Research Project: Antimicrobials for Biorefining and Agricultural Applications

Location: Renewable Product Technology Research

2023 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. 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. We have previously patented technology to produce improved antibiotic enhancers called modified tunicamycins, which can be combined with other antibiotics to improve efficacy and even overcome antibiotic resistance. Tunicamycins are produced by a group of bacteria called actinomyces, 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. We recently completed toxicity studies in fish embryos in collaboration with researchers at the Medical College of Wisconsin, Milwaukee, Wisconsin, and pharmacokinetic studies (i.e., absorption, distribution, metabolism, and excretion of a drug) in mice using modified tunicamycin. We are currently working with ARS collaborators at the National Animal Disease Center in Ames, Iowa, to examine the safety and effectiveness of these TunR2 products in dairy cattle that have contracted Johne’s disease. Johne's disease is an untreatable contagious and usually fatal infection of ruminants (e.g., cattle, sheep, goats). This work includes an ongoing pharmacokinetic analysis of Holstein cattle and testing efficacy if modified tunicamycin against other common pathogens. We have also filed a patent application for technology that improves drug delivery of antibiotics, including tunicamycins, by increasing solubility of antibiotics in water-based solutions. Another goal of this objective was to develop improved polyethylenimine (PEI) polymers for antimicrobial applications. PEI polymers enhance the activity of ß-lactam antibiotics (e.g., penicillin derivatives) by known mechanisms but they are limited by the relatively low solubility of the polymer in water. Our aim was to improve the solubility and the antimicrobial properties by chemically modifying PEI with sugar groups using ARS developed methods. The expectation was that this would lead to modified PEI with improved antimicrobial activities. However, attempts to utilize this technology for modified PEI resulted in limited success because of the poor solubility of the high molecular weight PEI polymers. 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. We used computer protein modeling to design modified endolysins with improved activity against these bacterial contaminants and demonstrated that ethanol production yields were substantially improved when using these novel antimicrobial agents. We have also designed yeast strains that can produce these endolysins against bacterial contamination. We are now working to develop yeast strains that can detect the presence of bacterial contaminants in fuel ethanol facilities and only produce these antimicrobial proteins when necessary. We continue to work with collaborators on producing endolysin against Clostridium perfringens. Clostridium perfringens is the causative agent of necrotic enteritis, which is a significant problem to the poultry, pig, and beef industry, as well as foodborne and non-foodborne human disease. Yeast strains that produce these endolysins were designed and used to supplement poultry feed. The yeast strains not only provide beneficial nutrients but the endolysins produced using ARS technology significantly reduced levels of Clostridium perfringens in poultry. We have made additional improvements on a previously developed ARS technology using antimicrobial peptides (AMPs), which are small proteins that can also be used to inhibit undesirable bacterial strains. We previously cloned an AMP called PEG446 that was identified though genome mining of a bacterial strain called Clostridium tyrobutyricum. Genetic engineering methods were utilized to improve production and scaleup of this AMP. The recovered product was shown to inhibit several different bacterial strains including a Listeria isolate that is a close relative of Listeria monocytogenes, an important foodborne pathogen, and causative agent of human listeriosis.

Accomplishments
1. Improved methods for controlling bacterial contamination associated with ethanol production. Commercial fuel ethanol facilities rely on baker’s yeast to convert agricultural sugars from corn and sugarcane to alcohol, but recurring bacterial contamination can result in expensive plant shutdowns and significant economic losses. Antibiotics are used with other chemical-based products to control contamination even though they are often ineffective or are associated with antibiotic resistance concerns. ARS researchers at Peoria, Illinois, developed an efficient method to control bacterial contamination with an ethanol producing yeast engineered to secrete an enzyme, called endolysin, that targets and kills bacteria commonly associated with contamination at fuel ethanol fermentation facilities. It restores ethanol production efficiency and is expected to reduce production cost associated with bacterial contamination. This innovative non-antibiotic method can potentially combat drug-resistant bacterial contamination by reducing antibiotic usage in fuel ethanol industries. This innovation should enhance ethanol production efficiency while reducing the use of antibiotics and improving the economics of ethanol production.

Review Publications
Lu, S.Y., Liu, S., Patel, M., Glenzinski, K.M., Skory, C.D. 2023. Saccharomyces cerevisiae surface display of endolysin LysKB317 for control of bacterial contamination in corn ethanol fermentations. Frontiers in Bioengineering and Biotechnology. 11. Article 1162720. https://doi.org/10.3389/fbioe.2023.1162720.
Moser, B.R., Doll, K.M., Price, N.P. 2022. Comparison of aliphatic polyesters prepared by acyclic diene metathesis and thiol-ene polymerization of alpha,omega-polyenes arising from oleic acid-based 9-decen-1-ol. Journal of the American Oil Chemists' Society. 100:149-162. https://doi.org/10.1002/aocs.12668
Eller, F.J., Vaughn, S.F., Price, N.P., Kenar, J.A., Jackson, M.A., Berhow, M.A., Brownstein, K.J., Selling, G.W. 2023. Extraction, purification and characterization of an arabinogalactan from frost (riverbank) grape (Vitis riparia michx.) stems. BioResources. 18(3): 4610-4635. https://doi.org/10.15376/biores.18.3.4610-4635.
Liu, S., Lu, S.Y., Qureshi, N., El Enshasy, H.A., Skory, C.D. 2022. Antibacterial property and metagenomic analysis of milk kefir. Probiotics and Antimicrobial Proteins. 14:1170-1183. https://doi.org/10.1007/s12602-022-09976-8.
Vaughn, S.F., Liu, S.X., Berhow, M.A., Moser, J.K., Peterson, S.C., Selling, G.W., Hay, W.T., Jackson, M.A., Skory, C.D. 2023. Production of an odor-reducing, low-dust, clumping cat litter from soybean hulls and soybean hull biochar. Bioresource Technology Reports. 21. Article 101317. https://doi.org/10.1016/j.biteb.2022.101317.
Qureshi, N., Liu, S., Saha, B.C. 2022. Butyric acid production by fermentation: Employing potential of the novel Clostridium tyrobutyricum strain NRRL 67062. Fermentation. 8(10). Article 491. https://doi.org/10.3390/fermentation8100491.
Fabian, M.L., Zhang, C., Sun, J., Price, N.P., Chen, P., Clarke, C.R., Jones, R.W., Stommel, J.R. 2023. Steroidal glycoalkaloids contribute to anthracnose resistance in solanum lycopersicum. Journal of Experimental Botany. 74(12):3700-3713. https://doi.org/10.1093/jxb/erad108.
Selvamani, S., Ramli, S., Dailin, D.J., Natasya, K.H., Varzakas, T., Abomoelak, B., Sukmawati, D., Nurjayadi, M., Liu, S., Gupta, V.K., El Enshasy, H.A. 2022. Extractive fermentation as a novel strategy for high cell mass production of hetero-fermentative probiotic strain Limosilactobacillus reuteri. Fermentation. 8(10). Article 527. https://doi.org/10.3390/fermentation8100527.