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Tracy B.P.,Elcriton, Inc | Jones S.W.,University of Delaware | Fast A.G.,University of Delaware | Indurthi D.C.,University of Delaware | Papoutsakis E.T.,University of Delaware
Current Opinion in Biotechnology | Year: 2012

Clostridia are anaerobic Firmicutes producing a large array of metabolites by utilizing simple and complex carbohydrates, such as cellulose, as well as CO2/H2 or CO. Their exceptional substrate diversity is enhanced by their ability to produce a broad spectrum of chemicals that can be used as precursors to or directly as biofuels and industrial chemicals. Genetic and genomic tools are under intense development, and recent efforts to metabolically engineer clostridia demonstrate their potential for biofuel and biorefinery applications. Pathway engineering to combine established substrate-utilization programs, such as for cellulose, CO2/H2 or CO, with desirable metabolic programs could lead to modular design of strains suitable for many applications. Engineering complex phenotypes - aerotolerance, abolished sporulation, and tolerance to toxic chemicals - could lead to superior bioprocessing strains. © 2011 Elsevier Ltd.


Jones M.A.A.,University of Delaware | Jones M.A.A.,Sultan Qaboos University | Papoutsakis E.T.,University of Delaware | Papoutsakis E.T.,Elcriton, Inc
Journal of Bacteriology | Year: 2014

Sporulation in the model endospore-forming organism Bacillus subtilis proceeds via the sequential and stage-specific activation of the sporulation-specific sigma factors, σH (early), σF, σE, σG, and σK (late). Here we show that the Clostridium acetobutylicum σK acts both early, prior to Spo0A expression, and late, past σG activation, thus departing from the B. subtilis model. The C. acetobutylicum sigK deletion (ΔsigK) mutant was unable to sporulate, and solventogenesis, the characteristic stationary-phase phenomenon for this organism, was severely diminished. Transmission electron microscopy demonstrated that the ΔsigK mutant does not develop an asymmetric septum and produces no granulose. Complementation of sigK restored sporulation and solventogenesis to wild-type levels. Spo0A and σG proteins were not detectable by Western analysis, while σF protein levels were significantly reduced in the ΔsigK mutant. spo0A, sigF, sigE, sigG, spoIIE, and adhE1 transcript levels were all downregulated in the ΔsigK mutant, while those of the sigH transcript were unaffected during the exponential and transitional phases of culture. These data show that σK is necessary for sporulation prior to spo0A expression. Plasmid-based expression of spo0A in the ΔsigK mutant from a nonnative promoter restored solventogenesis and the production of Spo0A, σF, σE, and σG, but not sporulation, which was blocked past the σG stage of development, thus demonstrating that σK is also necessary in late sporulation. sigK is expressed very early at low levels in exponential phase but is strongly upregulated during the middle to late stationary phase. This is the first sporulation-specific sigma factor shown to have two developmentally separated roles. © 2014, American Society for Microbiology.


Al-Hinai M.A.,Sultan Qaboos University | Al-Hinai M.A.,University of Delaware | Jones S.W.,Elcriton, Inc | Papoutsakis E.T.,University of Delaware | Papoutsakis E.T.,Elcriton, Inc
Microbiology and Molecular Biology Reviews | Year: 2015

Bacillus and Clostridium organisms initiate the sporulation process when unfavorable conditions are detected. The sporulation process is a carefully orchestrated cascade of events at both the transcriptional and posttranslational levels involving a multitude of sigma factors, transcription factors, proteases, and phosphatases. Like Bacillus genomes, sequenced Clostridium genomes contain genes for all major sporulation-specific transcription and sigma factors (spo0A, sigH, sigF, sigE, sigG, and sigK) that orchestrate the sporulation program. However, recent studies have shown that there are substantial differences in the sporulation programs between the two genera as well as among different Clostridium species. First, in the absence of a Bacillus-like phosphorelay system, activation of Spo0A in Clostridium organisms is carried out by a number of orphan histidine kinases. Second, downstream of Spo0A, the transcriptional and posttranslational regulation of the canonical set of four sporulation-specific sigma factors (σF, σE, σG, and σK) display different patterns, not only compared to Bacillus but also among Clostridium organisms. Finally, recent studies demonstrated that σK, the last sigma factor to be activated according to the Bacillus subtilis model, is involved in the very early stages of sporulation in Clostridium acetobutylicum, C. perfringens, and C. botulinum as well as in the very late stages of spore maturation in C. acetobutylicum. Despite profound differences in initiation, propagation, and orchestration of expression of spore morphogenetic components, these findings demonstrate not only the robustness of the endospore sporulation program but also the plasticity of the program to generate different complex phenotypes, some apparently regulated at the epigenetic level. Copyright © 2015, American Society for Microbiology. All Rights Reserved.


