Location: Renewable Product Technology Research
2025 Annual Report
Objectives
The broad goal of this project is to develop improved antimicrobial technologies that can be used in agricultural and biorefining industries. Technologies being investigated in this project not only target important agricultural problems, but they will result in the development of new value-added products made using renewable plant-based materials. We work closely with industrial collaborators, stakeholders, and customers to ensure that these goals are compatible with market needs and will strengthen available antimicrobial technologies, improve sustainable agriculture, and provide economic support to rural communities. Over the next 5 years, we will focus on the following objectives:
Objective 1: Develop technologies for production of small molecule antimicrobial agents and antibiotic adjuvants that enhance the activity of existing antibacterial agents.
Objective 2: Utilize alternative antimicrobial strategies for control of agricultural pathogens and bacterial contamination in biorefineries. Sub-Objective 2.1: Develop effective production and delivery systems for phage endolysins that can be utilized as novel antimicrobials. Sub-Objective 2.2: Identify and express new bacteriocins for control of biorefining contaminants and animal pathogens. Sub-Objective 2.3: Utilize genetically modified A. pullulans strains to generate novel liamocin structures and determine if these antimicrobial agents have the potential to be used for treatment of mastitis. Sub-objective 2.4: Conversion of food waste into microbial protein for food applications.
Objective 3: Resolve existing biocatalytic process issues to enable commercial production of novel biopolymers and oligomers that deliver alternative antimicrobial agents.
Approach
Antibiotics are perhaps one of the most significant medical breakthroughs of the last century, but emerging resistance represents a significant global threat to both the economy and health of humans and livestock. In addition, antibiotics are often used to control microbial contamination in biorefining processes. However, there is growing consensus that antibiotic use should be limited in biorefining and agricultural processes. It is therefore of critical importance that new antibiotic therapies and alternative antimicrobial agents are developed to combat this problem. This work will include continued efforts for commercialization of modified tunicamycins, which enhance the antibacterial activity of beta-lactam antibiotics, and thereby reduce the use of penicillins in agricultural applications. This research will also examine other uncharacterized products that can be used to augment the antibacterial and antifungal efficiency of existing antimicrobial agents. Alternative antimicrobial strategies will focus primarily on the use of microbial oils, bacterial hydrolases, phage endolysins, and antimicrobial peptides (e.g., bacteriocins) to control bacterial contamination in commercial biorefineries and pathogens that infect either plants or animals. Finally, genetically modified glucansucrase enzymes will be used to produce novel biopolymers and oligomers that can be utilized for numerous pharmaceutical, agricultural, and food applications. These efforts will concentrate on methods to optimize production of a unique non-reducing trisaccharide, called isomelezitose, that has been shown to stabilize proteins during desiccation and may be useful in improving the effectiveness of protein-based antimicrobials. Accomplishing these objectives will help overcome significant technical challenges for the development of new and improved antimicrobials. Most importantly, it will lead to better agricultural and biorefining practices by minimizing the reliance on antibiotics, which ultimately benefits both producers and everyday consumers.
Progress Report
This report summarizes progress for this project, which began September 24, 2020, and will terminate on September 23, 2025. Research will be continued under the new project 5010-30600-012-000D, “Precision Biotechnology for High-Value Bioproducts from Agricultural Feedstocks.”
Under Objective 1, significant progress was made on the development of new antimicrobial agents that enhance the activity of existing antibiotics for agricultural livestock. One product developed in this work is called tunicamycin, which can be combined with other antibiotics to improve efficacy and overcome antibiotic resistance. Tunicamycin is normally too toxic to be used for clinical applications but ARS researchers in Peoria, Illinois, developed novel methods to change the chemical structure of this compound so it is no longer toxic, but retains the ability to enhance antibiotic function.
