Project : USDA ARS

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Research Project: Antimicrobials for Biorefining and Agricultural Applications

Location: Renewable Product Technology Research

2021 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 to 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 antimicrobial product 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 have screened multiple strains of actinomyces bacteria resulting in the discovery of new tunicamycin producing isolates. It was also determined that the structure and function of these tunicamycins could be modified by altering growth conditions. These novel compounds enhanced the antimicrobial efficacy of the whole family of beta-lactam antibiotics, including penicillins, cephalosporins, cephems and penems. In collaboration with researchers in Sweden, we were able to gain new insight into how the structure of tunicamycins affect the antimicrobial activities, allowing us to better target more effective antibiotic enhancers. Our previously developed technology for modified tunicamycins with low toxicity continues to be improved. We were able to scale up production of these ARS modified tunicamycins in collaboration with an industrial partner and these products are now commercially available. We have also set up new collaborations with other partners to further test the toxicity of these modified tunicamycins. This information will be important in order to determine how these products can be used to treat animal diseases. We have also been able to combine two of our previously developed proprietary ARS technologies in order to synthesize novel antimicrobial compounds. This technology resulted in a patent application and a new collaborative agreement with an industrial partner to investigate the commercial potential of these compounds. We have been able to significantly scaleup production of this product for testing. Under Objective 2, considerable progress was made on identification of new alternative microbial strategies. Numerous bacterial genomes were sequenced and mined for antimicrobial products that could be used in biorefining and agriculture applications. Approximately ten different short proteins, called antimicrobial peptides (AMPs), were identified as having the potential to inhibit common contaminants of ethanol biorefining facilities. These AMPs were produced and are currently being screened for antimicrobial activity. We have isolated several new endolysins for use as novel antimicrobials. Endolysins 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 demonstrated that endolysins can be produced by yeast in order to stabilize and deliver these antimicrobial enzymes. Work has also continued 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. We have also partnered with several other collaborators to explore using similar technology to combat other common agricultural and biorefining problems associated with bacterial contamination or disease. Under Objective 3, we have collaborated with other researchers to identify several new rare sugars that have potential as important prebiotics. Prebiotics are non-digestible sugars that can be added to food products to stimulate growth of beneficial microorganisms in the gastrointestinal tract. These prebiotics were made using an enzyme called glucansucrase, which is able to modify cane or beet sugar to create distinct rare sugars with desired prebiotic characteristics. These prebiotics were shown to stimulate growth of favorable bacteria but did not allow growth of pathogenic bacteria, suggesting that they may be useful in animal feed and consumer products. We also utilized ARS proprietary technology to develop microbial strains for improved production of a polysaccharide polymer called pullulan that is used in a number of different food applications. The purity of pullulan made be these new strains is far superior to that obtained using traditional microbial isolates. These strains are being evaluated by a commercial partner for scaleup testing and analysis.

Accomplishments
1. Antibiotic alternative for microbial contamination associated with fuel ethanol production. Most fuel ethanol facilities use baker’s yeast to ferment sugars from agricultural products to alcohol. Bacterial contamination in large-scale production plants is unavoidable, so efforts usually focus on controlling levels of these bacteria. Contaminating bacteria compete for the same sugars that are used by the yeast and they often synthesize by-products that inhibit the ability of the yeast to grow. Chronic and acute contamination problems significantly reduce the economic viability of the U.S. fuel ethanol industry. While antibiotics can be used to control the contamination, alternatives are preferred to avoid overuse of antibiotics to combat these infections and eliminate the presence of antibiotic residues in fuel ethanol coproducts. ARS scientists at Peoria, Illinois, developed technology to control contamination using enzymes found in viruses that target contaminating bacteria. Studies have shown that these novel enzymes are able to reduce contamination 1,000-fold in a typical corn mash fermentation and restore ethanol productivity back to normal. These findings will allow ethanol producers to improve the efficiency of their fermentation and reduce the use of antibiotics in their plants.

2. Improved production of the polysaccharide pullulan for food applications. Pullulan is a commonly used versatile food ingredient because it is water soluble, tasteless and odorless, and has excellent film-forming properties for applications such as production of non-animal derived capsules. This natural polysaccharide is produced by a common fungus that produces melanin pigment as a byproduct, which contaminates the pullulan and must be removed through costly and difficult cleanup procedures. ARS scientists at Peoria, Illinois, developed technology to prevent the melanin from being produced, thereby eliminating the need for removal. Pullulan made using this technology had improved yield with significantly higher purity. This technology not only benefits pullulan producers but can also be applied to other microbial production methods, such as synthesis of antifungal agents, where melanin pigment is problematic.

3. Identification of microbial genes associated with alcohol tolerance. Yeasts are traditionally used for fuel ethanol production, but bacterial strains are often utilized for biomass fermentations of energy crops or agricultural residues due to their ability to utilize many types of sugars and produce alternative fuel alcohols, such as butanol. Most bacteria are inhibited by the presence of these alcohols, which limits their ability to accumulate product in high yields. ARS scientists at Peoria, Illinois, identified several key genes associated with microbial alcohol tolerance by studying the genomes and gene expression of two different bacteria in response of increased ethanol or butanol content in their growth environments. These findings will benefit research communities in better understanding alcohol tolerance and will aid in designing strategies to develop tolerant strains for improved production of biofuels.

U.S. DEPARTMENT OF AGRICULTURE

Renewable Product Technology Research: Peoria, IL