Probing the Natural Product Biosynthetic Potential of Actinomycetes Actinosynnema mirum

Natural products are complex organic molecules made by a variety of organisms that often possess bioactivities, making them useful as drugs, drug leads, and biological probes. The focus of my research is on studying natural products made by Actinosynnema mirum, an Actinobacterium whose genome has recently been sequenced. Using bioinformatics tools we have identified and annotated all natural product gene clusters in A. mirum genome, and have predicted the structures and molecular weights of six encoded compounds of interest. We have cultured this organism in large scale and have obtained fractionated natural product extracts which will be analyzed for the predicted compounds using analytical HPLC-ESI mass spectrometry. Once compounds of interest are identified, they will be purified using preparative HPLC, their structures determined by NMR, and their bioactivities assessed by our group and others. I have also developed real-time PCR assays for 13 genes – a GAPDH control and 1 to 2 genes from each of six natural product gene clusters of interest, isolated total mRNA and created corresponding cDNA libraries from each growth phase of A. mirum. These will be used to profile transcriptional activity of the six natural product gene clusters of interest by real-time PCR. From this, we will be able to determine whether, and in which growth phase, each of the six gene clusters is transcriptionally active.  Transcriptional activity suggests that the corresponding natural product is being produced. This data will provide a more global view of regulation of the natural product biosynthesis in A. mirum.