During this project, ARS worked with industrial partners to scale up production of these modified tunicamycins to make them commercially available. The team collaborated with researchers across the world to test these novel compounds against numerous bacterial pathogens and performed structural analysis to gain better insight on the mechanism of action. Toxicity and pharmacokinetic studies (i.e., absorption, distribution, metabolism, and excretion of a drug) were performed in mice using modified tunicamycin to determine the biological effects of this new compound. Most recently, methods were developed for drug delivery to animals, allowing the researchers to examine the safety and effectiveness of the modified tunicamycin in dairy cattle suffering from Johne’s disease. Johne's disease is an untreatable, contagious, and usually fatal infection of ruminants (e.g., cattle, sheep, goats). This work was performed with ARS collaborators in Ames, Iowa, and confirmed that modified tunicamycin is well-tolerated and holds promise as a new antibiotic treatment for numerous animal diseases.
ARS researchers also demonstrated that modified tunicamycins can be combined with polymyxin antibiotics to significantly enhance their efficacy against a large class of pathogens, called Gram-negative bacteria. These bacteria, which include E. coli, Salmonella, Pseudomonas, and Legionella, are particularly problematic because of their prevalence in animal infections and high resistance to antibiotics. Polymyxin antibiotics have been used to treat these pathogens with limited success. However, combining this treatment with modified tunicamycins allows polymyxins to be used at a much lower concentration or in combination with other antibiotics to effectively control the pathogens. This patented technology is expected to have numerous uses in veterinary and health care markets.
In support of Objective 2, considerable progress has been made in developing alternative antimicrobial strategies for control of agricultural pathogens and bacterial contamination in biorefineries. Much of this work focused on a unique group of enzymes called “endolysins” that target and eliminate bacteria by degrading their cell walls. Endolysins can be selected and engineered to only attack specific bacteria, which makes them an excellent alternative to antibiotics. In addition, bacteria are unable to develop resistance mechanisms to avoid endolysins. In this project, several novel endolysins were engineered to target common bacterial contaminants found in fuel ethanol facilities. These contaminants are problematic because they can reduce ethanol production efficiency and lower overall yield. This causes unexpected facility shutdowns and requires costly cleanup procedures.
Specific isolates were identified as having the greatest negative impact on ethanol productivity on brewer’s yeast (used for fermentation production of ethanol), allowing the team to design novel endolysins that target these strains. Endolysin expression and delivery systems were utilized to optimize production and target these harmful contaminants. This involved computational protein modeling and genetic engineering to design endolysins with improved stability and antimicrobial activity under fermentation conditions. The improved endolysins were more effective at targeting the desired contaminants and fully restored ethanol productivity with yields comparable to fermentation of corn mash without contamination.
This cutting-edge technology was also used to design endolysins for possible treatment methods with a poultry disease called necrotic enteritis. Necrotic enteritis is a prevalent and often fatal gastrointestinal disease in poultry caused by the bacterium Clostridium perfringens. Necrotic enteritis not only severely impacts the health and welfare of chickens, but global losses have been estimated to cost the poultry industry $6 billion annually. ARS scientists in collaboration with researchers at University of Maryland Eastern Shore, Princess Anne, Maryland, developed a novel approach to control Clostridium perfringens using an endolysin that eliminated up to 99.99% of the harmful bacteria in laboratory testing and feeding experiments using broiler chickens. These promising results suggests that incorporating this endolysin into chicken feed could serve as an effective strategy to control this devastating disease, thereby reducing dependency on antibiotics.
ARS researchers also worked with manufacturing partners to commercialize another antimicrobial agent called liamocins. Liamocins, discovered by the team, are a microbial oil secreted by yeast isolates. They have potent antimicrobial activity against certain animal pathogens, which makes them desirable for numerous applications. The scientist genetically modified the yeast strains and optimized production conditions for enhanced liamocin synthesis. Two different companies recently obtained co-exclusive license rights on this technology.
