Technologies for the rapid engineering of bacterial chromosomes with synthetic DN

DESCRIPTION (provided by applicant): The long term goal of the project is to develop enabling technology for the synthesis of long DNA sequences and for their insertion into bacterial chromosomes with concomitant replacement or deletion of unwanted or unneeded host DNA. Our initial studies will concentrate on developing the technology for the rapid metabolic engineering of E. coli for the production of pharmaceutically active polyketide natural products. The chromosome engineering approach that we will pursue will require the development of two technical components: (A) the high-throughput, economical synthesis of long (30-50 kb) DNAs and (B) the ability to efficiently and specifically insert these large DNAs into bacterial chromosomes. The specific aims of the proposal are to: (1) Use long synthetic gene gorging (lsGG) to replace a model singe-gene target in E. coli with varying lengths of synthetic DNA (5 to 32 kb) and to measure the efficiency of lsGG- mediated single- gene target replacement as a function of DNA length. The DNA donor sequence will be comprised of synthetic DNA encoding one to six modules of the DEBS polyketide synthase, the megaenzyme that catalyzes the biosynthesis of 6-deoxyerythronolide B (6- dEB), the macrolide core of the antibiotic erythromycin. The DEBS synthetic DNA has already been synthesized and is functionally expressed in E. coli. (2) Use lsGG to replace a large 82 kb chromosomal segment of E. coli with up to 32 kb of synthetic DNA. (3) Use lsGG to replace a second large chromosomal segment in the modified strain produced in aim #2. The synthetic DNA donor sequence, which has already been prepared and functionally expressed in E. coli, encodes the biosynthetic pathway for the hydroxylation and glycosylation of 6-dEB. The ability to rapidly engineer bacterial chromosomes with long synthetic DNAs would have broad implications in human health. Potential applications of the technology include the rapid development of engineered bacterial strains that either (a) abundantly produce pharmaceutically important natural products, available only in limited quantities from natural hosts or (b) catalyze the degradation or bioremediation of environmental toxins.