Random Mutagenesis of the Aspergillus oryzae Genome Results in Fungal Antibacterial Activity

Multidrug-resistant bacteria cause severe infections in hospitals and communities. Development of new drugs to combat resistant microorganisms is needed. Natural products of microbial origin are the source of most currently available antibiotics. We hypothesized that random mutagenesis of Aspergillus oryzae would result in secretion of antibacterial compounds. To address this hypothesis, we developed a screen to identify individual A. oryzae mutants that inhibit the growth of Methicillin-resistant Staphylococcus aureus (MRSA) in vitro. To randomly generate A. oryzae mutant strains, spores were treated with ethyl methanesulfonate (EMS). Over 3000 EMS-treated A. oryzae cultures were tested in the screen, and one isolate, CAL220, exhibited altered morphology and antibacterial activity. Culture supernatant from this isolate showed antibacterial activity against Methicillin-sensitive Staphylococcus aureus, MRSA, and Pseudomonas aeruginosa, but not Klebsiella pneumonia or Proteus vulgaris. The results of this study support our hypothesis and suggest that the screen used is sufficient and appropriate to detect secreted antibacterial fungal compounds resulting from mutagenesis of A. oryzae. Because the genome of A. oryzae has been sequenced and systems are available for genetic transformation of this organism, targeted as well as random mutations may be introduced to facilitate the discovery of novel antibacterial compounds using this system. 1. Introduction Antibiotic resistant microorganisms became a medical concern shortly after antibiotics became readily available in the 1940s. In fact, resistance has typically been identified within four years of Food and Drug Administration approval of antibacterial agents [1]. Currently, multidrug-resistant bacteria such as MRSA, coagulase negative Staphylococci, and Enterococci cause severe infections in both hospital and community settings [2]. Development of new drugs to combat the increasing host of drug resistant microorganisms is essential if we are to avoid the emergence of pathogens for which there exist no effective antimicrobial therapies. Most classes of antibiotics were developed from natural products produced by fungi or filamentous bacteria. Significantly, the majority of antibiotics still commonly used today are natural product compounds, or their derivatives, discovered during the “golden era” of antibiotic discovery from the 1940s through the 1960s [3]. More recent efforts to generate new antibiotics based on high-throughput, target-focused screening of large libraries of synthetic compounds have largely