Grant
Agency: National Science Foundation | Branch: | Program: STTR | Phase: Phase I | Award Amount: 225.00K | Year: 2014

This Small Business Technology Transfer Research Phase I project aims to enhance fermentation yield of four carbon (C4) chemicals by instating mixotrophic fermentation. In order to realize the most cost effective fermentation for commodity chemical and biofuel production, the process should achieve maximum conversion of feedstock. In carbohydrate fermentations, CO2 and H2 are commonly evolved, which negatively impacts yield of desired products. We hypothesize that mixotrophic fermentation can recapture that yield loss. We define mixotrophic fermentation as the simultaneous consumption of organic and inorganic substrates. Improvements in yield from mixotrophic fermentation can be very significant. Moreover, certain clostridial organisms in theory can perform such fermentation, but relatively little is known about this. Moreover, the genetic tools to manipulate these microorganisms are underdeveloped. Consequently, this Phase I STTR will develop a genetic toolbox for these microorganisms, interrogate their ability for simultaneous substrate utilization of both carbohydrate and gas, and demonstrate the potential to produce C4 chemicals from mixotrophic fermentation. The broader impact/commercial potential of this project is to develop renewable and domestic chemical production and transportation fuel technologies that are cheaper, greener and more sustainable. Project outcomes, have the potential to increase product yield 10 ? 50%, which greatly reduces production-operating expense. The potential to utilize CO2 in the fermentation, minimizes the carbon footprint of the process. Lastly, process sustainability is enhanced since a greater diversity of feedstocks can be concurrently used such as complex carbohydrates, five and six carbon sugar monomers, biodiesel waste, hydrolyzed biomass, syngas, waste gas, and activated methane molecules. Overall, the project has the commercial potential to improve the triple bottom line of many chemical companies. Furthermore, this project could significantly enhance scientific and technological understanding of microbial physiology and metabolism during gas and carbohydrate fermentation.


Grant
Agency: National Science Foundation | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 99.49K | Year: 2009

This Small Business Innovation Research Phase I project aims to develop platform clostridia strains suitable for industrial scale alcohol production from renewable feedstocks and also to improve metabolic engineering technologies for all clostridia. Clostridia are strictly anaerobic, endospore forming prokaryotes of major importance to cellulose degradation, human and animal health and physiology, anaerobic degradation of simple and complex carbohydrates. Obstacles for the industrial use of these organisms include the development of genetic and metabolic engineering tools and strategies that could lead to strains suitable for production of chemicals and fuels from renewable feedstocks. This project focuses on developing metabolic engineering strategies and strains of solventogenic clostridia for the production of chemicals and biofuels. Through novel approaches, this project aims to solve three important bioprocessing bottlenecks: 1) product formation characteristics, 2) product yield and selectivity, 3) and suitable characteristics for repeated fed-batch or continuous fermentations. Anticipated outcomes of this project are clostridia strains that overcome the aforementioned bioprocessing bottlenecks and improved metabolic engineering technologies that are applicable to all clostridia. Development of biorefinery and biofuel technologies has been on the scientific and technological agenda of our nation for over 35 years now but never quite with the urgency of the last 2-3 years. Oil supplies for producing chemicals and fuels are becoming increasingly limiting and unreliable. Moreover, use or combustion of non-renewable chemicals and fuels detrimentally impacts the climate of our planet. Biomass is a carbon-neutral renewable resource for producing chemicals and fuels and the basis for the biorefinery concept. Solventogenic, butyric-acid clostridia played a major industrial role in the production of acetone and butanol in the past. Metabolic engineering of solventogenic clostridia may lead to industrial processes for production of chemicals such as butyric acid, butanol, butanediol, propanol, and acetoin, and production of hydrogen. Some of these chemicals can serve as biofuels directly, while others can be used for chemical conversion to biofuels. A major advantage of these organisms is that they can directly ferment a large spectrum of simple and complex carbohydrates including lignocellulosics with minimal pretreatment. The commercial potential of metabolically engineered solventogenic clostridia is exceptional but remains largely unexplored. This project aims to capture and demonstrate part of this potential. This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).


