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Cell-Free Bioinnovations Inc.

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Zhang Y.-H.P.,Virginia Polytechnic Institute and State University | Zhang Y.-H.P.,Cell-Free Bioinnovations Inc. | Zhang Y.-H.P.,CAS Tianjin Institute of Industrial Biotechnology
Biotechnology Advances | Year: 2015

The largest obstacle to the cost-competitive production of low-value and high-impact biofuels and biochemicals (called biocommodities) is high production costs catalyzed by microbes due to their inherent weaknesses, such as low product yield, slow reaction rate, high separation cost, intolerance to toxic products, and so on. This predominant whole-cell platform suffers from a mismatch between the primary goal of living microbes - cell proliferation and the desired biomanufacturing goal - desired products (not cell mass most times). In vitro synthetic biosystems consist of numerous enzymes as building bricks, enzyme complexes as building modules, and/or (biomimetic) coenzymes, which are assembled into synthetic enzymatic pathways for implementing complicated bioreactions. They emerge as an alternative solution for accomplishing a desired biotransformation without concerns of cell proliferation, complicated cellular regulation, and side-product formation. In addition to the most important advantage - high product yield, in vitro synthetic biosystems feature several other biomanufacturing advantages, such as fast reaction rate, easy product separation, open process control, broad reaction condition, tolerance to toxic substrates or products, and so on. In this perspective review, the general design rules of in vitro synthetic pathways are presented with eight supporting examples: hydrogen, n-butanol, isobutanol, electricity, starch, lactate,1,3-propanediol, and poly-3-hydroxylbutyrate. Also, a detailed economic analysis for enzymatic hydrogen production from carbohydrates is presented to illustrate some advantages of this system and the remaining challenges. Great market potentials will motivate worldwide efforts from multiple disciplines (i.e., chemistry, biology and engineering) to address the remaining obstacles pertaining to cost and stability of enzymes and coenzymes, standardized building parts and modules, biomimetic coenzymes, biosystem optimization, and scale-up, soon. © 2014 Elsevier Inc.


Qi P.,Virginia Polytechnic Institute and State University | You C.,Virginia Polytechnic Institute and State University | Zhang Y.-H.P.,Virginia Polytechnic Institute and State University | Zhang Y.-H.P.,Cell-Free Bioinnovations Inc.
ACS Catalysis | Year: 2014

Synthetic amylose could be a very important compound as a precursor of low-oxygen diffusion biodegradable plastic films, a healthy food additive, and a potential high-density hydrogen carrier. In this study, one-pot reactions composed of sucrose phosphorylase and potato alpha-glucan phosphorylase or supplemented with the three other enzymes (i.e., glucose isomerase, glucose oxidase, and catalase) were carried out to convert cheap sucrose to synthetic amylose, whereas one glucose unit from sucrose was added into the nonreducing end of the primer-maltodextrin. A thermostable sucrose phosphorylase was cloned from a thermophilic bacterium Thermoanaerobacterium thermosaccharolyticum JW/SL-YS485. The values of kcat and Km on sucrose were 15.1 s-1 and 20.2 mM, respectively, at 37 °C. The half-life time of this enzyme was 3.1 h at 70°C. The yield of synthetic amylose was not significantly improved when glucose isomerase, glucose oxidase, and catalase were used to remove fructose, which was an inhibitor to sucrose phosphorylase. This result suggested that the two-enzyme system equipped with the sucrose phosphorylase with a high value of fructose dissociation constant (34.4 mM) did not require the three other enzymes to mitigate product inhibition. The number-average degree of polymerization of synthetic amylose was controlled from 33 to 262 by adjusting primer maltodextrin concentration and reaction time. © 2014 American Chemical Society.


