Location: Renewable Product Technology Research2016 Annual Report
1a. Objectives (from AD-416):
This project develops commercially targeted technologies for producing value added bioproducts, such as specialty/commodity chemicals and biopolymers made from renewable agriculture feedstocks or biomass. Materials being investigated in this project have potential for significant market expansion and address the growing demand for improved manufacturing of products made with renewable technology. We work closely with industrial collaborators, stakeholders, and customers to ensure that goals are compatible with market needs and will ultimately strengthen our energy independence, improve sustainable agriculture, and provide economic support to rural communities. Goals for this project include the following specific objectives: Objective 1: Enable, from a technological standpoint, fungal-based processes for the commercial production of carboxylic acids and microbial oils. Sub-Objective 1.1: Enhance productivity and yield of microbial oils synthesized by Aureobasidium pullulans. Sub-Objective 1.2: Improve current methods for the fermentative production of carboxylic acids by Rhizopus. Objective 2: Enable chemical and enzymatic processes for the commercial production of (1) sugar-based biopolymers/oligosaccharides and (2) ethers derived from sugars or polyols. Sub-Objective 2.1: Develop biocatalytic processes for the production of novel biopolymers and oligomers from agricultural feedstocks. Sub-Objective 2.2: Develop renewable chemical processes for the synthesis of valuable sugar/polyol-based ethers.
1b. Approach (from AD-416):
The objectives of this research are achieved using strategies that include microbial strain development, fermentation technology, bacterial/fungal/yeast biotechnology, microbial bioengineering, enzyme technology, chemical/biochemical syntheses, and analytical analyses using state of the art equipment. Approaches for this project currently include the following areas of research: Specialty Oils. In this project, we develop advanced technologies for the production of specialty microbial oils, called liamocins, which are produced by certain strains of the fungus Aureobasidium. Liamocins are a family of novel oils that have significant potential for numerous veterinary, medical, industrial and food applications. However, the technology for large-scale production of liamocin is currently underdeveloped and is only economical for high-value applications. This work provides further development towards the commercialization of liamocins by increasing the yield and desired type of product through a combination of specialized techniques. Carboxylic Acids. We utilize metabolic engineering technology to enhance the production of carboxylic acids by the fungus Rhizopus, which is used in industry to convert sugars obtained from agricultural crops to this important commodity chemical. Carboxylic acids, such as fumaric and lactic acid, are natural fermentation biochemicals that are utilized for the manufacture of several environmentally friendly products, such as biodegradable plastics and cleaning solvents. In order to allow the market potential to continue expanding, it is important that the production costs are minimized by the development of new and improved technologies. Novel Biopolymers and Oligomers. We work on technologies to synthesize unique water-insoluble biopolymers using enzymatic conversion of agriculturally-derived sugars. These polymers are similar to dextrans, which are utilized in a large number of industrial, medical, and food applications. We identify, characterize, and modify novel microorganisms/enzymes that have potential for production of biodegradable products (e.g., fibers, films, encapsulation materials) for a broad number of consumer applications. In addition, we develop novel oligomers (i.e., short sugar chains) that have potential to promote the growth of healthy intestinal bacteria and potentially inhibit pathogens. In order to bring this technology to maturity, we continue improving these processes and develop further novel products made with these methods. Chemical Conversion of Sugars. We develop environmentally-friendly technologies that are capable of converting sugars to a class of compounds, called ethers, which are used extensively in many industrial applications. Ethers made from sugars have valuable potential applications as drop-in renewable alternatives for solvents, lubricants, and waxes. Chemical based conversion of sugars has immense potential to synthesize these important compounds, but progress is hampered by difficulties with reactions that typically involve toxic compounds. Therefore, we continue to explore and develop safer technologies and examine additional applications and products.