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

This Small Business Technology Transfer Research Phase I project aims to enhance fermentation yield of four carbon (C4) chemicals by instating mixotrophic fermentation. In order to realize the most cost effective fermentation for commodity chemical and biofuel production, the process should achieve maximum conversion of feedstock. In carbohydrate fermentations, CO2 and H2 are commonly evolved, which negatively impacts yield of desired products. We hypothesize that mixotrophic fermentation can recapture that yield loss. We define mixotrophic fermentation as the simultaneous consumption of organic and inorganic substrates. Improvements in yield from mixotrophic fermentation can be very significant. Moreover, certain clostridial organisms in theory can perform such fermentation, but relatively little is known about this. Moreover, the genetic tools to manipulate these microorganisms are underdeveloped. Consequently, this Phase I STTR will develop a genetic toolbox for these microorganisms, interrogate their ability for simultaneous substrate utilization of both carbohydrate and gas, and demonstrate the potential to produce C4 chemicals from mixotrophic fermentation.

The broader impact/commercial potential of this project is to develop renewable and domestic chemical production and transportation fuel technologies that are cheaper, greener and more sustainable. Project outcomes, have the potential to increase product yield 10 ? 50%, which greatly reduces production-operating expense. The potential to utilize CO2 in the fermentation, minimizes the carbon footprint of the process. Lastly, process sustainability is enhanced since a greater diversity of feedstocks can be concurrently used such as complex carbohydrates, five and six carbon sugar monomers, biodiesel waste, hydrolyzed biomass, syngas, waste gas, and activated methane molecules. Overall, the project has the commercial potential to improve the triple bottom line of many chemical companies. Furthermore, this project could significantly enhance scientific and technological understanding of microbial physiology and metabolism during gas and carbohydrate fermentation.


Grant
Agency: National Science Foundation | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 149.71K | Year: 2012

This Small Business Innovation Research (SBIR) Phase I project aims to develop a duckweed system for poultry waste bioremediation and use of the duckweed as a renewable feedstock for Clostridium acetobutylicum (Cac) biofuel production. Runoff from agricultural, municipal and other sources leads to eutrophication of the Chesapeake and other bays, with approximately half of the loads from agriculture, particularly poultry production on Delmarva. This work will provide an alternative nutrient reduction strategy, while simultaneously providing feedstock for clostridial biobutanol fermentation and poultry feed supplements. Current biobutanol work relies on corn hydrolysate, and duckweed is an attractive renewable feedstock. Life cycle assessments of environmental impacts indicate the ideal systems combine strategic placements of small/medium-scale wastewater remediation with biofuel feedstock production. The broader/commercial impacts of this research are the development of poultry waste bioremedation strategies coupled with the development of that biomass for renewable chemical and biofuel technologies. The goal of this Phase I project is to maximize manure remediation while producing biomass for Cac biobutanol fermentation. Depletion of non-renewable energy sources, leading to high oil prices, highlights the importance of economically-viable technologies for production of biofuels from renewables. Renewable biomasses should be carbon-neutral and be efficiently used in biorefinery production. The economic potential of biorefineries is hindered by cellulosic materials that compete with food supply economics (i.e. corn). The commercial potential of a biomass from a nuisance plant, duckweed, combined with the use of clostridia biobutanol production is exceptional, but remains unexplored. This project aims to demonstrate this potential.


Grant
Agency: NSF | Branch: Standard Grant | Program: | Phase: SMALL BUSINESS PHASE I | Award Amount: 174.71K | Year: 2012

This Small Business Innovation Research (SBIR) Phase I project aims to develop a duckweed system for poultry waste bioremediation and use of the duckweed as a renewable feedstock for Clostridium acetobutylicum (Cac) biofuel production. Runoff from agricultural, municipal and other sources leads to eutrophication of the Chesapeake and other bays, with approximately half of the loads from agriculture, particularly poultry production on Delmarva. This work will provide an alternative nutrient reduction strategy, while simultaneously providing feedstock for clostridial biobutanol fermentation and poultry feed supplements. Current biobutanol work relies on corn hydrolysate, and duckweed is an attractive renewable feedstock. Life cycle assessments of environmental impacts indicate the ideal systems combine strategic placements of small/medium-scale wastewater remediation with biofuel feedstock production.