Grant
Agency: National Science Foundation | Branch: | Program: STTR | Phase: Phase I | Award Amount: 225.00K | Year: 2013

This Small Business Innovation Research Phase I project will scale up high-yield hydrogen production from maltodextrin and water mediated by cell-free enzymatic biosystems and develop prototype mobile electricity generators (MEGs). Cell-free biosystems for biomanufacturing (CFB2) implement complicated biochemical reactions in one pot by the in vitro assembly of more than three enzymes and/or cofactors. The Co-PI at Virginia Tech has demonstrated the production of nearly theoretical yields of hydrogen from sugars (including hexoses and pentoses) and water as CH2O (sugar) + H2O 2H2 + CO2 using CFB2. In this STTR I project, Cell-Free Bioinnovations Inc. will scale up enzymatic hydrogen production from a 2-mL bioreactor (current laboratory scale) to a 5-L bioreactor and integrate this process with a proton exchange membrane fuel cell stack for the high-efficiency generation of electricity. These integrated systems will charge numerous portable electronics and provide emergency power at low costs. The specific objectives are (i) scale-up of recombinant thermophilic enzyme production through high-cell density fermentation, (ii) discovery and production of more high-activity and ultra-thermostable enzymes, (iii) construction of synthetic enzyme complexes (metabolons) for easy purification and fast reaction rates, (iv) further enhancement of hydrogen generation rate by three fold, and (v) demonstration of prototype MEGs. The broader impact/commercial potential of this project is the scale-up of enzymatic production of hydrogen, which is mainly produced from natural gas and crude oil. Its satellite production facilities and its distribution are not widely available and are too costly. In the future, the production of low-cost, green hydrogen from local, renewable biomass sugars would create biomanufacturing and agricultural jobs in the bioeconomy, lower infrastructure costs for the hydrogen economy, decrease reliance on finite fossil fuels, and reduce net greenhouse gas emissions. Prior to large-scale production of economically competitive sugary hydrogen, several high-end applications are suggested for development to further improve the CFB2 platform, for example, MEGs, enzymatic fuel cells, and chiral compound synthesis. MEGs based on sugars will have some special markets. In addition, the new biotechnology platform CFB2 has unique advantages, such as higher product yields (i.e., neither by-product formation nor cell mass synthesis), greater engineering flexibility, faster reaction rates, broader reaction conditions (e.g., high temperature, low pH, organic solvent, toxic compound), and easier operation and control, compared with whole-cell fermentation. It is believed that the CFB2 platform could be used to produce jet fuels and long chain alcohols, store electricity, and fix CO2 in the future.


Grant
Agency: Department of Energy | Branch: | Program: STTR | Phase: Phase I | Award Amount: 150.00K | Year: 2015

A new high-value product zero calorie sweetener L-arabinose has been identified because it may be produced from the D-xylose-rich aqueous biomass intermediate stream at low costs. L-arabinose is an FDA approved healthy sweetener with dual functions as a sucrase inhibitor and a prebiotic, but its current high price ($30/kg) prevents wide use. The co-production of high-value products such as L-arabinose will be essential to the economic success of next generation biorefineries. The goal of this project is to decrease L-arabinose production costs by a factor of 10-15 via synergistic efforts in feedstock selection, novel biocatalysis, and product separation. Here, we will use a synthetic enzymatic pathway to convert the D-xylose in aqueous biomass hydrolysate to high concentration L-arabinose. This three-enzyme biocatalysis can be accomplished in an aqueous solution without the input of energy, chemicals, and cofactors. Also, a selective yeast fermentation will be used to remove other sugars for easy L-arabinose purification. The objective of this STTR I project to validate the feasibility of the isomerization of D-xylose to L-arabinose via this three-enzyme pathway.In this Phase I project, we will discover a promiscuous homolog of L-ribulose 5- phosphate 4-epimerase that works on L-ribulose, increase specific activities of L-ribulose 4- epimerase mutants by protein engineering, validate the production of L-arabinose from D-xylose, and file a provisional patent disclosure based on experimental data.Low-cost L-arabinose as a sucrose neutralizer could have a potential market of up to $10 billion/year. Its addition in numerous food and drinks will decrease risks of obesity, type 2 diabetes, cardiovascular diseases, hypertension, and cancer. The co-production of L-arabinose from D-xylose, along with the production of advanced biofuels from cellulose, will improve the economic viability of next generation biorefineries greatly and efficiently utilize abundant renewable biomass resources. The success of this project will promote the concept of cell-free biocatalysis as a green and low-energy intensive manufacturing process.


