Genetically Modified Bacterial Strains and Novel Bacterial Artificial Chromosome Shuttle Vectors for Constructing Environmental Libraries and Detecting Heterologous Natural Products in Multiple Expression Hosts
Open Access
- 1 April 2004
- journal article
- Published by American Society for Microbiology in Applied and Environmental Microbiology
- Vol. 70 (4), 2452-2463
- https://doi.org/10.1128/aem.70.4.2452-2463.2004
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.Keywords
This publication has 36 references indexed in Scilit:
- Recombinant Environmental Libraries Provide Access to Microbial Diversity for Drug Discovery from Natural ProductsApplied and Environmental Microbiology, 2003
- Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440Environmental Microbiology, 2002
- Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2)Nature, 2002
- POLYKETIDE PRODUCTION BY PLANT-ASSOCIATED PSEUDOMONADSAnnual Review of Phytopathology, 1999
- High efficiency intergeneric conjugal transfer of plasmid DNA from Escherichia coli to methyl DNA-restricting streptomycetesFEMS Microbiology Letters, 1997
- Characterization of a Genomic Locus Required for Synthesis of the Antibiotic 2,4-diacetylphloroglucinol by the Biological Control AgentPseudomonas fluorescensQ2-87Molecular Plant-Microbe Interactions®, 1996
- Site-specific recombination: developments and applicationsCurrent Opinion in Biotechnology, 1994
- Engineered Biosynthesis of Novel PolyketidesScience, 1993
- Cloning and expression in a heterologous host of the complete set of genes for biosynthesis of the Streptomyces coelicolor antibiotic undecylprodigiosinGene, 1990
- 16S rRNA sequences reveal numerous uncultured microorganisms in a natural communityNature, 1990