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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. Source

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

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.

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. Source

Chen H.-G.,Henan Agricultural University | 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
Renewable and Sustainable Energy Reviews | Year: 2015

Abstract The sustainability revolution is the defining challenge of our time to meet increasing needs in the energy-food-water nexus without compromising the ability of next generations. To feed the world, modern agriculture is primarily based on annual grain crops that replace native perennial plant communities on most of the arable land on the planet. This practice may not be sustainable due to high inputs of fresh water, fertilizers, and herbicides; soil erosion; and runoff water pollution. Recent biotechnology breakthroughs enable the fractionation of nonfood lignocellulosic biomass to multiple components, the conversion of nonfood cellulose to starch without sugar loss, the production of in vitro meat without slaughtering livestock, and the production of healthy oil from microbes, suggesting great opportunities of new biorefineries based on nonfood biomass. Perennial plant communities have higher biomass yield per hectare, have easily resource management, store more carbon, maintain better water quality, utilize nutrients more efficiently, tolerate more extreme weather events, and resist pests better than annual crops. Sustainable agriculture based on annual grains and perennial high-biomass yield plants along with new biorefineries could produce a myriad of products from biofuels (e.g., butanol and hydrogen), biomaterials, to food/feed. Sustainable agriculture and new biorefineries could be cornerstones of the coming sustainability revolution based on the most abundant renewable bioresource-biomass. © 2015 Elsevier Ltd. All rights reserved. Source

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