Grant
Agency: National Science Foundation | Branch: | Program: STTR | Phase: Phase I | Award Amount: 221.73K | Year: 2016

The broader impact/commercial potential of this Small Business Innovation Research Phase I project could be a significant decrease of sugar phosphate manufacturing costs, thereby creating opportunities for the low-cost production of numerous drugs and their derivatives used to treat cardiac diseases, cancers, and degenerative diseases. For example, fructose 1,6-bisphosphate (FBP) is a very important drug for treating cardiac diseases. Furthermore, less costly FBP, along with two thermostable enzymes (i.e., aldolase and triosephosphate isomerase), will provide affordable glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP) to both the pharmaceutical industry and academia. Both G3P and DHAP are important precursors for the synthesis of numerous carbon-carbon chiral compounds used for pharmaceutical production. The in-vitro synthetic biosystems platform to be developed in this project could be instrumental in unlocking the full potential of modified sugar phosphates in drug discovery and drug synthesis. Furthermore, this platform is much more environmentally friendly than typical chemical synthesis: modest reaction conditions and significant decreases in environmental footprint. This platform also has the potential to lead the next paradigm shift in biomanufacturing, by establishing a viable, environmentally friendly alternative for pharmaceutical manufacturing plants currently using chemical synthesis. The technical objectives in this Phase I research project are to validate the technological feasibility of the biosynthesis of fructose 1,6-bisphosphate, glyceraldehyde 3-phosphate and dihydroxyacetone phosphate via a non-natural ATP-free enzymatic pathway. In contrast to glycolysis, this novel pathway involves neither costly coenzymes (e.g., NAD+, ATP) nor requires ATP regeneration. Also, thermostable enzymes from hyperthermophilic microorganisms will be used to carry out reactions at 50-60 deg C in aqueous solution. Thermophilic enzymes have a longer lifetime than mesophilic enzymes, and the relatively high reaction temperature eliminates possible microbial contamination. The goal of this project is to demonstrate the biosynthesis of fructose 1,6-bisphosphate, glyceraldehyde 3-phosphate and dihydroxyacetone phosphate from low-cost substrates, that is, starch and pyrophosphate, via a novel ATP-free enzymatic pathway. The technical tasks are to (1) clone and express several recombinant pyrophosphate phosphofructokinases from various thermophilic microorganisms; (2) validate the feasibility of the synthetic pathway for biomanufacturing sugar phosphates; (3) optimize the synthetic pathway for cost-effective production; and (4) obtain grams of high-purity fructose 1,6-bisphosphate crystals via a series of purification steps. The most essential task is the efficient expression of a recombinant, high-activity pyrophosphate phosphofructokinase, which enables the omission of glycolysis? ATP-dependent phosphofructokinase from our pathway


Zhu Z.,Virginia Polytechnic Institute and State University | Zhu Z.,Cell-Free Bioinnovations Inc. | Kin Tam T.,Cell-Free Bioinnovations Inc. | Sun F.,Cell-Free Bioinnovations Inc. | And 3 more authors.
Nature Communications | Year: 2014

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. © 2014 Macmillan Publishers Limited. All rights reserved.


Grant
Agency: NSF | Branch: Standard Grant | Program: | Phase: | Award Amount: 225.00K | Year: 2013

This Small Business Innovation Research Phase I project will scale up high-yield hydrogen production from maltodextrin and water mediated by cell-free enzymatic biosystems and develop prototype mobile electricity generators (MEGs). Cell-free biosystems for biomanufacturing (CFB2) implement complicated biochemical reactions in one pot by the in vitro assembly of more than three enzymes and/or cofactors. The Co-PI at Virginia Tech has demonstrated the production of nearly theoretical yields of hydrogen from sugars (including hexoses and pentoses) and water as CH2O (sugar) + H2O 2H2 + CO2 using CFB2. In this STTR I project, Cell-Free Bioinnovations Inc. will scale up enzymatic hydrogen production from a 2-mL bioreactor (current laboratory scale) to a 5-L bioreactor and integrate this process with a proton exchange membrane fuel cell stack for the high-efficiency generation of electricity. These integrated systems will charge numerous portable electronics and provide emergency power at low costs. The specific objectives are (i) scale-up of recombinant thermophilic enzyme production through high-cell density fermentation, (ii) discovery and production of more high-activity and ultra-thermostable enzymes, (iii) construction of synthetic enzyme complexes (metabolons) for easy purification and fast reaction rates, (iv) further enhancement of hydrogen generation rate by three fold, and (v) demonstration of prototype MEGs.


