This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).
This Small Business Technology Transfer (STTR) Phase II project offers a novel route to finding critically needed new antibiotics. The emergence of antibiotic-resistant bacterial pathogens is a growing medical challenge, urgently requiring new drugs. Natural products, synthesized primarily by environmental microorganisms, have supplied most of the current arsenal of effective antibiotics. However, the discovery rate of new antibiotics has greatly diminished. With the recent understanding that the vast majority of environmental microorganisms have never been screened for the production of antibiotics because they cannot be easily cultured in the laboratory, EarthGenes has developed a technology to access these organisms, involving extracting environmental DNA, cloning large fragments into specialized vectors to create DNA libraries, expressing these libraries in suitable easily-grown surrogate hosts, and screening the libraries for antibiotics encoded by the environmental DNA. Professor Blaine Pfeifer at Tufts University has developed the most advanced bacterial host for expressing environmental DNA, potentially improving the efficiency of this technology. Thus, the EarthGenes-Tufts collaboration is designed to lead to the discovery of new, more potent antibiotic drugs.
The broader impacts of this research include a technology to provide a new, continuous supply of potent antibiotics to treat infectious diseases, thus addressing a critical health-related goal with technical innovation. The technology can also be extended to other disease areas. The impact is augmented by education and outreach, including the education of undergraduates, graduate students, and postdoctoral associates, with mechanisms in place to attract underrepresented students from diverse backgrounds.
1. Intellectual merit The increase in antibiotic resistance among microbial pathogens is an urgently growing medical challenge requiring novel antibiotic treatments. Natural products and their derivatives have been a primary source for antibacterial and other drugs, but the discovery rate of new antibacterial natural products from cultured microbes has greatly diminished. The knowledge that most environmental microorganisms have never actually been screened for new antibiotics because they cannot be cultured by standard methods has spurred development of new technologies to access their enormous potential to provide novel natural products. EarthGenes’ technology focuses on natural product discovery based upon screening environmental DNA libraries. In our approach, large DNA fragments taken directly from the environment (both soil and marine) are cloned into suitable vectors to create eDNA (environmental DNA) libraries. The libraries are then introduced into suitable bacterial hosts to express the cloned genes and screen them for the production of a natural product, thus bypassing the need to cultivate the original producing organisms in the laboratory (See Fig. 1). Our process thus allows access to the more than 99% of environmental organisms that have never been screened previously, providing a vast potential source of new natural product drugs. The bacterium E. coli has been used as a host to screen environmental libraries for new antibiotic activities because it is the easiest to handle and manipulate genetically. However, despite its ease of use, E. coli cannot efficiently express polyketides and non-ribosomal peptides, common classes of molecules often found in antibacterial natural products. However, the Pfeifer laboratory has created genetically engineered strains of E. coli ("advanced" E. coli strains) that synthesize precursors and modifying enzymes required for polyketide and nonribosomal peptide production, in an effort to improve the ability of E. coli to serve as an effective screening host for detecting new antibacterial compounds. Therefore, the EarthGenes-Pfeifer laboratory phase II objectives were to screen marine and soil-derived metagenomic libraries in the advanced E. coli bacterial host, with the goal of using this host as an aide in discovering new antibiotic hits. During the course of this grant we synthesized and validated bacterial strains and vectors required for building environmental DNA libraries and screening them in normal and advanced E. coli hosts, constructed 4 environmental libraries and screened one marine and one soil library for antibacterial activities, and identified 13 antibacterial hits, which will be analyzed further to determine their suitability for structural analysis and potential further development. 2. Broader impact. Development of health-related technology. The broader impact of this phase II STTR grant includes technology development and an industry-academic collaboration with the potential to provide new, more potent antibiotic drugs to treat infectious diseases. In addition to the obvious health-related impact, the technology feeds into a large commercial infrastructure, given the size and growing demand of the antibiotics market. Thus, the EarthGenes-Tufts partnership has provided technical innovation towards a critical health-related goal, with a strong need and market providing commercialization potential. Diversity and education. Several other broader impact components critical to the mission of the NSF also pertain. As a woman-owned small business, EarthGenes aligns with the diversity and outreach themes stressed by the NSF. Further, the STTR mechanism, by definition, has provided an avenue for education and additional outreach, and the grant has provided training at Tufts for undergraduate and graduate students of diverse backgrounds, and for postdoctoral associates.
