title: Genetically modified bacterial strains and novel bacterial artificial chromosome shuttle vectors for constructing environmental libraries and detecting heterologous natural products in multiple expression hosts.
abstract: The enormous diversity of uncultured microorganisms in soil and other environments provides a potentially rich source of novel natural products, which is critically important for drug discovery efforts. Our investigators reported previously on the creation and screening of an Escherichia coli library containing soil DNA cloned and expressed in a bacterial artificial chromosome (BAC) vector. In that initial study, our group identified novel enzyme activities and a family of antibacterial small molecules encoded by soil DNA cloned and expressed in E. coli. To continue our pilot study of the utility and feasibility of this approach to natural product drug discovery, we have expanded our technology to include Streptomyces lividans and Pseudomonas putida as additional hosts with different expression capabilities, and herein we describe the tools we developed for transferring environmental libraries into all three expression hosts and screening for novel activities. These tools include derivatives of S. lividans that contain complete and unmarked deletions of the act and red endogenous pigment gene clusters, a derivative of P. putida that can accept environmental DNA vectors and integrate the heterologous DNA into the chromosome, and new BAC shuttle vectors for transferring large fragments of environmental DNA from E. coli to both S. lividans and P. putida by high-throughput conjugation. Finally, we used these tools to confirm that the three hosts have different expression capabilities for some known gene clusters.
authors.affiliation: Cambridge Genomics Center, Aventis Pharmaceuticals Inc., Cambridge, Massachusetts 02139, USA. Asuncion.martinez@aventis.com
authors.forename: Asuncion
authors.initials: A
authors.lastname: Martinez
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Kolvek
authors.forename: Choi Lai Tiong
authors.initials: CL
authors.lastname: Yip
authors.forename: Joern
authors.initials: J
authors.lastname: Hopke
authors.forename: Kara A
authors.initials: KA
authors.lastname: Brown
authors.forename: Ian A
authors.initials: IA
authors.lastname: MacNeil
authors.forename: Marcia S
authors.initials: MS
authors.lastname: Osburne
mesh_terms: D000900:Anti-Bacterial Agents / Q000096:biosynthesis; D001483:Base Sequence; D001688:Biological Products / Q000302:isolation & purification; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D004783:Environmental Microbiology; D004926:Escherichia coli / Q000235:genetics; D017353:Gene Deletion; D015870:Gene Expression; D015723:Gene Library; D005798:Genes, Bacterial; D005822:Genetic Vectors; D005810:Multigene Family; D016958:Pseudomonas putida / Q000235:genetics; D013302:Streptomyces / Q000235:genetics
chemical_list: D000900:Anti-Bacterial Agents; D001688:Biological Products; D017931:DNA Primers