PROJ NO: 3620-41000-080-00D AGENCY: ARS 3620
START: 03 MAY 1999 TERM: 02 MAY 2004 FY: 1999




OBJECTIVES: Improve technologies for conversion of renewable agricultural biomass into value-added products. Emphasis will include several areas identified as being economically and technologically constrained. General objectives are: (1) Identify novel cellulase enzymes to improve rates that cellulose is converted to fermentable sugars, (2) engineer selected plants to express a thermal stable cellulase, and (3) metabolic engineering of the fungus Rhizopus to increase the production efficiency of lactic acid.

APPROACH: Innovative biotech and metabolic engineering approaches are being used to find better processes to convert renewable material into value-added bioproducts. Specific approaches over the next year will include: (1) Continue with gene analysis and modification of the cloned cellulase gene from the shipworm bacterium Teredinidae turnerae, (2) finish constructing plant expression vectors containing the Acidothermus cellulolyticus endoglucanase, (3) transform the recombinant thermal stable cellulase into selected plants, (4) develop Rhizopus strains containing either multiple copies of lactate dehydrogenase (ldh) gene or the ldh gene containing a stronger promoter, (5) test the above strains in shake flask and identify those having higher production of lactate dehydrogenase activity, (6) and find new methods to improve efficiency of site-directed transformation and integration of specific genes into Rhizopus. Peoria, IL, NCAUR, Rms 3300-3318; BL-1, IBC Certified 5/20/99.

PROGRESS: 1999/01 TO 1999/09
1. What major problem or issue is being resolved and how are you resolving it? The purpose of this research is to improve the technologies for the conversion of renewable agricultural biomass into value-added products (e.g., fuel ethanol, lactate, and enzymes). Such conversions are currently possible but are cost prohibitive if using plant biomass other than starch. This is because agricultural material is made of many different polymers that must first be hydrolyzed into simple sugars, called saccharification, that microorganisms can then use for the formation of products possessing higher value. Starch is easily transformed to simple fermentable sugars using low cost enzyme. However, the more prevalent polymer, cellulose, is much more recalcitrant to saccharification by the group of enzymes known as cellulase. The production cost of making these enzymes and the efficiency of hydrolyzing agricultural material must be improved, if conversion of cellulose to higher value products is to become a viable technology. Furthermore, it is necessary to create new uses and improve the efficiency of microbial conversions of these sugars to the desired end-products. Emphasis of this project will be multifaceted and approach the problem from several areas identified as being economically and technologically constrained. Specific objectives will be to 1) identify and characterize novel cellulase enzymes to improve the rate at which cellulose can be converted into fermentable sugars, 2) genetically engineer selected plants to express a thermal stable cellulase as a way to decrease cost of these enzymes, and 3) develop novel metabolic engineering technologies in the fungus Rhizopus oryzae to increase the production efficiency of industrial chemicals, such as lactic acid.

2. How serious is the problem? Why does it matter? Fossil fuels are by far the most exploited natural resource used today. The whole infrastructure of modern society is built upon oil, which undebatably has a limited reserve. Petroleum serves as the foundation for not only most of the USA's energy needs but also for many products, such as solvents, organic acids, and numerous polymers and plastics. In 1994, the USA consumed about 748 million gallons per day. Approximately 64% was converted to motor gasoline, and 2-5% was used for the production of plastics. Although many benefits to society are derived from the use of petroleum, potential problems, in the form of litter, landfill depletion, environmental pollution, global warming, and increased atmospheric carbon dioxide, are associated with its use. Means to help alleviate these problems include the development of viable technologies for the production of industrial chemicals using renewable feedstocks (e.g., starch or cellulosic biomass) and the development of more efficient fermentation processes. Finding new uses for agricultural crops and improving the efficiency of how we utilize them is imperative for competing in today's global market. The transformation of low-value plant residues and byproducts into valuable commodities is not currently economically feasible, largely due to the costs and technical problems associated with cellulose saccharification. Methods that use chemical means for saccharification have many problems with the formation of byproducts that are inhibitory to microorganisms and the creation of environmental pollutants. Enzymatic saccharification of cellulose by the concerted action of cellulase is non-polluting and generates few, if any, toxins. However, the reaction times by the commonly used fungal cellulase are often so slow that large amounts of enzyme must be added, thereby offsetting the advantage of using low cost cellulose as a feedstock. Production of more effective cellulases and ways to produce these enzymes at less cost is the most direct route to overcoming these technical hurdles. This technology is expected to benefit numerous industries that require low cost sugars as a fermentation feedstock. The potential sources of the agricultural biomass that could serve as feedstock are endless and include herbaceous and woody plants, agricultural and forestry residues, as well as municipal solid waste and industrial waste streams. The biofuels sector, which currently uses starch as a source of fermentable sugars, would probably be the first to embrace this technology. The production of ethanol in the U.S. was in excess of 1.4 billion gallons in 1998. Most of this ethanol was made from over 550 million bushels of domestically grown corn. Demand for fuel ethanol is expected to increase because of concerns related to national security, economic stability, environmental impact, and global warming. If ethanol is to become competitive with petroleum derived fuels, the ability to economically convert cellulose into fermentable sugars must become a reality. Equally important, is the development of technologies to improve the efficiency of industrial fermentations, whether the sugars come from cellulose or starch. Much of our research will focus on the production of lactic acid because of the potential market growth. This organic acid is commonly used as a food additive for preservation, flavor, acidity, and for the manufacture of the biodegradable plastic, polylactic acid (PLA). The lactic acid market for the U.S. is currently about 50,000 tons per year and could increase substantially if the market for PLA develops as expected. Another market that may grow substantially is the biodegradable solvent ethyl lactate. This ester is considered non-toxic and has many applications that include electronic manufacturing, paints and coatings, textiles, cleaners and degreasers, adhesives, printing, and de-inking. It has been estimated that lactate esters could potentially replace as much as 80% of the 3.8 million tons of solvents used each year in the USA. However, fermentation efficiency must be improved to ensure the economic feasibility of the anticipated market expansion and to ensure that the U.S. maintains its leadership role.