The broader impact/commercial potential of this project is the scale-up of enzymatic production of hydrogen, which is mainly produced from natural gas and crude oil. Its satellite production facilities and its distribution are not widely available and are too costly. In the future, the production of low-cost, green hydrogen from local, renewable biomass sugars would create biomanufacturing and agricultural jobs in the bioeconomy, lower infrastructure costs for the hydrogen economy, decrease reliance on finite fossil fuels, and reduce net greenhouse gas emissions. Prior to large-scale production of economically competitive sugary hydrogen, several high-end applications are suggested for development to further improve the CFB2 platform, for example, MEGs, enzymatic fuel cells, and chiral compound synthesis. MEGs based on sugars will have some special markets. In addition, the new biotechnology platform CFB2 has unique advantages, such as higher product yields (i.e., neither by-product formation nor cell mass synthesis), greater engineering flexibility, faster reaction rates, broader reaction conditions (e.g., high temperature, low pH, organic solvent, toxic compound), and easier operation and control, compared with whole-cell fermentation. It is believed that the CFB2 platform could be used to produce jet fuels and long chain alcohols, store electricity, and fix CO2 in the future.


Grant
Agency: National Science Foundation | Branch: | Program: SBIR | Phase: Phase II | Award Amount: 677.75K | Year: 2014

This Small Business Innovation Research Phase II project seeks the further development of metal-free enzymatic fuel cells (EFCs) that can completely oxidize sugar (maltodextrin) to yield electricity. Sugar-powered EFCs have received increasing interest as next-generation, environmentally friendly bio-batteries. These bio-batteries, which will be fully biodegradable, non toxic, and easily refillable, are envisioned as next-generation green power sources, particularly for portable electronic devices (e.g., smartphones, portable computers, tablets, and GPS systems to name a few). These Sugar-powered EFCs with a feed of 20% maltodextrin utilizing a proprietary synthetic cocktail of enzyme catalysts for sugar oxidation, have an energy storage density of approximately 800 Ah/kg, nearly 20-times that of lithium batteries. The technical objectives of this project are the following: (i) to further increase their power density to 10 mW/cm2, (ii) to prolong their lifetimes to months, and (iii) to develop a commercial cost competitive product. The use of a low-cost chemical ingredient along with the use of engineered redox enzymes in sugar biobatteries, could increase their power output, prolong their lifetime, and decrease their production cost. In this SBIR II project, Cell Free Bioinnovations Inc. will make prototype high energy density power banks that could charge smartphones directly by integrating synthetic biology, protein engineering, nanobiotechnology, and electrochemistry. The broader impact/commercial potential of this project is the demonstration of the prototype bio-inspired sugar-powered biobatteries featuring high-energy storage density (e.g., 800 Ah electricity/kg of 20% sugar solution, nearly twenty times that of lithium ion batteries), fast refilling by the addition of a sugar solution, non toxic and safe to handle and use. Because sugar-powered EFCs are based on low-cost biocatalysts and do not require costly or rare metals, they are disposable and biodegradable devices. High-energy storage density EFCs would have broad potential applications as rechargeable battery chargers (e.g., cellular phone chargers for outdoor use or for portable military devices), educational toy kits, and disposable (primary) batteries. In the future, sugar-powered EFCs could potentially replace some secondary (rechargeable) batteries and primary batteries. In addition, the innovation of EFCs equipped with this cell-free synthetic pathway would greatly promote the concept of cell-free biomanufacturing composed of synthetic enzymatic pathways.


Grant
Agency: NSF | Branch: Standard Grant | Program: | Phase: STTR PHASE I | Award Amount: 221.73K | Year: 2016

The broader impact/commercial potential of this Small Business Innovation Research Phase I project could be a significant decrease of sugar phosphate manufacturing costs, thereby creating opportunities for the low-cost production of numerous drugs and their derivatives used to treat cardiac diseases, cancers, and degenerative diseases. For example, fructose 1,6-bisphosphate (FBP) is a very important drug for treating cardiac diseases. Furthermore, less costly FBP, along with two thermostable enzymes (i.e., aldolase and triosephosphate isomerase), will provide affordable glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP) to both the pharmaceutical industry and academia. Both G3P and DHAP are important precursors for the synthesis of numerous carbon-carbon chiral compounds used for pharmaceutical production. The in-vitro synthetic biosystems platform to be developed in this project could be instrumental in unlocking the full potential of modified sugar phosphates in drug discovery and drug synthesis. Furthermore, this platform is much more environmentally friendly than typical chemical synthesis: modest reaction conditions and significant decreases in environmental footprint. This platform also has the potential to lead the next paradigm shift in biomanufacturing, by establishing a viable, environmentally friendly alternative for pharmaceutical manufacturing plants currently using chemical synthesis.