3. Progress Report:
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. This project addresses the National Program (NP) 306 (Quality and Utilization of Agricultural Products) Action Plan, Statement 2B-Enable technologies for (1) expanding market applications for existing biobased products, or (2) producing new marketable non-food biobased products derived from agricultural products and byproducts, and ensure that these technologies will generate economic impact by estimating their potential economic value. In addition, we continue to develop new technologies that support these efforts and lead to new areas of research. Specific examples of significant developments in FY2016 include the following: • Numerous isolates of the yeast Aureobasidium pullulans were screened for production of novel antimicrobial compounds, called liamocins. These naturally occurring bioactive agents have significant potential for applications, such as antibacterial veterinary treatment and agricultural pathogen control. Identifying organisms that produce liamocins in high yields is important for developing a commercially viable technology. • The genomes of two Aureobasidium pullulans strains that produce significant quantities of liamocin were sequenced. This new information is critical to discovering genes that are involved in the biosynthesis of this important compound and now enables the development of new methods for increasing production through genetic manipulation. • Aureobasidium strains were genetically modified to produce novel types of liamocin. The antimicrobial activity is highly dependent on the specific structure of the liamocin. This technology allows one to produce unique structures, which we expect will be superior for some application, especially for those requiring specific antibacterial spectrums. • Enzymes that synthesize biopolymers from sucrose were genetically modified to optimize production of short sugars chains, called oligosaccharides, that are valuable for stimulating the growth of “good” bacteria (i.e., probiotics) in the intestinal tracts of humans and animals. These enzymes produced high yields of a potentially valuable oligosaccharide that was shown to support the growth of probiotic bacterium Bifidobacterium. Similar oligosaccharides that promote the growth of beneficial bacteria are gaining significant interest as more producers look for ways to reduce antibiotics in animal feed. • The efficiency of enzymes that produce water-insoluble biopolymers was significantly improved by adding certain types of soluble polysaccharide to the reaction. This method yields significantly higher amounts of product and allows the synthesis of novel biopolymers with potential applications for films and fibers. • A novel enzyme that produces high-viscosity dextrans was discovered. Dextran is a complex polysaccharide that is used in numerous industrial and medical applications. The ability to produce dextran with significantly greater viscosity than currently available products will allow new commercial application to be developed. A bacterial strain that produces such a dextran was found and the genome was sequenced. The glucansucrase gene responsible for dextran biosynthesis was identified and will be used in future studies. • Improved methods were developed for synthesizing industrially important chemicals, called ethers, from agricultural sugars. This improved technology resulted in higher product yields with less expensive reagents. • A novel viscous polysaccharide produced by North American grape species Vitis riparia (frost grape) was found to have properties that would be valuable in numerous food applications. Purification methods have been developed and the molecular structure has been determined for this potential food ingredient. • In collaboration with scientists at Wayne State University, Detroit, Michigan, a grant proposal “Shaping Next Generation Aminoglycoside Antibiotics for Treatment of Multidrug-Resistant Diseases” was funded. Aminoglycoside antibiotics have long been used as potent broad spectrum antibiotic, but have limitations due to toxicity and bacterial resistance. This research will evaluate novel aminoglycoside antibiotics for improved efficacy.
1. Modified production of antimicrobial compounds. The yeast Aureobasidium pullulans is able to convert agricultural sugars to a family of related compounds called liamocins that have significant promise as selective antibacterial agents against certain organisms that are important in veterinary and clinical medicine. However, the yeast often produces multiple chemical forms of liamocin, which have varying degrees of antimicrobial activity. ARS scientists in Peoria, Illinois, developed genetic methods to control the type of liamocin that is produced depending on the sugar that is used to grow the strain. This technology allows for the production of novel liamocin structures for applications that require specific antibacterial properties and will benefit veterinary care with non-antibiotic treatment options.
5. Significant Activities that Support Special Target Populations:
Bischoff, K.M., Leathers, T.D., Price, N.P.J., Manitchotpisit, P. 2015. Liamocin oil from Aureobasidium pullulans has antibacterial activity with specificity for species of Streptococcus. Journal of Antibiotics. 68:642-645. doi: 10.1038/ja.2015.39.
Cote, G.L., Skory, C.D. 2016. Effect of a single point mutation on the interaction of glucans with a glucansucrase from Leuconostoc mesenteroides NRRL B-1118. Carbohydrate Research. 428:57-61.
Cote, G.L., Skory, C.D. 2015. Water-insoluble glucans from sucrose via glucansucrases. Factors influencing structures and yields. In: Cheng, H.N., Gross, R.A., Smith. P.B., editors. Green Polymer Chemistry: Biobased Materials and Biocatalysis. Washington, DC: ACS Symposium Series. p. 101-112.
