Progress 05/04/15 to 05/03/20
Outputs
Progress Report 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. 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. This is the final report for this project, which terminated in May 2020. Most of this work will continue under the replacement project ⿿Antimicrobials for Biorefining and Agricultural Applications. This project addressed the National Program 306 (Quality and Utilization of Agricultural Products) Action Plan, Statement 2B-Enable technologies to produce new and expanded marketable nonfood, nonfuel biobased products derived from agricultural feedstocks. Progress was made on both objectives of the research 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. In addition, ARS scientists in Peoria, Illinois, continue to develop new technologies that support these efforts and lead to new areas of research. Specific examples of significant developments in FY 2020 include the following: Under Objective 1, technology developed for genetic modification of Aureobasidium yeast strains producing the antimicrobial compound, called liamocins, was applied to related Aureobasidium isolates that are used for production of the polysaccharide, pullulan. Pullulan is commonly used in the food and pharmaceutical industries. Strains were modified to eliminate production of contaminating pigments called melanin. These contaminants require post-production removal and results in increased processing costs. Under Objective 2, continued progress has been made on increasing production of a novel sugar called isomelezitose. This rare sugar is often produced as a minor byproduct by a group of enzymes called glucansucrases during the conversion of sucrose (such as table sugar) to polysaccharides. ARS scientists in Peoria, Illinois, have used genetic engineering to modify one of these enzymes to significantly improve synthesis of isomelezitose. Technology for production of this modified enzyme and conversion of sucrose to isomelezitose was optimized and scaled up. In addition, several different alternative production methods were investigated to minimize synthesis of unwanted byproducts in order to simplify subsequent purification procedures. Significant progress was also made in developing processes to convert sugars from crop residues into valuable industrial chemicals. Previous ARS technology on chemical modification of sugars was utilized to develop new methods for synthesizing biobased surfactants or detergents from cuphea oil using only environmentally friendly methods. These new surfactants have been shown to have antimicrobial properties. The procedure uses seed oil purified from Cuphea, which is recognized as a new valuable crop alternative. Crop rotation with Cuphea has previously been shown to improve the yield of agricultural crops such as corn or wheat. Progress has also been achieved in cooperation with an industry partner on a research project to enable the production of the antibiotic tunicamycin at a commercially relevant scale. Tunicamycin is a natural product that enhances the antimicrobial activity of penicillins, but it is too toxic to be used clinically. ARS researchers in Peoria, Illinois, developed technology to chemically convert tunicamycin into a less toxic derivative, known as TunR2, which still retains its antimicrobial action. Methods were also developed to scale up the conversion of tunicamycin to this less toxic derivative. This has allowed for the expansion of ARS research into the use of TunR2 to address other agricultural problems. ARS is currently examining the tunicamycin derivatives against the causative agents of specific animal and plant diseases. This final report of the project plan concludes a successful research undertaking that accomplished all of the project plan objectives and yielded many important discoveries, new technologies, and industrial partners. During the course of this work, ARS researchers in Peoria, Illinois, developed numerous microbial and enzymatic approaches for the conversion of biomass feedstocks to value-added products. Examples include the development of new enzyme technologies for the production of carbohydrate-based polymers made from cane or beet sugars that can be used for coatings and encapsulation of compounds for controlled-release. These same enzymes were further modified to produce a novel sugar, which has potential applications in long-term storage stability of foods, drugs, vaccines, and agricultural biocontrol agents. Methods were then developed to scale-up production of this sugar so they can be tested by commercial partners. A new polysaccharide isolated from grape vines was discovered and shown to have applications as a thickener in the food industry. We also developed technology to chemically convert corn-based sugars to value-added compounds that can be used as detergents and solvents. Finally, several new antibiotic alternatives were developed that can be used to combat problems associated with antimicrobial resistance. These include sugar-based compounds that have been shown to inhibit several bacterial pathogens. In addition, we developed several new non-toxic antibiotic adjuvants that can be combined with traditional penicillin-based antibiotics to significantly enhance the antimicrobial activity and often overcome antibiotic resistance. These new compounds have been shown to be effective against bacteria associated with animal diseases and are also showing promising results with some plant pathogens. Methods to scale-up production of these new antimicrobial compounds have also been developed and we are currently working with several commercial partners to assist with transfer of this technology. The new technologies developed in this work not only help farmers by creating new agricultural markets and offering improved antimicrobials for animal health, but also provide economic benefits to producers and ultimately the consumers. Accomplishments 01 Commercial scale production of antibiotic enhancers. Penicillins are a class of antibiotics that are used to treat a wide range of human and veterinary bacterial infection, but their effectiveness has decreased with the development of penicillin-resistant pathogens. Tunicamycin is a natural product that can be combined with penicillins to overcome this resistance, but its toxicity has prevented it from being used for therapeutic applications. ARS researchers in Peoria, Illinois, developed procedures to chemically modify tunicamycin to make it less harmful while still retaining the ability to enhance penicillins. However, large-scale production of modified tunicamycin has been difficult because the commercial market for tunicamycin is minimal due to the toxicity problems. Working with industrial partners, ARS scientists optimized methods for the fermentative production and purification of native tunicamycins and developed improved conversion techniques using chemical catalysts to produce the less toxic version at a larger scale. This technology will allow stakeholders to potentially reduce the use of traditional antibiotics to treat livestock, which will alleviate antibiotic resistance, and potentially allow the use of older antibiotics previously abandoned due to bacterial resistance.