In support of Objective 3, researchers developed new methods to improve production of a rare sugar called isomelezitose, which is found in honey. Isomelezitose has been shown to stabilize proteins during drying, which makes it suitable for numerous food and pharmaceutical applications. ARS developed technology to convert cane or beet sugar into this valuable sugar. The team is working with industry partners on scale up methods for isomelezitose and testing it for new applications. They have also begun efforts to convert agricultural sugars from cheese processing into human milk oligosaccharides (HMOs), a key component of infant formula that supports the establishment of a healthy gut microbiome in nursing infants. In addition, there is evidence that HMOs have significant antimicrobial properties against numerous pathogens. Researchers have developed several new rapid detection methods for HMOs that will help with future research efforts.
This research project has been extremely successful in developing innovative biobased products and technologies. The bioproducts developed by this project will create new markets for agricultural producers, job opportunities for rural populations, and support growth of domestic industrial, pharmaceutical, and food businesses.
Accomplishments
1. Improved fuel ethanol production by reducing bacterial contamination without the use of antibiotics. Commercial fuel ethanol facilities utilize brewer’s yeast to convert agricultural sugars from corn, sorghum, sugarcane and sugar beets to ethanol. However, bacterial contamination in fermentation facilities often reduces ethanol yield and productivity, which can result in unexpected biorefinery shutdowns and cause significant economic losses of more than $4.5 million annually for a typical facility. ARS researchers in Peoria, Illinois, developed an innovative and effective method to mitigate bacterial contamination without the use of antibiotics using an engineered enzyme that targets and eliminates the problematic bacteria. This novel antibacterial protein is designed to be produced by the brewer’s yeast, so it is unnecessary to add it during the fermentation. Using this technology fully restores ethanol productivity and yields comparable to fermentation of corn mash without contamination. This technology is expected to reduce production cost of fuel ethanol and helps meet volume requirements for American grown biofuels in the updated Renewable Fuel Standard.
Review Publications
Lu, S.Y., Patel, M., Hector, R.E., Bowman, M.J., Skory, C.D. 2024. Saccharomyces cerevisiae secretion of recombinant bacteriophage endolysin LysKB317 inhibits Limosilactobacillus fermentum in corn mash fermentation. Biofuel Research Journal. https://doi.org/10.18331/BRJ2024.11.4.4.
Compton, D.L., Pero, B.A., Radloff, G.H., Evangelista, R.L., Winkler-Moser, J.K., Kenar, J.A., Cermak, S.C., Appell, M., Evans, K.O., Wegener, E.C., Rheay, H.T., Skory, C.D. 2025. Lipase-catalyzed transesterification of virgin and refined hemp seed oil with ferulic acid ethyl ester. Journal of the American Oil Chemists' Society. https://doi.org/10.1002/aocs.12849.
Bruni, G.O., Terrell, E.C., Klasson, K.T., Qi, Y. 2025. Control of industrially relevant microbial isolates by antimicrobial agents: Implications for sugar factories. Journal of Industrial Microbiology and Biotechnology. 52. Article kuaf001. https://doi.org/10.1093/jimb/kuaf001.
Rheay, H.T., Compton, D.L., Brownstein, K.J., Skory, C.D. 2025. Necessary reporting of reaction yield for method evaluation: Considering Knoevenagel synthesis of ferulic acid. Results in Chemistry. https://doi.org/10.1016/j.rechem.2025.102349.
Colombatti Olivieri, M.A., Price, N.P., Jackson, M.A., Bannantine, J.P. 2025. Evaluation of the cytotoxicity and antibacterial activity of a synthetic tunicamycin derivative against Mycobacterium avium complex. Frontiers in Microbiology. https://doi.org/10.3389/fmicb.2025.1604400.
Qi, Y., Patel, M.H., Lu, S.Y., Skory, C.D. 2025. Genomic insights into persistence, antibiotic resistance, and intraspecific diversity of lactic acid bacterial contaminants at corn dry-grind fuel ethanol facilities. Journal of Bioresource Technology. https://doi.org/10.1016/j.biortech.2025.132836.