3. How does it relate to the National Program(s) andNational Component(s) to which it has been assigned? National Program 306, New Uses, Quality & Marketability of Plant Products (100%). This research will result in new uses and expanding markets for agricultural products and co-products. New technologies will convert commodities and processing byproducts into important value-added products, such as lactic acid, acetic acid, and enzymes with desirable properties. Much of the technology for saccharification by cellulase will be important for fuel ethanol production using renewable resources. Corn and its processing byproducts (e.g., corn stover and corn fiber) will initially serve as the primary agricultural crop which this research will emphasize.

4. What were the most significantaccomplishments this past year? This project was created on May 29, 1999, to continue successful aspects of the project from our previous project (3620-41000-058-00D), which was terminated due to the 60-month time limitation. The development of this new project was to continue investigating the conversion of renewable materials into bioproducts through fungal, enzymatic, and plant technologies. Additionally, we identified and incorporated novel areas of biomass conversion research that we believe will contribute significantly to improving current saccharification technologies. 1. Three-way collaborations, with Department of Energy and North Carolina State University, have been initiated to study protein expression of a high-temperature resistant cellulase in genetically modified plants. We have already constructed an expression system for this protein and have introduced it into tobacco to confirm functionality. Strains of the aquatic plant Lemna gibba, or duckweed, that we believe are more well suited for on-site production at biomass conversion facilities have been obtained for further expression work with this enzyme. Successful production of this cellulase in plants could result a considerable savings over conventional methods, thereby increasing the total value of agricultural crops. 2. Production of plant secondary metabolites were found to be greatly influenced by levels of carbon dioxide, thereby increasing the evidence that secondary metabolites can be manipulated by a variety of atmospheric gases, including carbon dioxide levels. Interest in plant secondary metabolites as valuable pharmaceuticals and health supplements has been increasing in recent years. This work may provide a new use for low value carbon dioxide which is produced during fuel ethanol fermentations and has already resulted in a CRADA that will soon be implemented. 3. Genes have been isolated from the fungus R. oryzae and determined to be involved in the production and utilization of lactic acid. This is the first description of lactate dehydrogenase genes in a fungal species and fills a significant void between higher eukaryotic (e.g., plant, mammalian, fish, etc.) and prokaryotic (e.g., bacterial) lactate dehydrogenase genes which have been extensively studied. This accomplishment represents a crucial step in developing genetically engineered strains that will improve efficiency of fermentations using fungal strains currently employed in industry. The isolation of these genes and the development of methods to reintroduce DNA back into the fungus has resulted in considerable interest in future research collaborations and has already yielded a Cooperative Research & Development Agreement (CRADA) with a major industrial partner.

