The discovery of new enzyme pathways and enzymatic targets is a vital part of antimicrobial drug discovery. The inability to culture many microbial strains under laboratory conditions has led to a general lack of access to bacteriological information, including an understanding of microbial metabolism. This high-risk, high-reward program explores a new tool that adapts unites single cell genomic analyses with chemoenzymatic live cell imaging as a means to rapidly identify new enzymatic and biosynthetic features within typically unavailable microorganisms.
. The understanding of primary and secondary metabolic pathways in bacteria plays a dual role for human health by serving as therapeutic targets (primary metabolism) and also as a template for drug discovery (secondary metabolism). While molecular methods to study living systems have advanced significantly in recent years, our ability to access the wealth of metabolic systems in microbes is starkly limited because most microbial species cannot be addressed in the laboratory. This program explores a new `high risk / high reward' approach to uncover features of microbial metabolism by probing at the single microbial cell level. In this application the UC San Diego team begins by exploring the development of optimized fluorescent probes that are used to detect positive enzyme activity, namely phosphopantetheinyltransferase (PPTase), within microbial cells. Next the team then applies synthetic PPTase inhibitors as feedback tools to properly validate the identified microbial species. During this development phase, concurrent efforts at Bigelow Laboratory explore the development and optimization of fluorescently labeled microbes. Once complete, the teams will unite to explore the convergence of these tools to identify novel PPTase enzymes from microbial species present in seawater collected by the Bigelow Laboratory. While marine microbes are known to offer a diversity of PPTases to regulate biosynthetic processes, the majority of marine PPTases have yet to be discovered, and hence they offer an ideal starting point to test this new approach to enzyme and biosynthetic discovery. Overall, this program provides an important step for combining in vivo screening and whole genome sequencing. We have identified a realizable path to rapidly select and address organisms without culturing. Currently, the ability to develop new antibiotics and antibiotic targets has been limited by the culture of specific organisms. The methods described here are not limited to PPTases ? indeed this workflow can be broadly implemented to rapidly screen, identify and characterize new biosynthetic functions at the single cell level using other targets. We anticipate this tool will have an immediate impact for a wide array of applications in the microbial field, and hence presents a valuable advance toward understanding of and treatment of infectious disease.
