor of succinic acid, malic acid, L-aspartic acid, and biodegradable polymers. B. subtilis strains have numerous advantages in industrial chemical production, such as their extensive usage as industrially-safe microorganisms, an inherent ability to use monomeric and oligomeric C5 and C6 sugars, tolerance to organic acids, low medium nutrient needs, and fast growth rates. The goal of this project is to develop an industrially-safe recombinant cellulolytic Bacillus subtilis strain that can produce high-yield fumarate from cellulosic materials in a two- step fermentation. Aerobic fermentation is used first, for fast cell mass synthesis and cellulase production, followed by anaerobic fermentation, which produces two moles of fumarate from two moles of CO2 and one mole of glucose (released from cellulose), with a theoretical yield of 1.29 g fumarate per g glucose. Compared to other dicarboxylic acids (e.g., succinic acid), the production of fumarate from cellulosic materials has balanced cofactors between the substrate and desired product, along with potentially high product yields, easy regulation of complex metabolic pathways, and low separation costs. Different from other consolidated bioprocessing (CBP) microorganisms, such as Gram- positive Clostridium spp., Gram-negative Escherichia coli, and yeast, B. subtilis strains have lots of advantages for industrial fermentation. Furthermore, we have developed several new techniques so that we can modify B. subtilis strains rapidly and easily, such as rapid scar-free chromosome knockout or knockin, directed evolution for enhanced activity secretory cellulases, and creation of the first generation recombinant cellulolytic B. subtilis. To produce high-yield fumarate from cellulosic materials and simplify product separation, the specific objectives are to (i) redirect the carbon flux to a fumarate-producing pathway, by knocking out other side-product pathways, (ii) over-express key enzymes, such as phosphoenolpyruvate carboxykinase, and (iii) co-express family 5, 9 and 48 cellulase genes for fast microbial cellulose utilization. This project provides a new route for generating high-yield fumarate from biomass with an industrially-safe B. subtilis strain, without the addition of costly fungal cellulase or expensive medium nutrients.

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