Impacts
(N/A)
Publications
- Price, N.P., Jackson, M.A., Vermillion, K., Blackburn, J.A., Hartman, T.M. 2019. Rhodium-catalyzed reductive modification of pyrimidine nucleosides, nucleotide phosphates, and sugar nucleotides. Carbohydrate Research. 488(2020): 107893.
- Ispirli, H., Yüzer, M., Skory, C.D., Colquhoun, I., Sagdiç, O., Dertli, E. 2019. Characterization of a glucansucrase from Lactobacillus reuteri E81 and production of malto-oligosaccharides. Biocatalysis and Biotransformation. 37:6, 421-430..
- Zhang, M., Zhang, P., Xu, G., Zhou, W., Gao, Y., Gong, R., Cai, Y., Cong, H., Deng, Z., Price, N.P.J, Chen, W., Mao, X. 2019. Comparative investigation into formycin A and pyrazofurin A biosynthesis reveals branch pathways for the construction of C-nucleoside scaffolds. ACS Chemical Biology.
- Leathers, T.D., Saunders, L.P., Bowman, M.J., Price, N.P.J., Bischoff, K.M. , Rich, J.O., Skory, C.D., Nunnally, M.S. 2020. Inhibition of Erwinia amylovora by Bacillus nakamurai. Current Microbiology. 77:875⿿881.
- Price, N.J.P., Jackson, M.A., Singh, V., Hartman, T.M., Dowd, P.F., Blackburn, J.A. 2019. Synergistic enhancement of beta-lactam antibiotics by modified tunicamycin analogs TunR1 and TunR2. Journal of Antibiotics. 72(11):807-815.
- Berhow, M.A., Singh, M., Bowman, M.J., Price, N.P.J., Vaughn, S.F., Liu, S. X. 2020. Quantitative NIR determination of isoflavone and saponin content of ground soybeans. Food Chemistry. 317:126373.
- Kong, L., Xu, G., Liu, X., Wang, J., Tang, Z., Cai, Y., Shen, K., Tao, W., Zheng, Y., Deng, Z., Price, N.P.J., Chen, W. 2019. Divergent biosynthesis of C-Nucleoside minimycin and indigoidine in bacteria. iScience. 22:430⿿440.
- Xu, G., Kong, L., Xu, L., Gao, Y., Jiang, M., Cai, Y., Hong, K., Deng, Z., Price, N.P.J., Chen, W., Yu, Y. 2018. Coordinated biosynthesis of the purine nucleoside antibiotics aristeromycin and coformycin in the actinomycetes. Applied and Environmental Microbiology. 34(22):e01860-18.
- Dowd, P.F., Naumann, T.A., Johnson, E.T., Price, N.P.J. 2020. A maize hydrolase with activity against maize insect and fungal pests. Plant Gene. 21:100214.
