As antibiotic resistance continues to rise, the development of new antibiotics is becoming increasingly necessary. TLN-05220 is a natural product antibiotic biosynthesized by a strain of soil-derived Actinobacteria, Micromonospora echinospora. Researchers have found that TLN-05220 exhibits more potent bioactivity against methicillin-resistant Staphylococcus aureus (MRSA) than vancomycin, the current “last-resort” antibiotic. Hence, this molecule could be valuable for new antibiotic development. The purpose of this study is to investigate which genes in the M. echinospora genome are responsible for biosynthesis of the unique polyketide core and nitrogenated-ring system found in TLN-052250’s structure. To accomplish this, we are employing two techniques in parallel. The first technique, Transformation Associated Recombination (“TAR”) Capture, uses homologous recombination in yeast to clone the gene cluster of interest, which can then be expressed in a biosynthetically null strain of Streptomyces sp. Products are characterized to determine whether TLN-05220 is produced. We have cloned four constructs to capture different combinations of clustered genes, which will help determine the minimal genes necessary for production of TLN-05220. These constructs will be transformed into yeast with genomic DNA fragments to capture the desired genes. To optimize this intensive transformation protocol and investigate yeast’s ability to recombine linear DNA fragments, we developed a screen using a green fluorescent protein (GFP) construct. Successful transformants are analyzed for recombination under ultraviolet light, and the results of this screen inform our process with M. echinospora genomic DNA. The second technique is an amino acid feeding study, in which M. echinospora colonies are supplemented with radiolabeled amino acids and products are analyzed via MALDI-TOF spectrometry. To date, this technique has confirmed the incorporation of alanine and glycine, and additional building blocks are being investigated. Ultimately, the deep understanding of TLN-05220 biosynthesis gained from this investigation is critical for use of TLN-05220 in future antibiotic development.

Cloning strategies and feeding studies to investigate antibiotic biosynthesis

2019 Fall National Meeting

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