Khalil, S., Ali, T.A., Skory, C., Slininger, P.J., Schisler, D.A. 2016. Evaluation of economically feasible, natural plant extract-based microbiological media for producing biomass of the dry rot biocontrol strain Pseudomonas fluorescens P22Y05 in liquid culture. World Journal of Microbiology and Biotechnology. 32(2):25. doi: 10.1007/s11274-015-1984-1.
Leathers, T.D., Price, N.P.J., Bischoff, K.M., Manitchotpisit, P., Skory, C.D. 2015. Production of novel types of antibacterial liamocins by diverse strains of Aureobasidium pullulans grown on different culture media. Biotechnology Letters. 37(10):2075-2081. doi: 10.1007/s10529-015-1892-3.
Liu, S., Skory, C., Qureshi, N., Hughes, S. 2016. The yajC gene from Lactobacillus buchneri and Escherichia coli and its role in ethanol tolerance. Journal of Industrial Microbiology and Biotechnology. 43(4):441-450. doi: 10.1007/s10295-015-1730-6.
Price, N.P.J., Labeda, D.P., Naumann, T.A., Vermillion, K.E., Bowman, M.J., Berhow, M.A., Metcalf, W.W., Bischoff, K.M. 2016. Quinovosamycins: New tunicamycin-type antibiotics in which the alpha, beta-1", 11'-linked N-acetylglucosamine residue is replaced by N-acetylquinovosamine. Journal of Antibiotics. 69(8):637-646. doi: 10.1038/ja.2016.49.
Price, N.P.J., Hartman, T.M., Vermillion, K.E. 2015. Nickel-catalyzed proton-deuterium exchange (HDX) procedures for glycosidic linkage analysis of complex carbohydrates. Analytical Chemistry. 87(14):7282-7290. doi: 10.1021/acs.analchem.5b01505.
Price, N.P.J., Vermillion, K.E., Eller, F.J., Vaughn, S.F. 2015. Frost grape polysaccharide (FGP), an emulsion-forming arabinogalactan gum from the stems of native North American grape species Vitis riparia Michx. Journal of Agricultural and Food Chemistry. 63(32):7286-7293. doi: 10.1021/acs.jafc.5b02316.
Rich, J.O., Leathers, T.D., Bischoff, K.M., Anderson, A.M., Nunnally, M.S. 2015. Biofilm formation and ethanol inhibition by bacterial contaminants of biofuel fermentation. Bioresource Technology. 196:347-354.
Rich, J.O., Manitchotpisit, P., Peterson, S.W., Liu, S., Leathers, T.D., Anderson, A.M. 2016. Phylogenetic classification of Aureobasidium pullulans strains for production of feruloyl esterase. Biotechnology Letters. 38(5):863-870.
Skory, C.D., Cote, G.L. 2015. Secreted expression of Leuconostoc mesenteroides glucansucrase in Lactococcus lactis for the production of insoluble glucans. Applied Microbiology and Biotechnology. 99(23):10001–10010.
Sutivisedsak, N., Leathers, T.D., Biresaw, G., Nunnally, M.S., Bischoff, K.M. 2016. Simplified process for preparation of schizophyllan solutions for biomaterial applications. Preparative Biochemistry and Biotechnology. 46(3):313-319.
Turgeman, T., Shatil-Cohen, A., Moshelion, M., Teper-Bamnolker, P., Skory, C.D., Lichter, A., Eshel, D. 2016. The role of aquaporins in pH-dependent germination of Rhizopus delemar spores. PLoS One. 11(3)e0150543. doi: 10.1371/journal.pone.0150543.
Turner, T.L., Zhang, G.C., Oh, E.J., Subramaniam, V., Adiputra, A., Subramaniam, V., Skory, C.D., Jang, J.Y., Yu, B.J., Park, I., Jin, Y.S. 2015. Lactic acid production from cellobiose and xylose by engineered Saccharomyces cerevisiae. Biotechnology and Bioengineering. 113(5):1075-1083. doi: 10.1002/bit.25875.