2013 Annual Report
1a.Objectives (from AD-416):
The broad goal of this project is to develop technologies for producing specialty/commodity chemicals and polymers from agriculturally derived carbohydrates. Objective 1: Develop commercially viable biocatalytic and chemical processes that enable the production of chemicals and monomers from agricultural feedstocks. Objective 2: Develop commercially viable biocatalytic processes that enable the production of novel biopolymers from agricultural feedstocks.
1b.Approach (from AD-416):
This research will specifically include the following strategies to achieve our objectives: (1) sequential metabolic engineering improvements of key metabolic steps of the fungus Rhizopus to enhance the production of carboxylic acids, including malic, fumaric, and lactic acid, which are all used as chemical feedstock in numerous manufacturing applications; (2) modification of carbohydrates through novel water based methods for the production of functional products, such as surfactants or detergents; (3) screening for superior isolates of the fungus Aureobasidium and fermentation optimization for improved bioproduction of the polyester, poly malic acid, which has the potential to be used as a biocompatible polymer; and (4) strain selection, fermentation optimization, and genetic modification of the bacterium Leuconostoc to enable enhanced production of a water insoluble polymer, alpha D glucan, which can be used in the production of polymer fibers and films. Accomplishing these objectives will allow for the development of new and improved methods for producing sustainable chemicals and polymers that can be employed in everyday consumer products.
Annual progress was made on all four subobjectives of this project, which addresses research needs to discover and develop commercially viable biobased materials and conversion processes; and to improve biobased material performance and processing through enhanced knowledge of their structure/property relationships. These technologies will ultimately strengthen our energy independence, improve sustainable agriculture, and provide economic support to rural communities.
In FY13, ARS Renewable Product Technology Research Unit scientists at the National Center for Agricultural Utilization Research, Peoria, Illinois made significant progress toward the objectives of this research project, as demonstrated by the following activities:
• A key enzyme involved in production of fumaric acid by the fungus Rhizopus was modified through gene manipulation so that the enzyme would accumulate in a different location inside of the cell where it can more efficiently carry out this conversion step. This work is expected to be a significant contribution towards the development of improved technologies for producing fumaric acid, thereby benefiting the agricultural grower and ultimately the consumer.
• Microbial biosurfactants called sophorolipid that are produced by an ARS patented yeast strain were found to be highly effective for improving the efficacy of commercial herbicides. This discovery may lead to enhanced methods to combat invasive weed species that decrease yield of agricultural crops.
• An ARS developed method of linking chemical groups to sugars was utilized to study a newly synthesized milk-derived sugar that is thought to protect infants from pathogens. This technology will facilitate purification and metabolic studies by scientists working to improve infant formula and develop new pharmaceuticals.
• The biochemical mechanisms for microbial production of certain of antibiotics made from carbohydrate-based fermentations was discovered through an international collaboration with Chinese and U.S. scientists.
• A novel biopolymer, called poly-malic acid, which may have applications in second-generation bioplastics was produced from agricultural biomass substrates. This work has potential impact for research to develop value-added products from agricultural commodities and byproducts.
• A glucansucrase gene was engineered to produce several variant enzymes that synthesize glucan polymers having significantly altered and desired characteristics. This has led to the ability to custom-tailor the glucans with different properties for use in a variety of applications.
• The modified glucansucrase enzymes were characterized and key differences were found that allow us to further understand the molecular mechanism that control the synthesis of insoluble glucans.
• It was discovered that the above native and modified glucansucrase enzymes differ from previously known enzymes of this class in their relative ability to form short sugars chains, called oligosaccharides, via acceptor reactions. They thus represent a significant improvement in the technology available to produce novel carbohydrates for food and feed applications.
Production of poly-malic acid from agricultural biomass substrates. Poly (malic acid) or PMA is a water-soluble biopolymer that has numerous potential applications in the biomedical and pharmaceutical industries. PMA can be produced from sugars obtained from agricultural crops by a yeast-like fungus called Aureobasidium. USDA, ARS scientists with the National Center for Agricultural Utilization Research (NCAUR), Renewable Product Technology Research Unit in Peoria, Illinois, in collaboration with a visiting scientist from Rangsit University, Thailand, further improved this process by developing fermentation methods that allow the Aureobasidium to produce the PMA directly from the agricultural biomass substrates. This work provides new information that allows further development towards the commercialization of microbial production of PMA from agricultural biomass.