The broader/commercial impacts of this research are the development of poultry waste bioremedation strategies coupled with the development of that biomass for renewable chemical and biofuel technologies. The goal of this Phase I project is to maximize manure remediation while producing biomass for Cac biobutanol fermentation. Depletion of non-renewable energy sources, leading to high oil prices, highlights the importance of economically-viable technologies for production of biofuels from renewables. Renewable biomasses should be carbon-neutral and be efficiently used in biorefinery production. The economic potential of biorefineries is hindered by cellulosic materials that compete with food supply economics (i.e. corn). The commercial potential of a biomass from a nuisance plant, duckweed, combined with the use of clostridia biobutanol production is exceptional, but remains unexplored. This project aims to demonstrate this potential.


Grant
Agency: National Science Foundation | Branch: | Program: SBIR | Phase: Phase II | Award Amount: 667.38K | Year: 2015

The broader impact/commercial potential of this Small Business Innovation Research Phase II project is to develop renewable and domestic chemical production and transportation fuel technologies that are cheaper, greener and more sustainable. Additionally, chemical companies are constantly looking for ways to improve business sustainability by reducing carbon footprints along with cost of manufacturing. Mixotrophic fermentation can be a step-change improvement in microbial fermentation for the production of numerous intermediate and commodity chemicals. Yields from feedstock can be increased substantially, and CO2 gas produced from the fermentation, as a waste byproduct can instead be captured and assimilated into valuable chemicals, which translates into a significant improvement in yield and productivity for any applicable commodity or intermediate chemical production process. Consequently, the commercial and environmental implications of this innovative technological approach are tremendous. Furthermore, this project could significantly enhance scientific and technological understanding of microbial physiology and metabolism during gas and carbohydrate fermentation. The objectives of this Phase II research project are to develop and scale-up platform strains for C3 and C4 chemical production using carbon efficient pathways. The approach being developed is referred to as Anaerobic Non-Photosynthetic (ANP) mixotrophic fermentation, and process advancements will focus on bioreactor operation parameters, media formulations, and integration with product separation. By the end of this project, the project will likely have demonstrated and validated enhanced mass yield of C3 and/or C4 metabolite production at pilot-scale using ANP mixotrophic fermentation.


Grant
Agency: NSF | Branch: Standard Grant | Program: | Phase: | Award Amount: 20.00K | Year: 2010

This Small Business Innovation Research (SBIR) Phase IB project aims to develop platform clostridia strains suitable for industrial scale alcohol production from renewable feedstocks and also to improve metabolic engineering technologies for all clostridia. Clostridia are strictly anaerobic endospore forming prokaryotes of major importance to cellulose degradation, human and animal health and physiology, and anaerobic degradation of simple and complex carbohydrates. Obstacles for the industrial use of these organisms include the development of genetic and metabolic engineering tools and strategies that could lead to strains suitable for production of chemicals and fuels from renewable feedstocks. This project focuses on developing metabolic engineering strategies and strains of solventogenic clostridia for the production of chemicals and biofuels. Through novel approaches, this project aims to solve three important bioprocessing bottlenecks: 1) product formation characteristics, 2) product yield and selectivity, 3) and suitable characteristics for repeated fed-batch or continuous fermentations. Anticipated outcomes of this project are clostridia strains that overcome the aforementioned bioprocessing bottlenecks and improved metabolic engineering technologies that are applicable to all clostridia.

The borader/commercial impacts of this project include the development of improved biorefinery and biofuel technologies. Oil supplies for producing chemicals and fuels are becoming increasingly limiting and unreliable. Moreover, use or combustion of non-renewable chemicals and fuels detrimentally impacts the climate of our planet. Biomass is a carbon-neutral renewable resource for producing chemicals and fuels and the basis for the biorefinery concept. Solventogenic, butyric-acid clostridia played a major industrial role in the production of acetone and butanol in the past. Metabolic engineering of solventogenic clostridia may lead to industrial processes for production of chemicals such as butyric acid, butanol, butanediol, propanol, and acetoin, and production of hydrogen. Some of these chemicals can serve as biofuels directly, while others can be used for chemical conversion to biofuels. A major advantage of these organisms is that they can directly ferment a large spectrum of simple and complex carbohydrates including lignocellulosics with minimal pretreatment. The commercial potential of metabolically engineered solventogenic clostridia is exceptional but remains largely unexplored. This project aims to capture and demonstrate part of this potential.

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