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Blacksburg, VA, United States

Yang C.-K.,Georgia State University | Zhang X.-Z.,Georgia State University | Zhang X.-Z.,Gate Fuels Incorporated | Lu C.-D.,Georgia State University | Tai P.C.,Georgia State University
Biochemical and Biophysical Research Communications | Year: 2014

Many cytoplasmic proteins without a cleavable signal peptide, including enolase, are secreted during the stationary phase in Bacillus subtilis but the molecular mechanism is not yet clear. We previously identified a highly conserved embedded membrane domain in an internal hydrophobic α-helix of enolase that plays an important role in its secretion. In this study, we examined the role of the helix in more detail for the secretion of enolase. Altering this helix by mutations showed that many mutated forms in this domain were not secreted, some of which were not stable as a soluble form in the cytoplasm. On the other hand, mutations on the flanking regions of the helix or the conserved basic residues showed no deleterious effect. Bacillus enolase with the proper hydrophobic helical domain was also exported extracellularly in Escherichia coli, indicating that the requirement of the helix for the secretion of enolase is conserved in these species. GFP fusions with enolase regions showed that the hydrophobic helix domain itself was not sufficient to serve as a functional secretion signal; a minimal length of N-terminus 140 amino acids was required to mediate the secretion of the fused reporter GFP. We conclude that the internal hydrophobic helix of enolase is essential but is not sufficient as a signal for secretion; the intact long N-terminus including the hydrophobic helix domain is required to serve as a non-cleavable signal for the secretion of Bacillus enolase. © 2014 Elsevier Inc. All rights reserved. Source


Grant
Agency: NSF | Branch: Standard Grant | Program: | Phase: | Award Amount: 225.00K | Year: 2014

This Small Business Technology Transfer (STTR) Phase I project proposes to develop a new low-cost platform for the production of methyl ethyl ketone directly from pretreated cellulosic biomass in a single step using a novel recombinant cellulolytic Bacillus subtilis strain. Methyl ethyl ketone (MEK), also referred to as 2-butanone, is the second most important commercial ketone after acetone. MEK is currently only produced by the oxidation of 2-butanol. However, this industrial synthesis process uses starting materials derived from petrochemicals and is generally expensive as well as not environmentally friendly. There is an urgent need to develop a novel cost-effective and environmentally friendly method to produce MEK other than through 2-butanol. The cellulosic biomass is the most abundant natural renewable resource and has great potential for the production of valuable biocommodities for both short- and long-term sustainability. However, the process for converting non-food lignocellulosic material into MEK is not yet economically feasible due to the high cost of the cellulase involved in cellulose hydrolysis and the use of fastidious culture media. Using synthetic pathway and metabolic engineering, this project will convert noncellulose-utilizing B. subtilis into an efficient cellulose utilizer to produce MEK with high yield and titer, suitable for industrial fermentation.

The broader impact/commercial potential of this project, if successful, will be a low-cost platform for producing MEK from nonfood biomass in a process called consolidated bioprocessing. MEK may then be used as a solvent for paint, and serve as an intermediate in the production of other chemicals. Therefore, MEK could easily find a market in the paint industry and in plastics manufacturing. More importantly, MEK could be converted by subsequent hydrogenation into octane isomers that can be used to produce high-grade aviation fuel. Currently, MEK is synthesized from petroleum-derived chemicals via a method involving greenhouse gas emissions. So far, few efforts have been made to produce bio-based MEK due to low process economics. The proposed recombinant cellulolytic B. subtilis would have advantages over developing other microorganisms. In addition, the novel green technology will satisfy operational cost considerations, environmental concerns, and health and safety regulations. Compared to traditional mechanism, this novel route will be more cost-effective and environmentally friendly. If successfully commercialized after the completion of the Phase II project, this bio-based MEK production technology will have a significant competitive advantage over traditional methods because it is more commercially attractive and supports sustainable societal development.


Grant
Agency: NSF | Branch: Standard Grant | Program: | Phase: | Award Amount: 150.00K | Year: 2012

This Small Business Innovation Research Phase I project will develop high-power and high-energy-density enzymatic fuel cells (EFCs) that can completely oxidize low-cost maltodextrin (i.e., a partially hydrolyzed starch fragment). EFCs have received increasing interest as a next-generation, environmentally friendly (micro-)power source. Compared to microbial fuel cells, EFCs have much higher power densities suitable for more applications. However, current EFCs are limited by the partial oxidization of hexose molecules by one or two redox enzymes (i.e., 2-4 mol of electrons produced per mol of glucose) and a short enzyme lifetime. The goal of this project is to demonstrate the technical feasibility of the complete oxidation of maltodextrin in EFCs through a patent-pending synthetic enzymatic pathway. The technological innovation of this project is the construction of an ATP-free and CoA-free pathway by an assembly of thermostable enzymes to generate 24 electrons per glucose unit and increase power density. As a result, EFCs are expected to feature high energy density due to the complete oxidization of the fuel, high-power density due to substrate channeling among cascade enzymes and the mitigation of product inhibition of the enzymes, and a long lifetime due to the use of thermostable enzymes.

