Location: Renewable Product Technology Research
2022 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
Under Objective 1, significant progress was made on work with the antibiotic enhancer called tunicamycin, 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. We developed patent pending technology to introduce new fatty acid chains into the tunicamycin molecules, resulting in novel antibiotics called OATs (omega-alicyclic tunicamycins). These compounds were produced by tunicamycin-producing Streptomyces strains grown with various non-natural small acids. The result is uniquely modified OATs with altered fatty acids attached. These were shown to have improved antibacterial activities and lower toxicity.
We continue to work with industrial partners to improve scale up of ARS technology related to tunicamycins. Commercial sales of these ARS modified tunicamycins for research have generated further interest in the potential use of these antibiotic enhancers. We are working with collaborators to examine the safety of these products in animals and efficacy against common pathogens. This work includes an ongoing pharmacokinetic study (absorption, distribution, metabolism, and excretion of a drug) of modified tunicamycins in mice and toxicity studies in fish embryos.
We demonstrated that a novel microbial oil, called liamocins, can inhibit growth of the highly contagious mastitis pathogen Streptococcus in milk samples. Liamocin is produced using proprietary ARS technology from the yeast Aureobasidium. We previously showed that this compound is an effective antimicrobial agent against Streptococcus, a common pathogen of humans and animals. Mastitis is prevalent in dairy cattle and a source of economic loss for the industry. This study demonstrates that liamocins may be an effective treatment.
We are working with industrial partners to test proprietary antimicrobial compounds made from carbohydrates using ARS technology. These unique compounds, called C-glycoside carbohydrate amines, also have detergent properties that make them attractive for some specialty applications. Production of these compounds have been scaled up for further product testing.
Under Objective 2, considerable progress was made on development of new alternative microbial strategies. Much of this work focused on endolysins, which are a unique group of enzymes isolated from bacterial viruses that attack the bacterial cell wall and can reduce the presence of unwanted organisms without the use of antibiotics. We previously showed that that endolysins are an effective strategy to target common contaminants associated with fuel ethanol facilities. We recently identified numerous novel endolysins by sequencing over 100 microbial genomes. Expression systems for many of these antimicrobial proteins were optimized to improved production and testing. One of these expression systems utilized production of these endolysin in the same yeast strains that are used to produce fuel ethanol. Endolysin were produced in a manner where they are anchored to the yeast cells for more effective targeting and eradication of contaminating organisms.
We are using computer protein modeling to design endolysins with improved activity and stability. Portions of different endolysins are combined to create chimeric proteins with optimal function. We have examined a number of these novel endolysins for potential control of contaminating organisms associated with fuel ethanol facilities.
We continue to work with collaborators on producing endolysin with antibacterial activity 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. Preliminary results suggest that endolysins produced using ARS technology can significantly reduce levels of Clostridium perfringens in poultry.
We have been working on expressing antimicrobial peptides (AMPs) that have inhibitory activity against foodborne pathogens, such as Campylobacter. Inhibiting this bacterium in poultry, cattle, and swine, without the use of antibiotics is a high priority in the feed industry. We have developed methods for scaling up production of several promising AMPs and are working with other ARS collaborators to test their efficacy.
Under Objective 3, we have developed new methods for improved production of the rare sugar isomelezitose, which is produced using proprietary ARS technology. An enzyme called glucansucrase is used to convert cane or beet sugar into a novel sugar that has been shown to stabilize proteins during drying. This attribute makes it beneficial for numerous applications in the pharmaceutical and food industries. This research also involved the development of new enzyme systems to remove contaminating sugars. We are currently working with industrial collaborators on scale up methods for isomelezitose and testing for new applications.
Accomplishments
1. Produced novel antibiotic enhancers to overcome penicillin-resistant pathogens. Penicillins are a class of antibiotics that are used to treat a wide range of human and veterinary bacterial infection. Unfortunately, their effectiveness has decreased over the years with development of penicillin-resistant pathogens. Tunicamycin is a natural product that can be combined with penicillins to overcome this resistance, but 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. ARS scientists further improved this technology by discovering that the structure of tunicamycins can also be modified by altering growth conditions of the tunicamycin-producing Streptomyces strains. These novel antibiotics called OATs (omega-alicyclic tunicamycins) have improved antibacterial activities and lower toxicity. ARS is working with collaborators to improve scaleup of these antibiotics and perform testing on animal and human pathogens. This discovery further expands available ARS technology of low toxicity antibiotic enhancers that have potential to treat animal diseases.
Review Publications
Yu, L., Zhou, W., She, Y., Ma, H., Cai, Y., Jiang, M., Deng, Z., Price, N.P., Chen, W. 2021. Efficient biosynthesis of nucleoside cytokinin angustmycin A containing an unusual sugar system. Nature Communications. 12. Article 6633. https://doi.org/10.1038/s41467-021-26928-y.
Jackson, M.A., Evans, K.O., Price, N.P.J., Blackburn, J.A., Ward, C.J., Ray, K.J., Vermillion, K. 2021. New family of surfactants from biobased materials. ACS Sustainable Chemistry & Engineering. 9(41):13842-13850. https://doi.org/10.1021/acssuschemeng.1c04703.
Lu, S.Y., Skory, C.D., El Enshasy, H.A., Liu, S. 2021. Fermentative production of alternative antimicrobial peptides and enzymes. Biocatalysis and Agricultural Biotechnology. 37. Article 102189. https://doi.org/10.1016/j.bcab.2021.102189.
Ispirli, H., Bowman, M.J., Skory, C.D., Dertli, E. 2021. Synthesis and characterization of cellobiose-derived oligosaccharides with bifidogenic activity by glucansucrase E81. Food Bioscience. 44(Part A). Article 101388. https://doi.org/10.1016/j.fbio.2021.101388.
