The broad objective of the proposal is to test whether E. coli hosts genetically altered to support complex natural product biosynthesis improve the success of screening environmental DNA libraries for novel and useful antibiotics and consists of the following Specific Aims: (1) To further modify both the E. coli strains engineered to produce polyketides and non-ribosomal peptides, and our BAC vector in which libraries are constructed, to allow screening of large- insert environmental DNA BAC clones in both single and multiple copy number; (2) To construct an environmental library in the modified BAC vector and to screen for antibiotic activity from environmental DNA libraries, in both low and high copy, transferred to native and genetically modified E. coli hosts. In Specific Aim 1, both the E. coli strains currently used for complex natural product production and the vector system used for high-throughput environmental DNA antibiotic screening will be modified and made compatible for efforts to integrate these advanced E. coli heterologous hosts into environmental DNA screening schemes. Aim 2 will then test for advanced antibiotic screening success based upon use of advanced E. coli heterologous hosts. Success will point to a new level of accessibility to as- yet-undiscovered antibiotic agents. Success will provide new antibiotic compounds to be used in the continual battle against antibiotic-resistant bacteria. More broadly, a method will be developed to access more chemically diverse natural products (antibiotics, anticancer agents, immunosuppressants, or others) from previously inaccessible terrestrial and marine natural environments