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Progress 10/01/18 to 09/30/19
Outputs
Progress Report 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. 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. Progress was made on both objectives of the research 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, ARS scientists in Peoria, Illinois, continue to develop new technologies that support these efforts and lead to new areas of research. Specific examples of significant developments in FY 2019 include the following: Under Objective 1, progress was made on improving genetic modification methods for the fungus Aureobasidium. This allowed us to more easily delete genes involved in the production of a dark pigment, called melanin, which often contaminants bioproducts made by this organism. Eliminating the requirement to perform post-production cleanup to remove this impurity facilitates the commercial development of liamocin and other important Aureobasidium bioproducts such as pullulan. ARS scientists are now utilizing this technology in collaboration with industrial partners to improve existing production methods of commercially available bioproducts. Under Objective 2, progress was made on increasing production of a novel sugar called, isomelezitose. This rare sugar is often produced as a byproduct in small amounts by a group of called glucansucrases. ARS scientists have used genetic engineering to modify one of these enzymes to significantly improve synthesis of isomelezitose. Optimization of production and purification methods have further enhanced yields of this product to levels far in excess of any previously reported studies. ARS scientists have also demonstrated that this unique sugar may be useful in numerous food and biotechnology applications. We are currently working with industrial partners to accelerate the commercialization of this product. ARS scientists have also identified several unique glucansucrase enzymes through our previous genome sequencing efforts. These enzymes were shown to be very efficient for synthesis of novel oligosaccharides, which are comprised of a small number of sugars linked together. These types of oligosaccharides are important because they often encourage growth of probiotics (i.e. healthy bacteria) and have been shown to have anti- inflammatory properties. Efforts are now focused on increasing production of these oligosaccharides for further testing. Other significant progress: 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. ARS scientists recently developed technology to chemically modify tunicamycin so that it has drastically reduced toxicity, while still increasing the efficacy of penicillin- based drugs up to 128-fold. They are now working with other ARS researchers to test these modified tunicamycins in combination with penicillins for the treatment of Johne⿿s Disease, a highly contagious and usually fatal infection in cattle, has been conducted. Accomplishments 01 Improving antimicrobial activity of modified antibiotics. The alarming growth of antibiotic resistance threatens agriculture and human health, so development of new antimicrobial strategies is critical to combating this problem. Tunicamycin is a powerful antibiotic that can be combined with other antibiotics in order to improve their efficacy and often overcome antimicrobial resistance, but toxicity in human and animal cells prevents it from being used for therapeutic applications. ARS scientists in Peoria, Illinois, have developed technology to chemically modify tunicamycin to have significantly less toxicity while still retaining the antimicrobial properties. These same researchers also recently determined that natural structural variants of tunicamycin, which differ in length and branching of an attached fatty acid chain, have significantly altered binding to bacterial cell wall components and distinct antimicrobial activities. This significant discovery will now allow ARS scientists to specifically focus research efforts on synthesis of tunicamycin structures that have the desired characteristics. This will further improve the use of these modified antibiotics and is an important step towards combating drug resistance.
Impacts
(N/A)
Publications
- Ispirli, H., Simsek, ÿ., Skory, C.D., Sagdic, O., Dertli, E. 2018. Characterization of a 4,6-a-glucanotransferase from Lactobacillus reuteri E81 and production of malto-oligosaccharides with immune-modulatory roles. International Journal of Biological Macromolecules. 124:1213-1219.
- Leathers, T.D., Rich, J.O., Bischoff, K.M., Skory, C.D., Nunnally, M.S. 2019. Inhibition of Streptococcus mutans and S. sobrinus biofilms by liamocins from Aureobasidium pullulans. Biotechnology Reports. 21:e00300.
- Hay, W.T., Fanta, G.F., Felker, F.C., Peterson, S.C., Skory, C.D., Hojilla- Evangelista, M.P., Biresaw, G., Selling, G.W. 2019. Emulsification properties of amylose-fatty sodium salt inclusion complexes. Food Hydrocolloids. 90:490-499.
- Saunders, L.P., Bischoff, K., Bowman, M.J., Leathers, T.D. 2018. Inhibition of Lactobacillus biofilm growth in fuel ethanol fermentations by Bacillus. Bioresource Technology. 272:156-161.
- Price, N.P.J., Hartman, T.M., Vermillion, K.E. 2018. Thiazolidine peracetates: carbohydrate derivatives that readily assign cis-, trans-2,3- monosaccharides by gas chromatography - mass spectrometry analysis. Analytical Chemistry. 90(13):8044-8050. doi:10.1021/acs.analchem.8b00976.
- Naumann, T.A., Price, N.P.J. 2018. Purification and in vitro activities of a chitinase-modifying protein from the corn ear rot pathogen Stenocarpella maydis. Physiological and Molecular Plant Pathology 106:74-80.
