Historically, small molecule metabolites produced by bacteria have been a major source of lead compounds in the development of clinically used antibiotics - representing more than 60% of the FDA approved anti-infective agents currently on the market. Many of these clinically useful antibiotics are the metabolites of cultured bacteria bu the majority of environmental bacteria cannot be explored for antibiotic production using traditional laboratory techniques. It is possible to access the untapped biosynthetic potential of this uncultured majority of environmental bacteria by isolating and directly cloning their high molecular weight DNA from the environment (eDNA) and expressing the DNA in heterologous bacterial hosts. Cosmid libraries constructed from eDNA typically contain tens of millions of clones, a number that ensures the capture of the genomes of the majority of previously inaccessible bacteria but which presents a financial and logistical challenge to those wishing to screen these libraries for the production of metabolites. This proposal will address these aforementioned issues by developing and improving sequence-targeting tools to allow for the rapid exploration of environmental DNA for novel antibiotic production. As a proof of concept, these new approaches will be evaluated for their ability to identify new derivatives with potentially improved antibiosis activity for the recently discovered antibiotic, teixobactin. New methods will also be developed and employed in the discovery of structurally novel antibiotics linked only to teixobactin by the biosynthesis of its rare non-proteinogenic amino acid, enduracididine.
For Aim 1 of this proposal, the trainee will first sequence and clone a diverse set eDNA samples identified to be rich in the target gene clusters.
In Aim 2, the trainee will screen and annotate the metagenomic libraries for teixobactin-like NRPS genes and enduracididine biosynthesis genes and the recovery of gene clusters containing these genes.
In Aim 3, the trainee will heterologous express the recovered and reassembled gene clusters and complete the functional analysis of antibiotics encoded by these clusters. All together, these endeavors will not only result in innovative tools to fully explore the environment for novel and otherwise inaccessible antibiotics, but will also tangibly generate new natural teixobactin derivatives and medically significant enduracididine-containing molecules.
Bacterial natural products have led to the development of more than 60% of the FDA approved anti-infective agents, but only about 1% of environmental bacteria can be cultured in the laboratory setting. It is possible to access the untapped biosynthetic potential of this uncultured majority of environmental bacteria by isolating and cloning their DNA into an amenable host strain for heterologous expression. This proposal will develop and improve metagenomic screening tools to allow for the rapid exploration of environmental DNA for novel antibiotic production.
| Hover, Bradley M; Kim, Seong-Hwan; Katz, Micah et al. (2018) Culture-independent discovery of the malacidins as calcium-dependent antibiotics with activity against multidrug-resistant Gram-positive pathogens. Nat Microbiol 3:415-422 |
