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State College, PA, United States

Jana B.,Pennsylvania State University | Manning M.,Pennsylvania State University | Manning M.,Expansyn Technologies Inc. | Postle K.,Pennsylvania State University
Journal of Bacteriology | Year: 2011

The TonB system of Gram-negative bacteria provides passage across the outer membrane (OM) diffusion barrier that otherwise limits access to large, scarce, or important nutrients. In Escherichia coli, the integral cytoplasmic membrane (CM) proteins TonB, ExbB, and ExbD couple the CM proton motive force (PMF) to active transport of iron-siderophore complexes and vitamin B 12 across the OM through high-affinity transporters. ExbB is an integral CM protein with three transmembrane domains. The majority of ExbB occupies the cytoplasm. Here, the importance of the cytoplasmic ExbB carboxy terminus (residues 195 to 244) was evaluated by cysteine scanning mutagenesis. D211C and some of the substitutions nearest the carboxy terminus spontaneously formed disulfide cross-links, even though the cytoplasm is a reducing environment. ExbB N196C and D211C substitutions were converted to Ala substitutions to stabilize them. Only N196A, D211A, A228C, and G244C substitutions significantly decreased ExbB activity. With the exception of ExbB(G244C), all of the substituted forms were dominant. Like wild-type ExbB, they all formed a formaldehyde cross-linked tetramer, as well as a tetramer cross-linked to an unidentified protein(s). In addition, they could be formaldehyde cross-linked to ExbD and TonB. Taken together, the data suggested that they assembled normally. Three of four ExbB mutants were defective in supporting both the PMF-dependent formaldehyde cross-link between the periplasmic domains of TonB and ExbD and the proteinase K-resistant conformation of TonB. Thus, mutations in a cytoplasmic region of ExbB prevented a periplasmic event and constituted evidence for signal transduction from cytoplasm to periplasm in the TonB system. © 2011, American Society for Microbiology. Source


Grant
Agency: Environmental Protection Agency | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 69.67K | Year: 2008

Fuels derived from cellulosic biomass offer an alternative to conventional energy sources that supports national economic growth, national energy security, and environmental goals. In the 2007 State of the Union address, President Bush called for 35 billion gallons of alternative fuel use in the United States by 2017, which would replace 20 percent of the nation¿s petroleum consumption. Cellulosic biomass is an attractive energy feedstock because supplies are abundant domestically and globally. Current methods to break down biomass into simple sugars and convert them into ethanol are inefficient and constitute the core barrier to producing ethanol at volumes and costs competitive with gasoline. This project seeks to demonstrate the feasibility of the use of a novel plant protein, HED2, to improve the performance of enzymes in the deconstruction of cellulosic biomass. Preliminary experimentation indicated that HED2 proteins act synergistically with cellulose in the hydrolysis of crystalline cellulose. Expansyn Technologies objective for Phase I will focus on producing gram-scale levels of recombinant Zea m3 (ZM3), a maize HED2, and comprehensive testing of the recombinant protein for cellulose synergism with various forms of cellulose and cellulosic biomass. Once feasibility of the HED2 protein has been demonstrated, Expansyn Technologies will follow up with a Phase II proposal in which the synthesis and use of the protein will be demonstrated at a pilot scale. This technology has the potential to dramatically improve the digestibility of cellulosic feedstocks, thereby enabling commercial-scale production and use of an alternative fuel from renewable resources while reducing greenhouse gas emissions.


Grant
Agency: National Science Foundation | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 99.75K | Year: 2008

This Small Business Innovation Research Phase I project will explore the feasibility of recombinant expression and use of a selected group of plant proteins to enhance cellulase performance in the conversion of cellulosic biomass to simple sugars for production of biofuels such as ethanol. Cellulosic biomass is an attractive energy feedstock because supplies are abundant both domestically and globally. Current methods to break down biomass into simple sugars for fermentation into ethanol are inefficient and constitute a significant barrier to producing ethanol at volumes and costs competitive with gasoline. A primary research objective for Phase I is to achieve gram-scale expression of a representative sample of naturally-occurring and artificially-constructed homologs of Expansin Domain-2 (HED2) proteins in a heterologous expression system. The recombinant proteins will be assayed for cellulase synergism using commercial cellulases and biomass samples under conditions that approximate industrial processes. The broader impacts of this research will be to significantly improve the breakdown of cellulosic feedstocks by improving enzyme performance. It is widely recognized that measurable improvements in enzyme performances are required in order to reach an economically viable, biomass-based, fuel production process. While significant strides have been made to reduce production-related enzyme costs, overall cellulase performance must be improved in order to achieve a cost-effective process. An accessory protein, which enhances cellulase activity, would be a key development in unlocking the energy potential of recalcitrant cellulose to potentially reduce the nation's dependence of fossil fuels.


Grant
Agency: Department of Agriculture | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 80.00K | Year: 2008

In the 2007 State of the Union address, President Bush called for 35 billion gallons of alternative fuel use in the US by 2017 which would replace 20% of the nation's petroleum consumption. It is apparent that this goal cannot be achieved with existing technology and existing feedstocks alone. New technology is needed to improve the conversion of non-grain (cellulosic) feedstocks. In the US, total fuel ethanol production in 2006 reached 4.86 billion gallons - a 24% increase over 2005. Domestic demand for fuel ethanol rose 33% over 2005 to 5.4 billion gallons (Renewable Fuels Association, March 2007.) There are 114 ethanol biorefineries in operation in the US with an aggregate capacity of 5.6 billion gallons. Nearly all fuel ethanol is produced by fermentation of corn glucose (from corn grain) in the US. Presently, there are no commercial cellulosic ethanol biorefineries in operation (Renewable Fuels Association, February 2007.) Commercialization of ETI's proprietary technology will address present and future needs for the growing biofuels market. In the short-term, ETI's biocatalyst will be developed as an accessory protein used in conjunction with the most advanced cellulase technologies. Successful implementation of the proposed Phase I and subsequent research and development effort will result in the use of an ETI protein as a separate, plant-derived additive to cellulase during the hydrolysis of recalcitrant cellulose to glucose. Longer term protein development will shift from use in biomass conversion to functionality in a dedicated, genetically modified biofuel crop. Since expansin proteins are ubiquitous in plant cell walls, it is possible that, through genetic engineering, expression of a select expansin protein will enhance the accessibility of plant cellulose to enzymatic attack. Developments in this arena could lead to proprietary plant crops which encode the ETI protein. The present Phase I application will provide important fundamental insight into the technical feasibility of this approach.


Grant
Agency: Department of Energy | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 100.00K | Year: 2008

Due to the economics, politics and environmental impacts of petroleum use, there is growing interest in the use of biomass to partially replace petroleum as an energy source. However, in order to utilize biomass as a feedstock for the production of fuel or chemical products, its structure needs to be significantly altered to facilitate the efficient completion of downstream steps. This alteration typically is achieved by different pretreatment technologies, which usually involve temperature and pH extremes. The severity of these pretreatment conditions results in increased cost, loss of useful product, and inhibition of downstream steps. This project will evaluate the technical feasibility of the use of novel plant proteins, known as expansins, to reduce the severity of biomass pretreatment conditions. In Phase I, a parametric evaluation of processing conditions will be conducted to assess the utility of expansins in the dilute acid pretreatment of corn stover. Commercial Applications and other Benefits as described by the awardee: The technology should further the nation¿s effort to develop and execute an alternative energy plan based upon renewable energy sources.

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