5. Describe the major accomplishments over the life of the project including their predicted or actual impact. This project is relatively new, although accomplishments from our previous project serve as a foundation for much of our ongoing work. As an example, it was discovered that ultra-high carbon dioxide levels promote rooting and enhance shoot growth by as much as 10-fold for numerous plants propagated by tissue culture techniques. This led to a practical means to reduce production costs and shorten growth times in the nursery for Sweetgum and Loblolly pine trees. It has also been demonstrated that production of secondary metabolite production in plants can be increased under high carbon dioxide conditions, and it is believed that this will be important in accomplishing the goals of a recently developed CRADA to produce secondary metabolites in sterile culture conditions. Lastly, our work has demonstrated that growth of the duckweed can be substantially increased in the presence of carbon dioxide, which will aid in the rapid production of cellulase for modified duckweed strains that we are currently developing. All of this research will enable a low-value byproduct of the fuel ethanol industry to be utilized in a practical and economic way. Much of our project also relies on previous work that demonstrated that it was possible to increase lactic acid production in the fungus Rhizopus. Mutagenesis and selection were used to obtain mutants with impaired alcohol production to minimize ethanol accumulation associated with growth in low oxygen conditions. Eliminating the need for aeration could result in considerable savings in the production of lactic acid by R. oryzae. One of these mutants had a 10-fold increase in lactic acid production, under low oxygen conditions, when compared to production by the parent strain. This was presumably because the organism's ability to ferment ethanol was minimized and carbon flow was diverted to lactic acid production. However, the stability of the mutant has prevented industrial application of this technology. Recent work has taken on a more direct strategy of controlling lactic acid and ethanol production by using molecular biology techniques. We have isolated several genes from Rhizopus that involved the metabolic scheme of growth and lactate production. We have developed methods to introduce genetically modified genes back into the fungal host and are using this system to decrease production of the byproducts and increase the accumulation of lactic acid. Each modification of a particular enzymatic conversion step is expected to result in varying degrees of improvement that will hopefully lead to significant economic gains for industrial fermentations using R. oryzae.

6. What do you expect to accomplish, year by year, over the next 3 years? During the next three years, we will continue with our investigations of novel cellulases, metabolite accumulation, and enzyme production. This will include the identification of those duckweed strains that are amenable to genetic modification for the expression of recombinant cellulase and screening for isolates that are expressing maximal enzyme activity. Additionally, we will continue to study the effects of high carbon dioxide levels to increase production of valuable metabolites. A CRADA has recently been approved for funding beginning in October. This CRADA will concentrate on determining those factors that stimulate plant production of an industrially important secondary metabolite. We will further characterize the genes involved in the synthesis of lactic acid, ethanol, and glycerol. This work is necessary to further identify key rate-limiting steps that will allow us to more carefully control the flow of various metabolites. We will continue to put modified genes back into the fungal host to determine the effects on lactic acid production. Additionally, we will find new ways to introduce these engineered genes into the organism so that we can include different industrial Rhizopus strains into our research. Recombinant strains exhibiting improved lactic acid production will be tested in pilot scale as part of an established CRADA.

7. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end user (industry, farmer, other scientists)? What are the constraints ifknown, to the adoption & durability of the technology product? A successful CRADA has been ongoing for 4 years to study methods to accelerate the generation of newly planted Sweetgum and Loblolly pine, which are important to the paper industry. Paper products are a multi-billion dollar industry in the U.S., and supply is directly related to the ability to replace existing forest populations. Our partner, as well as several commercial greenhouses, have adopted methods developed in this laboratory for plant propagation with tissue culture and ultra-high levels of carbon dioxide. This work will help to satisfy the demand for tens of thousands of acres of trees planted yearly. For the last year, we have had a CRADA with private industry so that they may assist in our efforts to increase lactic production in Rhizopus. This CRADA employs a unique and innovative arrangement since it involves a company employee working in our laboratory in an effort to most efficiently achieve our common research goals. Our cooperator is currently one of the world's largest producers of lactic acid. Their experience with fungal fermentation will be invaluable in testing our recombinant strains and should hasten the transfer of this technology to the marketplace. Ultimately, the farmer will benefit from this research since it relies on renewable agricultural materials for the final product. But, other scientists will also benefit from the knowledge of the genetic control mechanisms for such an industrially important fungus.

8. List your most important non-peer reviewed publications and presentations to non-scientific organizations, and articles written about your work(NOTE: this does not replace your peer reviewed publications which arelisted below). "Produce lactic acid anaerobically and save," September 1997. Technical Insights: Ind. Bioprocessing. p. 3.

PUBLICATIONS: 1999/01 TO 1999/09
1. SKORY, C.D. and BOTHAST, R.J. 1999. Genetic control of metabolic functions in Rhizopus oryzae. Soc. Ind. Microbiol. Annu. Meet. Abstr. p. 83.
2. FREER, S.N., SKORY, C.D., BOTHAST, R.J. and GREENE, R.V. 1999. Gene structure of a bifunctional cellulase ... Teredinobacter turnerae. Am. Chem. Soc. Annu. Meet. Abstr. p. Cell 10.
3. TISSERAT, B. and VAUGHN, S. 1999. Influence of ultra-high levels of carbon dioxide on secondary metabolite production in vitro. HortScience 34:543.
4. TISSERAT, B. and Berhow, M. 1998. Citrus ploidy level influence on the flavonoid composition in juice and leaf tissue in grapefruit cultivars. Proc. Fla. State Hort. Soc. 111:257-260.

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