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Growing Fuel and Medicine: Advancing Biofuels and Plant-Produced Therapeutics

Scientists from WPI and the Arkansas Bioscience Institute Present their Latest Research on Cellulosic

30-10-2008: Can biofuels produced from non-food plant products like corn stalks or wood chips ever become a commercial reality? Can plants be engineered to grow vaccines or anti-cancer drugs? These and other questions were explored by researchers from Worcester Polytechnic Institute (WPI) and the Arkansas Bioscience Institute (ABI) at a symposium at WPI’s Life Sciences and Bioengineering Center at Gateway Park.

In general, the answer to these questions may soon be “yes” and teams from WPI and ABI are advancing the science and technology needed to reach those ends. “For most of human history, plants and microorganisms were the source for medicinal products, fuels, and specialty chemicals. So in a very real sense, what we’re doing is a back-to-the-future approach,” says Pamela J. Weathers, PhD, professor of biology and biotechnology at WPI, organizer of the symposium and a leader in the emerging collaboration with ABI.

“With our colleagues in Arkansas, we are making good progress on developing the technology and understanding the biology that will allow us to use plants and microbes to help meet our energy needs and to create new pharmaceuticals and other chemical building blocks essential for a healthy society and environment.”

The abstracts from the presentations at today’s symposium are:

“Plant produced small- and large-molecule therapeutics" (Pamela J. Weathers, PhD Professor, Department of Biology and Biotechnology, WPI): Plants naturally produce many valuable small molecules like terpenes and alkaloids that have for centuries been used in treatment of disease and as pesticides. More recently plants have also been proposed as suitable production vehicles for large molecules such as therapeutic proteins. Harnessing plants to yield both large and small molecular weight products on a larger scale has been challenging. Although field plants are the least expensive mode of production, conditions can’t be controlled and there is often political resistance to field culture of transgenic plants. Here we show two examples of how both native and transgenic plants can be used in inexpensive bioreactors for the production of the therapeutic protein, murine interleukin-12 (m-IL-12) in transgenic tobacco roots, and the sesquiterpene lactone antimalarial drug, artemisinin, in shoot cultures.

“Biofuels: Fuels Controversy” (Alex DiIorio, PhD, Director, Bioprocess Center, and Chris McPhee, MS Lab Manager, Bioprocess Center, WPI): Biofuels production, specifically, ethanol production in the United States has increased dramatically since the year 2000, ranging from 1 percent of the overall gasoline pool or 1.63 billion gallons in 2000, up to 2.85 percent and 3.9 billion gallons in 2005. Total production potential in 2007 was almost 6.5 billion gallons, leading the world in ethanol output. The United States and Brazil account for over 90 percent of the world’s total ethanol production, but is it enough? And are we headed in the right direction? Ethanol from corn, which is the basis of ethanol production in the United States, is a grossly inefficient process as compared to other sources such as sugar cane which is the basis for ethanol production in Brazil. Corn yields only 320 to 420 gallons of ethanol per acre, while sugar cane yields in Brazil range from 720 to 870 gallons per acre. Ethanol from corn, also a food crop, would eventually compete with food markets and cause prices to rise. There is conflicting evidence that this is currently happening. Cellulosic ethanol promises much higher efficiencies than are currently available from plants possessing inherently large concentrations of sugar as this method uses most of the available biomass. Complex carbohydrate structures such as lignin and hemi-cellulose pose a formidable technical barrier to releasing fermentable sugars from plant-derived feedstock. Technologies specifically designed to overcome these barriers are currently in development, from mechanical/chemical techniques designed to destroy the lignin barrier, to enzyme cocktails, and finally to genetic engineering techniques to design a “super digester” of these complex structural materials. In the WPI Bioprocess Center, naturally occurring organisms have been isolated and their genes modified to generate enhanced enzymes for improved and cost-effective cellulolytic breakdown. As part of our ongoing efforts, starting with dissected termite gut; native organisms have been cultivated and modified to yield enhanced enzymatic activity.

“The Arkansas Bioscience Institute: What it is and its research activities” (Carole Cramer, PhD Executive Director, Arkansas Bioscience Institute at Arkansas State University): The Arkansas Biosciences Institute is a five-member research consortium funded by Arkansas’ Tobacco Settlement Proceeds Act of 2000. The consortium is focused on cutting edge research at the interface of agriculture and medicine with the long term goal of enhancing the health of Arkansans and the nation. At Arkansas State University, a new state-of-the-art research building was constructed to house this new endeavor with a grand opening held in September 2004. Since 2004, dynamic cross-disciplinary research clusters have been developed in four target areas: plant-based bioproduction of proteins for medical and biofuels applications; plant metabolic engineering; molecular innovations in food sciences; and the interface of environment, agriculture, and human disease. The institute provides excellent support facilities in plant transformation and propagation, analytical instrumentation, and microscopy. It also provides a unique interface between researchers focused on plant biology and plant-based bioproduct and those involved with research on human diseases, addiction, neurobiology, and vaccine development. Examples of “discovery at the interface” and efforts to move science toward commercialization will be highlighted.