The broader impact/commercial potential of this project is developing bio-inspired sugar biobatteries featuring four appealing advantages: (i) biodegradability, (ii) safety, (iii) high energy storage density (e.g., 400 Wh electricity/kg for a 20% (w/v) maltodextrin solution, nearly three times that of lithium ion batteries), and (iv) fast refilling by adding a sugar solution. EFCs would have broad potential applications, such as rechargeable battery chargers (e.g., cellular phone chargers for outdoor uses or portable military devices), educational toy kits, and disposable (primary) batteries. In the future, miniaturized sugar-powered EFCs could potentially replace some secondary (rechargeable) batteries. Sugar-powered EFCs would be nearly 100% biodegradable, with the exception of the electrodes and wires, and are based on non-toxic and earth-abundant elements. The maltodextrin solution is neither toxic nor flammable. The innovation of EFCs equipped with this in vitro synthetic pathway would greatly promote the concept of in vitro synthetic biology and demonstrate another advantage a faster reaction rate than that of microbes due primarily to the absence of a cellular membrane. In addition, the generation of electricity from renewable and low-cost sugars, namely maltodextrin or future cellulosic materials, would decrease greenhouse gas emissions, increase national energy security, and promote rural economies.


Grant
Agency: National Science Foundation | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 149.98K | Year: 2011

This Small Business Innovation Research Phase I project will develop a new ultra-low-cost platform for the production of lactic acid directly from pretreated lignocellulosic biomass in a single step by using a novel recombinant cellulolytic Bacillus subtilis strain. Lactic acid is the precursor of polylactic acid (PLA), an environmentally friendly biodegradable plastic. Currently, lactic acid is commercially produced through bacterial fermentation based on corn starch or cane sugar, which are food and animal feed. Cellulosic biomass is the most abundant natural renewable resource, which has great potential in the production of valuable biocommodities for both short- and long-term sustainability. However, the process for converting non-food lignocellulosic material into lactic acid is not feasible yet due to the high cost of cellulase involved in cellulose hydrolysis and also to the use of fastidious culture media. Through the systematic genetic engineering and metabolic engineering, this project will convert noncellulose- utilizing B. subtilis to an efficient lignocellulose utilizer and to produce lactic acid at high yield and titer, suitable for industrial fermentation. The broader impact/commercial potential of this project is that the proposed recombinant cellulolytic B. subtilis would be an ultra-low-cost platform for producing lactic acid from non-food biomass, with obvious advantages over other developing CBP microorganisms. Also, this effort would serve as a model system to convert other industrially important microorganisms into cellulose utilizers and result in use of renewable and less expensive substrates for the production of valuable products. Lactic acid was identified by the U.S. DOE as one of the top 30 value-added and potential buildingblock chemicals made from biomass. There are many potential derivatives of lactic acid, some of which are new chemical products and others represent biobased alternatives to chemicals currently produced from petroleum. The use of lactic acid for making biodegradable PLA is growing rapidly, given the rising demand for environmentally friendly packaging. The production of PLA releases fewer toxic substances than making petroleum plastic, consumes less energy, and releases an estimated two-thirds less greenhouse gas. PLA can be composted, incinerated or recycled. There is no doubt that the consumption of the biodegradable plastic products derived from lactic acid would decrease the growing environmental pollution and attract greater consumer interest towards the use of green products.


Zhu Z.,Virginia Polytechnic Institute and State University | Wang Y.,Virginia Polytechnic Institute and State University | Wang Y.,CAS Shanghai Advanced Research Institute | Minteer S.D.,Saint Louis University | And 3 more authors.
Journal of Power Sources | Year: 2011

Enzymatic fuel cells (EFCs) use a variety of fuels to generate electricity through oxidoreductase enzymes, such as oxidases or dehydrogenases, as catalysts on electrodes. We have developed a novel synthetic enzymatic pathway containing two free enzymes (maltodextrin phosphorylase and phosphoglucomutase) and one immobilized glucose-6-phosphate dehydrogenase that can utilize an oligomeric substrate maltodextrin for producing electrons mediated via a diaphorase and vitamin K3 electron shuttle system. Three different enzyme immobilization approaches were compared based on electrostatic force entrapment, chemical cross-linking, and cross-linking with the aid of carbon nanotubes. At 10 mM glucose-6-phosphate (G6P) as a substrate concentration, the maximum power density of 0.06 mW cm-2 and retaining 42% of power output after 11 days were obtained through the method of chemical cross-linking with carbon nanotubes, approximately 6-fold and 3.5-fold better than those of the electrostatic force-based method, respectively. When changed to maltodextrin (degree of polymerization = 19) as the substrate, the EFC achieved a maximum power density of 0.085 mW cm-2. With the advantages of stable, low cost, high energy density, non-inhibitor to enzymes, and environmental friendly, maltodextrin is suggested to be an ideal fuel to power enzymatic fuel cells. © 2010 Elsevier B.V. All rights reserved. Source

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