Location: Renewable Product Technology Research
2017 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
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
1. 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.
Review 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.
