Project : USDA ARS

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Research Project: Antimicrobials for Biorefining and Agricultural Applications

Location: Renewable Product Technology Research

2024 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
In support of Objective 1, significant progress was made on the development of novel antibiotic enhancers. ARS researchers developed and patented technology to produce improved antibiotic enhancers called modified tunicamycins, which can be combined with other antibiotics to significantly improve efficacy and even overcome antibiotic resistance. Tunicamycins are produced by a group of bacteria called actinomycetes, but they are not used clinically because of their toxicity. ARS modified tunicamycins called TunR1, TunR2, and OATs (omega-alicyclic tunicamycins) have reduced toxicity with broader antibacterial activities (i.e., target more bacteria). Production of these novel compounds have been scaled up with industrial partners who also license and market tunicamycin made with this ARS technology. In collaboration with ARS researchers in Ames, Iowa, these modified tunicamycins have been examined for safety and effectiveness in mice and bovine. Ongoing data analysis confirms that TunR2 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 concerning because of their prevalence in infections and high resistance to antibiotics. Polymyxin antibiotics have been used to treat these pathogens with limited success. However, combining this treatment with 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 the veterinary and health care markets. Labelled tunicamycins were developed to better study the mode of action for antibiotic enhancers. A novel type of chemical synthesis, called click chemistry, was used to attach a fluorescent molecule to modified tunicamycins. This addition now allows researchers to use microscopic visualization techniques to monitor the interaction of tunicamycin with bacterial pathogens in the laboratory. This method will provide valuable data to further enhance the effectiveness of these compounds. In support of Objective 2, substantial progress was made on development of new antimicrobial protein technology that can be used to control bacterial contamination in commercial biorefineries and pathogens that infect animals. Much of this work focused on a group of enzymes called endolysins that attack the bacterial cell wall and can reduce the presence of unwanted organisms without the use of antibiotics. We previously demonstrated that endolysins are an effective strategy to target common bacterial contaminants associated with fuel ethanol facilities. These contaminants are problematic because they reduce the efficiency of ethanol production and result in lower product yield. Protein engineering was utilized to further improve the stability and activity of these enzymes. These optimized enzymes were tested on corn mash fermentations typical of a bioethanol production facility. They were also tested against biofilms, which often serve as a reservoir of contaminants in holding tanks and pipes. We are also working with collaborators to perform technoeconomic analysis (TEA) and life cycle assessment (LCA) on the use of endolysins for contamination control in a biorefining facility. These efforts include growth medium optimization, bioreactor scaleup, and long-term stability testing. These studies will be important to facilitate commercial transfer of this technology. In support of Objective 3, we began efforts to investigate production of human milk oligosaccharides (HMOs) from agricultural sources. HMOs are sugar chains that are critical for gut health in newborn babies. In addition, there is evidence that HMOs have significant antimicrobial properties against numerous pathogens. We have developed several new rapid enzymatic detection methods for HMOs that will help with future research efforts.

Accomplishments
1. Improved methods for control of bacterial pathogens that can result in serious infection to humans. Polymyxin antibiotics are often used to treat a class of pathogens, called Gram-negative bacteria, which include E. coli, Salmonella, Pseudomonas, and Legionella. These bacteria are challenging because of their prevalence in serious infections and high resistance to antibiotics. ARS researchers at Peoria, Illinois, discovered that polymixin antibiotics can be combined with modified tunicamycin or TunR2, an antibiotic enhancer synthesized using ARS technology, to significantly increase the antimicrobial activity of the polymixin. Combining this treatment with tunicamycins allows polymyxins to be used at much lower concentration or in combination with other antibiotics to effectively eradicate the pathogens. This patented technology provides critically important tools to the veterinary and healthcare markets for combating an ever-growing problem of emerging antibiotic resistant pathogens.

2. New treatment methods to help eliminate a $6 billion poultry disease called necrotic enteritis. Necrotic enteritis (NE) is a prevalent and often fatal gastrointestinal disease in poultry caused by the bacterium Clostridium perfringens. NE not only impacts the health and welfare of chickens, but global losses have been estimated to cost the poultry industry $6 billion annually. ARS scientists in Peoria, Illinois, and Beltsville, Maryland, in collaboration with researchers at University of Maryland Eastern Shore, Princess Anne, Maryland, developed a novel approach to control Clostridium perfringens using an enzyme, called phage endolysin PlyCp41, that has the unique ability to selectively target and destroy the harmful bacterium. This enzyme was produced using modified yeast so that it could eventually be included in animal feed to control NE. This modified yeast was able to eliminate up to 99.99% of the harmful bacteria in laboratory testing and experiments using the contents from chicken intestines. These promising results suggests that incorporating this modified yeast into chicken feed could serve as an effective strategy to control this devastating disease, reducing dependency on antibiotics.

U.S. DEPARTMENT OF AGRICULTURE

Renewable Product Technology Research: Peoria, IL