The technical objectives in this Phase I research project are to validate the technological feasibility of the biosynthesis of fructose 1,6-bisphosphate, glyceraldehyde 3-phosphate and dihydroxyacetone phosphate via a non-natural ATP-free enzymatic pathway. In contrast to glycolysis, this novel pathway involves neither costly coenzymes (e.g., NAD+, ATP) nor requires ATP regeneration. Also, thermostable enzymes from hyperthermophilic microorganisms will be used to carry out reactions at 50-60 deg C in aqueous solution. Thermophilic enzymes have a longer lifetime than mesophilic enzymes, and the relatively high reaction temperature eliminates possible microbial contamination. The goal of this project is to demonstrate the biosynthesis of fructose 1,6-bisphosphate, glyceraldehyde 3-phosphate and dihydroxyacetone phosphate from low-cost substrates, that is, starch and pyrophosphate, via a novel ATP-free enzymatic pathway. The technical tasks are to (1) clone and express several recombinant pyrophosphate phosphofructokinases from various thermophilic microorganisms; (2) validate the feasibility of the synthetic pathway for biomanufacturing sugar phosphates; (3) optimize the synthetic pathway for cost-effective production; and (4) obtain grams of high-purity fructose 1,6-bisphosphate crystals via a series of purification steps. The most essential task is the efficient expression of a recombinant, high-activity pyrophosphate phosphofructokinase, which enables the omission of glycolysis? ATP-dependent phosphofructokinase from our pathway


Grant
Agency: NSF | Branch: Standard Grant | Program: | Phase: SMALL BUSINESS PHASE II | Award Amount: 685.75K | Year: 2014

This Small Business Innovation Research Phase II project seeks the further development of metal-free enzymatic fuel cells (EFCs) that can completely oxidize sugar (maltodextrin) to yield electricity. Sugar-powered EFCs have received increasing interest as next-generation, environmentally friendly bio-batteries. These bio-batteries, which will be fully biodegradable, non toxic, and easily refillable, are envisioned as next-generation green power sources, particularly for portable electronic devices (e.g., smartphones, portable computers, tablets, and GPS systems to name a few). These Sugar-powered EFCs with a feed of 20% maltodextrin utilizing a proprietary synthetic cocktail of enzyme catalysts for sugar oxidation, have an energy storage density of approximately 800 Ah/kg, nearly 20-times that of lithium batteries. The technical objectives of this project are the following: (i) to further increase their power density to 10 mW/cm2, (ii) to prolong their lifetimes to months, and (iii) to develop a commercial cost competitive product. The use of a low-cost chemical ingredient along with the use of engineered redox enzymes in sugar biobatteries, could increase their power output, prolong their lifetime, and decrease their production cost. In this SBIR II project, Cell Free Bioinnovations Inc. will make prototype high energy density power banks that could charge smartphones directly by integrating synthetic biology, protein engineering, nanobiotechnology, and electrochemistry.

The broader impact/commercial potential of this project is the demonstration of the prototype bio-inspired sugar-powered biobatteries featuring high-energy storage density (e.g., 800 Ah electricity/kg of 20% sugar solution, nearly twenty times that of lithium ion batteries), fast refilling by the addition of a sugar solution, non toxic and safe to handle and use. Because sugar-powered EFCs are based on low-cost biocatalysts and do not require costly or rare metals, they are disposable and biodegradable devices. High-energy storage density EFCs would have broad potential applications as rechargeable battery chargers (e.g., cellular phone chargers for outdoor use or for portable military devices), educational toy kits, and disposable (primary) batteries. In the future, sugar-powered EFCs could potentially replace some secondary (rechargeable) batteries and primary batteries. In addition, the innovation of EFCs equipped with this cell-free synthetic pathway would greatly promote the concept of cell-free biomanufacturing composed of synthetic enzymatic pathways.

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