Location: Renewable Product Technology Research
2020 Annual Report
Objectives
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
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 Report
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
1. 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.
Review 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. Article 107893. https://doi.org/10.1016/j.carres.2019.107893.
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.. https://doi.org/10.1080/10242422.2019.1593969.
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. https://doi.org/10.1101/728154.
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. https://doi.org/10.1007/s00284-019-01845-y.
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. https://doi.org/10.1038/s41429-019-0220-x.
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. https://doi.org/10.1016/j.foodchem.2020.126373.
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. https://doi.org/10.1016/j.isci.2019.11.037.
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. https://doi.org/10.1128/aem.01860-18.
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. https://doi.org/10.1016/j.plgene.2019.100214.