- Jackson, M.A., Price, N.P., Blackburn, J.A., Peterson, S.C., Kenar, J.A., Haasch, R., Chen, C. 2019. Partial hydrodeoxygenation of corn cob hydrolysate over palladium catalysts to produce 1-hydroxy-2-pentanone. Applied Catalysis A: Genera. 577:52-61.
- Bantchev, G.B., Cermak, S.C., Durham, A.L., Price, N.P. 2019. Estolide molecular weight distribution via gel permeation chromatography. Journal of the American Oil Chemists' Society. 96(4):365-380.
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Progress 10/01/17 to 09/30/18
Outputs
Progress Report 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. 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. 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 �-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. Accomplishments 01 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. 02 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.
Impacts
(N/A)
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 �-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. doi: 10.1016/j.biortech. 2017.09.067.
- 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.
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Progress 10/01/16 to 09/30/17
Outputs
Progress Report 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. 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. 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 2017 include the following: � Aureobasidium strains were genetically modified to produce novel types of the antimicrobial compounds, called liamocins. These naturally occurring bioactive agents have significant potential for applications, such as antibacterial veterinary treatment and agricultural pathogen control. This work was accomplished by genetically altering the metabolism of a cell in order to control the structure of the molecules that are chemically linked to the liamocin backbone. Utilizing various media compositions for the growth of these modified strains further allowed control of the structure. We have previously shown that the antimicrobial activity is highly dependent on the specific structure of the liamocin. This technology allows the production of unique chemical structures, which should improve the biological activity, especially for those situations requiring specific antibacterial spectrums. � Ribonucleic acid (RNA) expression studies were performed on an Aureobasidium pullulans strain that produces significant quantities of liamocin. This new information is critical in discovering genes that are involved in the biosynthesis of this important compound and provides routes for further increasing production through genetic manipulation. � Aureobasidium produces a dark pigment, called melanin, which often contaminates bioproducts. We developed genetic technology for eliminating melanin synthesis in Aureobasidium pullulans, resulting in a superior product for liamocin production. This technology can also be used to improve purification of other Aureobasidium pullulans bioproducts (e.g., pullulan, polymalic acid, enzymes) by eliminating the need to remove contaminating pigments. � Carbonic anhydrase is an enzyme that is involved in converting dissolved carbon dioxide to bicarbonate, which is required for the synthesis of carboxylic acids. We cloned the gene for carbonic anhydrase from the fungus Rhizopus and developed a system for producing the enzyme for further characterization. This work will provide important information that can be used to improve the efficiency of this conversion. � Certain bacteria produce a type of enzyme, called glucansucrase, which is able to synthesize water-insoluble biopolymers from sucrose (i.e., sugar obtained from sugarcane or sugar beets). We discovered that removing part of the enzyme eliminated the ability to produce these polymers and the sucrose was instead converted into short sugar chains, called oligosaccharides. These oligosaccharides are important food ingredients that have potential as low-glycemic carbohydrates and for stimulating the growth of �good� bacteria (i.e., probiotics) in the intestinal tracts of humans and animals. We are currently investigating the potential of these oligosaccharide mixtures with the food industry. � We developed another modification to these glucansucrase enzymes that shifts polymer formation into a valuable trisaccharide (i.e., three sugar molecules linked together), called isomelezitose. This technology allows isomelezitose to be produced in high yields and paves the way towards commercialization. Previous methods for producing this trisaccharide were inefficient, but allowed investigators to demonstrate the importance of this compound for numerous agricultural and pharmaceutical applications. � We demonstrated that isomelezitose can be utilized to stabilize biological cells during freezing and subsequent thawing in a process called cryoprotection. This shows that it will be useful in many biotechnology applications, such as food preservation, biocontrol agents, drug development, and vaccines. � New potential food applications were studied using a novel viscous polysaccharide produced