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ARS Home » Midwest Area » Peoria, Illinois » National Center for Agricultural Utilization Research » Renewable Product Technology Research » Research » Research Project #427981

Research Project: Technologies for Producing Renewable Bioproducts

Location: Renewable Product Technology Research

2018 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 sub-objectives of research project 5010-41000-172-00D, 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 FY 2018 include the following: • Statistical methods were used to develop an optimized growth medium for liamocin production using Aureobasidium strains that were genetically modified to eliminate a dark pigment, called melanin, which often contaminants this bioproduct. Liamocin yields from optimized medium were nearly double those from standard medium, up to 22 g liamocin/L. This represents the highest yields of liamocin reported to date. Furthermore, analyses showed that the quality of the liamocins from the optimized medium was improved because the pigment was eliminated. Improving the productivity of liamocins will facilitate their commercial development. • Resistance to a variety of antibiotics, including ß-lactams and aminoglycosides, has been widely reported in pathogenic Streptococcus isolates. Consequently, the search for new antimicrobials is a high priority. Liamocins have been shown to have antibacterial activity specific for species of Streptococcus, including important pathogens of cattle, swine and humans. The mode of action for liamocins acting against these strains was studied and it was determined that they likely act via disruption of the cell membrane. Further efforts are underway to determine additional uses for these unique polyol lipids. • Enzymes that normally synthesize long glucose polymers, called glucans, were genetically modified to instead produce a novel small sugar molecule known as isomelezitose. Similar types of sugars, such as trehalose, are known to have bioprotective properties that minimize damage to proteins from heat, freezing, or drying; and are therefore extremely important to the pharmaceutical, agricultural, and food industries. Previous efforts to produce isomelezitose were hampered by inefficient synthesis methods, therefore we engineered several different enzymes and identified one that was capable of making high levels of this compound. This technology finally allows this valuable sugar to be produced in commercial quantities and enables evaluation of the sugar in a variety of industrial applications. • Isomelezitose was shown to have excellent properties that help maintain viability of organisms during drying. We demonstrated that isomelezitose was far superior to melezitose and trehalose (common bioprotectants) for drying a bacterial strain used as a biological control agent. Cells dried in the presence of isomelezitose were more viable and grew faster upon hydration than the other cells. It is expected that this novel sugar will be useful in protecting a number of different types of cells and proteins. • Genome sequencing was completed on several novel lactic acid bacteria isolated from traditional Turkish sourdough. These strains were sequenced as part of a collaboration because of their potential in the food industry as probiotics, and to identify novel bacteriocins and exopolysaccharides made by the organisms. As part of this work, several unique glucansucrase genes have already been cloned and used to produce novel polysaccharides. • Novel reduced molecular weight derivatives of frost grape polysaccharide were developed. Frost grape polysaccharide is a highly viscous gum produced by North American wild frost grapes. Reduced molecular weight derivatives have lower viscosities, extending their potential uses in food and prebiotic applications. Frost grape polysaccharide may be an alternative stabilizer and emulsifier for U.S. food producers, since gum arabic can only be imported and suffers from price volatility. • New rapid methods for the detection and identification of aminoglycoside antibiotics (e.g., kanamycin, tobramycin, gentamycin, etc.) were developed in collaboration with scientists at Wayne State University, Detroit, Michigan. These antibiotics can cause deafness in humans and are also used in veterinary medicine. These new methods will facilitate efforts to screen for novel aminoglycosides with improved efficacy and less toxicity. • Modification of tunicamycin antibiotics for reduced toxicity. Tunicamycin is a unique antibiotic that that can be combined with other antibiotics as a method to overcome antibiotic resistance in certain types of bacteria, but the toxicity of this antibiotic prevents it from being used for clinical applications. We recently showed that chemically modified tunicamycin has reduced toxicity, while still increasing the efficacy of penicillin based drugs up to 64-fold. Collaborations with several laboratories are ongoing to facilitate further evaluation of this novel tunicamycin derivative. • New methods were developed to analyze the composition and structure of carbohydrates. Current methods to study carbohydrates involve multiple complex steps in order to identify what types of sugars are present and to determine how they are linked together. A completely novel method that is much easier to perform was developed and shown to have broad applications in the field of carbohydrate chemistry.

4. Accomplishments
1. Overcoming antibiotic resistance using a novel antibiotic modified to have reduced toxicity. Beta-lactam antibiotics are a class of broad-spectrum (i.e., effective against a large variety of organisms) antimicrobials, which include penicillin derivatives and cephalosporins. The use of these important drugs has been limited over the years with the development of antibiotic resistant bacterial strains. Tunicamycin is a powerful antibiotic that can be combined with beta-lactam antibiotics in order to overcome this resistance. Scientists have known about this antibiotic for decades, but toxicity in human and animal cells prevented it from being used for therapeutic application. Recently, ARS researchers in Peoria, Illinois, have chemically modified tunicamycin into less harmful derivatives. The modified tunicamycins did not show any toxicity to human and hamster cells, but were still capable of increasing the efficacy of clinical penicillin based drugs by 32 to 64 times. This significant discovery now allows older type antibiotics to once again be effective and is an important step towards combating drug resistance.