“The Many Reasons Why Plants Also Need Their Vitamin C!” (Argelia Lorence, PhD Assistant Professor of Metabolic Engineering, Arkansas Bioscience Institute, Arkansas State University): Humans and several other animals are unable to synthesize vitamin C (ascorbate, AsA) and thus they are dependent on dietary sources, mainly fresh fruits and produce, to fulfill their requirements for this nutrient. In plants, AsA is one of the major carbohydrates and antioxidants that play a role in essential physiological processes such as photosynthesis, cell division, and stress tolerance. Although ascorbate as a chemical entity is an old molecule, the elucidation of how plants make this vitamin is a recent discovery. Four different pathways are known to be functional in plants, one of them proposed by our group involving myo-inositol (MI) as the main precursor. We have engineered elevated AsA levels in Arabidopsis by over-expressing MI oxygenase (MIOX4) and L-gulono-1,4-lactone oxidase (GLOase), enzymes involved in the MI pathway to AsA. One of our current objectives is to study the stress tolerance and growth of these high AsA lines. MIOX4 and GLOase over-expressers were challenged with various types of abiotics stresses and their growth and performance was compared to the one of wild type controls. MIOX4 or GLOase lines containing higher AsA content (2 to 3-fold), were more tolerant to salt, cold, heat, high light, and methyl viologen when compared to controls. They also displayed enhanced growth of both aerial and underground tissues. In addition, these lines exhibited tolerance to common environmental pollutants such as trichloroethylene, a chlorinated hydrocarbon, and pyrene, a model polycyclic aromatic hydrocarbon. These broad stress-tolerance responses are most likely due to the ability of AsA to detoxify reactive oxygen species. In addition, I will also present our progress on the characterization of two novel enzymes involved in the MI pathway to AsA and well as our efforts in investigating how these high AsA mustard lines respond when challenged with herbivores. Engineering crops to have elevated vitamin C may lead to delayed senescence, increased biomass, stress tolerance, and enhanced phytoremediation capabilities.

“Cellulase enzymes for biomass conversion from the transgenic maize production system” (Elizabeth Hood, PhD, Associate Vice Chancellor for Research and Technology Transfer, Arkansas Bioscience Institute, Arkansas State University): One of the major constraints with production of lignocellulosic ethanol is the cost and volume of enzymes required to digest the feedstock. Enzymes produced in commodity crops can resolve both of these issues. We have developed transgenic maize plants that produce E1 -D-glucosidase and cellobiohydrolase I in seed. Enzyme amounts in the first generation seed for both enzymes were as high as17 percent of total soluble protein. Our results suggest that enzyme amount is affected by several factors including subcellular localization and the promoter used to drive gene expression. Southern hybridization analysis indicates that the gene is present at one copy per genome, simplifying breeding and regulatory issues. Protein accumulation can be increased several fold over multiple generations through breeding (Hood et al., 2003), thus it is our expectation that cellulase accumulation can be improved to 3-5 percent of dry weight. This concentration of cellulase can provide for a cost-effective enzyme supply for the developing biomass to ethanol industry.

"The awesome power of yeast" (Reeta Prusty Rao, PhD, Assistant Professor, Department of Biology and Biotechnology, WPI): “The awesome power of yeast (genetics)” — an adaptation of a quote by Prof. David Botstein, currently professor of integrative genomics at Princeton University. Prof. Botstein was referring to the elegant genetics possible in Bakers (or brewers) yeast in the 1980s, which made it an object of envy of scientists not working with yeast. Saccharomyces cerevisiae continues to push the limits of genomics and molecular genetic research because it is a simple, safe, fast, and facile eukaryotic organism. My laboratory uses S. cerevisiae to understand the fundamental mechanisms of fungal pathogenesis. For example: how fungi perceive secondary metabolites as cues and integrate these in a way that is beneficial to them. A clear understanding of this signaling pathway might allow us to disrupt microbial communication thereby interrupting the infection process. In addition we use genomic approaches to identify novel virulence factor as targets for antifungal drug development. Potential drug targets are subjected to high throughput screens to identify small molecules that inhibit their function. We also use the powerful genetic and genomic tools available in S. cerevisiae to understand the mode of action of ill-understood drugs at the molecular level. Such information can be used to increase efficacy or decrease side effects of drugs. Finally we are genetically modifying yeast to increase ethanol production from cellulosic feedstock.

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