Microbial biosurfactants to improve effectiveness of herbicides. Certain microorganisms are able to synthesize compounds known as biosurfactants that can be used for production of detergents for cleaning, emulsification, foaming, wetting, and softening. Biosurfactants have several advantages over surfactants chemically synthesized from petro-chemicals, such as lower toxicity and higher biodegradability, but have traditionally been limited by the high cost of production due to low yields by existing microbial strains. USDA, ARS scientists with the National Center for Agricultural Utilization Research (NCAUR) in Peoria, Illinois, have been studying a newly identified yeast strain that produces large quantities of a highly effective biosurfactant called sophorolipid. This biosurfactant was recently shown to drastically improve the effectiveness of several herbicides against invasive weed species that otherwise decrease agricultural yield. This research not only allows production of biosurfactants from renewable agricultural material, but also may help improve agricultural weed control methods by utilizing a biobased compound with significantly lower toxicity.
Improved enzymes that produce water-insoluble gel-like polymers from sugar. Certain bacteria used in fermented food produce an enzyme called glucansucrase that are able to produce long polymers of glucose from cane or beet sugars. These polymers are typically water-soluble and utilized in a large number of industrial, medical, and food applications. USDA, ARS scientists with the National Center for Agricultural Utilization Research (NCAUR), Renewable Product Technology Research Unit in Peoria, Illinois, identified a novel glucansucrase enzyme from a food-grade bacterium that produces a unique gel-like polymer that is insoluble in water. They further strategically modified this enzyme so it is capable of synthesizing glucan polymers having significantly altered and desired characteristics. Using these enzymes individually and in combination with other enzymes has led to the ability to custom-tailor glucans with different properties for use in a variety of applications. These polymers have potential for production of biodegradable fibers and films that can be used in a broad number of consumer applications and provide the foundation for developing new eco-friendly materials derived from renewable agricultural materials that expand economic opportunity and decrease dependence on foreign oil.
Leathers, T.D., Manitchotpisit, P. 2012. Production of poly(beta-L-malic acid) (PMA) from agricultural biomass substrates by Aureobasidium pullulans. Biotechnology Letters. 35(1):83-89.
Cote, G.L., Skory, C.D., Unser, S.M., Rich, J.O. 2012. The production of glucans via glucansucrases from Lactobacillus satsumensis isolated from a fermented beverage starter culture. Applied Microbiology and Biotechnology. 97(16):7265-7273.
Price, N.P., Manitchotpisit, P., Vermillion, K., Bowman, M.J., Leathers, T.D. 2013. Structural characterization of novel extracellular liamocins (mannitol oils) produced by Aureobasidium pullulans strain NRRL 50380. Carbohydrate Research. 370(4):24-32.
Peter, G., Dlauchy, D., Price, N.P., Kurtzman, C.P. 2012. Diddensiella caesifluorescens gen. nov., sp. nov., a riboflavin-producing yeast species of the family Trichomonascaceae. International Journal of Systematic and Evolutionary Microbiology. 62(12):3081-3087.
Dowd, P.F., Johnson, E.T., Price, N.P. 2012. Enhanced pest resistance of maize leaves expressing monocot crop plant derived ribosome inactivating protein and agglutinin. Journal of Agricultural and Food Chemistry. 60(1):10768-10775.
McCormick, S.P., Price, N.P., Kurtzman, C.P. 2012. Glucosylation and other biotransformations of T-2 toxin by yeasts of the Trichomonascus clade. Applied and Environmental Microbiology. 78(24):8694-8702.
Peterson, S.W., Manitchotpisit, P., Leathers, T.D. 2013. Aureobasidium thailandense, a new species isolated from leaves and wooden surfaces. International Journal of Systematic and Evolutionary Microbiology. 63:790-795. DOI: 10.1099/ijs.0.047613-0.