2. New techniques to analyze carbohydrate composition and structure. Carbohydrates are the most abundant biomolecule on earth and they include simple sugars (e.g., glucose, mannose, and galactose), oligosaccharides consisting of short chains of sugar monomers, or complex branched sugar chains that may be composed of numerous types of sugars. Current methods to analyze the composition and structure of these carbohydrates have limitations. They often require multiple steps and usually have to be performed in combination with other analyses in order to fully characterize the material. ARS researchers in Peoria, Illinois, have developed a new technique for carbohydrate analysis that is much easier to perform and provides more detailed information about the carbohydrate structure. This method first involves a simple way to convert the sugars to a class of compounds called thiazolidine peracetates and then analyzing them by gas chromatography-mass spectrometry. In addition, we have shown that this technique can be combined with methods that label specific carbon molecules in the sugars with unique tags to obtain even more information about the composition and structure. This work will greatly benefit research laboratories where carbohydrate analysis is required and will facilitate the discovery of new agricultural derived sugars.

Review Publications
Leathers, T.D., Price, N.P.J., Vaughn, S.F., Nunnally, M.S. 2017. Reduced-molecular-weight derivatives of frost grape polysaccharide. International Journal of Biological Macromolecules. 105:1166-1170. doi: 10.1016/j.ijbiomac.2017.07.143.
Price, N.P.J., Jackson, M.A., Vermillion, K.E., Blackburn, J.A., Li, J., & Yu, B. 2017. Selective catalytic hydrogenation of the N-acyl and uridyl double bonds in the tunicamycin family of protein N-glycosylation inhibitors. Journal of Antibiotics. 70:1122-1128. doi: 10.1038/ja.2017.141.
Leathers, T.D., Skory, C.D., Price, N.P.J., Nunnally, M.S. 2017. Medium optimization for production of anti-streptococcal liamocins by Aureobasidium pullulans. Biocatalysis and Agricultural Biotechnology. 13:53-57. doi: 10.1016/j.bcab.2017.11.008.
Cote, G.L., Dunlap, C.A., Vermillion, K.E., & Skory, C.D. 2017. Production of isomelezitose from sucrose by engineered glucansucrases. Amylase. 1(1):82-93. doi: 10.1515/amylase-2017-0008.
Price, N.P.J., Hartman, T.M., Li, J., Velpula, K.K., Naumann, T.A., Guda, M.R.,Yu, B., Bischoff, K.M. 2017. Modified tunicamycins with reduced eukaryotic toxicity that enhance the antibacterial activity of ß-lactams. Journal of Antibiotics. 70(11):1070-1077. doi: 10.1038/ja.2017.101.
Hay, W.T., Vaughn, S.F., Byars, J.A., Selling, G.W., Holthaus, D.M., Price, N.P. 2017. Physical, rheological, functional and film properties of a novel emulsifier: Frost grape polysaccharide (FGP) from Vitis riparia Michx. Journal of Agricultural and Food Chemistry. 65(39):8754-8762.
Leathers, T.D., Rich, J.O., Nunnally, M.S., Anderson, A.M. 2017. Inactivation of virginiamycin by Aureobasidium pullulans. Biotechnology Letters. 40(1):157-163. doi: 10.1007/s10529.
Rich, J.O., Bischoff, K.M., Leathers, T.D., Anderson, A.M., Liu, S., Skory, C.D. 2017. Resolving bacterial contamination of fuel ethanol fermentations with beneficial bacteria – an alternative to antibiotic treatment. Bioresource Technology. 247:357-362.
Dertli, E., Colquhoun, I.J., Cote, G.L., Le Gall, G., Narbad, A. 2018. Structural analysis of the alpha-D-glucan produced by the sourdough isolate Lactobacillus brevis E25. Food Chemistry. 242:45-52. doi: 10.1016/j.foodchem.2017.09.017.
Dowd, P.F., Naumann, T.A., Price, N.P., Johnson, E.T. 2017. Identification of a maize (Zea mays) chitinase allele sequence suitable for a role in ear rot fungal resistance. AGRI GENE. 7:15-22.
Gong, R., Qi, J., Wu, P., Cai, Y., Ma, H., Liu, Y., Duan, H., Wang, M., Deng, Z., Price, N.P.J., Chen, W. 2018. An ATP-dependent ligase with substrate flexibility involved in assembly of the peptidyl nucleoside antibiotic polyoxin. Applied and Environmental Microbiology. doi: 10.1128/AEM.00501-18.
Bischoff, K.M., Brockmeier, S.L., Skory, C.D., Leathers, T.D., Price, N.P.J., Manitchotpisit, P., Rich, J.O. 2018. Susceptibility of Streptococcus suis to liamocins from Aureobasidium pullulans. Biocatalysis and Agricultural Biotechnology. 15:291-294. doi: 10.1016/j.bcab.2018.06.025.