by North American grape species Vitis riparia (frost grape). It was determined that this unique polysaccharide, which can be obtained as an agricultural waste product, has food emulsification properties similar to the commonly used gum arabic. Frost grape polysaccharide may be a better alternative for U.S. food producers, since gum arabic can only be imported and suffers from price volatility. � Cystinosis is a rare disease, usually diagnosed in young children, that causes the amino acid cystine to accumulate in the body and usually results in end stage kidney failure without treatment. Cysteamine is a drug used to treat cystinosis, but it is frequently associated with numerous side effects. We recently developed technology to chemically modify cysteamine in an attempt to minimize some of these side effects. Studies in collaboration with researchers at the University of Leuven, Leuven, Belgium, have looked at the efficacy of using these modified cysteamines in cell culture studies. � Apramycin is an aminoglycoside antibiotic used in veterinary medicine that is produced by the bacterium, Streptoalloteichus tenebrarius. Many of the known antibiotics in this family (e.g., kanamycin, tobramycin, gentamycin, etc.) can cause deafness in susceptible individuals, but it was recently found that apramycin causes very little hearing loss. In collaboration with scientists at Wayne State University, Detroit, Michigan, we have identified novel chemical forms of apramycin and are investigating methods to improve production. Accomplishments 01 Production of a new sugar for food and biomedical applications. As part of an ongoing investigation aimed at using bacteria and enzymes to create new high-value products from cane or beet sugar, ARS researchers in Peoria, Illinois, have engineered bacterial enzymes that are capable of producing high yields of a novel type of sugar for use in the pharmaceutical, agricultural, and food industries. This sugar, named isomelezitose, was previously found in trace amounts in honey, but efforts at commercialization were hampered by production costs. This new enzyme can produce isomelezitose in yields around 50% of the theoretical maximum, a major improvement over previous methods. A patent application has been filed on this invention, and researchers are currently demonstrating potential applications of the novel sugar. These include prebiotic food ingredients for improved intestinal health, and cryopreservatives for improving the long-term storage stability of foods, drugs, vaccines, and agricultural bio-control agents.
Impacts
(N/A)
Publications
- Leathers, T.D., Price, N.P.J., Manitchotpisit, P., Bischoff, K.M. 2016. Production of anti-streptococcal liamocins from agricultural biomass by Aureobasidium pullulans. World Journal of Microbiology and Biotechnology. 32(12):199. doi: 10.1007/s11274-016-2158-5.
- Cote, G.L., Skory, C.D. 2017. Isomelezitose formation by glucansucrases. Carbohydrate Research. 439:57-60.
- Leathers, T.D., Nunnally, M.S., Stanley, A.M., Rich, J.O. 2016. Utilization of corn fiber for production of schizophyllan. Biomass and Bioenergy. 95:132-136.
- Dunlap, C.A., Saunders, L.P., Schisler, D.A., Leathers, T.D., Naeem, N., Cohan, F.M., Rooney, A.P. 2016. Bacillus nakamurai sp. nov., a black pigment producing strain. International Journal of Systematic and Evolutionary Microbiology. 66(8):2987-2991. doi: 10.1099/ijsem.0.001135.
- Price, N.P.J., Bischoff, K.M., Leathers, T.D., Cosse, A.A., Manitchotpisit, P. 2016. Polyols, not sugars, determine the structural diversity of anti- streptococcal liamocins produced by Aureobasidium pullulans strain NRRL 50380. Journal of Antibiotics. 70(2):136-141. doi: 10.1038/ja.2016.92.
- Jackson, M.A., Blackburn, J.A., Price, N.P.J., Vermillion, K.E., Peterson, S.C., Ferrence, G.M. 2016. A one-pot synthesis of 1,6,9,13- tetraoxadispiro(4.2.4.2)tetradecane by hydrodeoxygenation of xylose using a palladium catalyst. Carbohydrate Research. 432:9-16. doi: 10.1016/j. carres.2016.06.003.
- Finkenstadt, V.L., Bucur, C., Cote, G.L., Evans, K.O. 2017. Bacterial exopolysaccharides for corrosion resistance on low carbon steel. Journal of Applied Polymer Science. doi: 10.1002/app.45032.
- Ramazani, Y., Levtchenko, E.N., Van Den Heuvel, L., Van Schepdael, A., Prasanta, P., Ivanova, E.A., Pastore, A., Hartman, T.M., Price, N.P.J. 2017. Evaluation of carbohydrate-cysteamine thiazolidines as pro-drugs for the treatment of cystinosis. Carbohydrate Research. 439:9-15.
- Naumann, T.A., Bakota, E.L., Price, N.P.J. 2017. Recognition of corn defense chitinases by fungal polyglycine hydrolases. Protein Science. 26(6) :1214-1223.
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Progress 10/01/15 to 09/30/16
Outputs
Progress Report 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. 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. 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. Accomplishments 01 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.
Impacts
(N/A)
Publications
- 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.
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