Aflatoxin detoxification

Abstract

Aflatoxins (AFs) are secondary fungal metabolites that contaminate common food crops and are harmful to humans and animals. The ability to remove AFs from feed commodities will improve health standards and counter the economic drain inflicted by AF contamination. Strategies to mitigate AF contamination fall into three categories: physical, chemical, and biological. In this thesis, I explore the identification of degraders and degradation mechanisms, as well as their enhancement, within the context of chemical and biological strategies. Known chemical strategies have used strong acids and bases to remove contaminating AF, but these methods often lead to ecological waste issues downstream. Chapter 3 investigates the application of weaker acidic and alkaline conditions to remove two types of AFs, AFB1 and AFG2. I find that a weakly alkaline environment is sufficient to degrade AF, providing an alternative solution for chemical decontamination. Biodetoxification is a promising solution to AF contamination because of its low cost and few undesired environmental side-effects. Microbes possess a rich potential for removing toxins and pollutants from the environment. Despite the fairly wide availability of this potential, identifying suitable candidates and improving them remain challenging. In Chapter 2, I explore the use of computational tools to discover strains and enzymes that detoxify harmful toxins. Of focus is the detoxification of mycotoxins by biological enzymes. Existing computational tools can be used to address questions in the discovery of new detoxification potential, the investigation the cellular processes that contribute to detoxification, and the improvement of detoxification potential in discovered enzymes. I showcase open bioremediation questions where computational researchers can contribute and highlight relevant existing and emerging computational tools that could benefit bioremediation researchers. In Chapter 4, I screen several environmental isolates for their AF detoxification ability, using AFG2. I used different carbon sources (glucose and starch) as isolation and culturing media to examine the effect of the environment on degradation ability. Overall, I find that starch medium expedites the screening process and generally improves the performance of isolates, making this a promising method for identifying new degraders and enhancing their performance. Chapter 5 highlights the characterization of degradation by two promising Rhodococcus species, R. erythropolis and R. pyridinivorans. While previous work has identified their degradation ability, further investigation into degradation mechanisms has been understudied. Here, I explore the characterization of degradation mechanisms toward enzyme identification. Finally, the appendix starts to broach the question of enhancing degradation of known degrading enzymes, the example here is laccase from the fungus Trametes versicolor. Using molecular dynamic and quantum mechanics simulations to identify mutations of interest in increasing the affinity of laccase toward AF, I create five mutants to test their degradation against the performance of wildtype. These mutants show a range of improvements against AF and showcase the efficacy of this approach to enhancement. Together, this body of work highlights the importance of understanding AF degradation for the creation of new strategies of AF mitigation. My thesis provides a framework for developing AF decontamination strategies, from identifying degraders and unlocking their mechanisms to enhancing their performance.