Recombinant Environmental Libraries Provide Access to Microbial Diversity for Drug Discovery from Natural Products

ABSTRACT To further explore possible avenues for accessing microbial biodiversity for drug discovery from natural products, we constructed and screened a 5,000-clone “shotgun” environmental DNA library by using an Escherichia coli-Streptomyces lividans shuttle cosmid vector and DNA inserts from microbes derived directly (without cultivation) from soil. The library was analyzed by several means to assess diversity, genetic content, and expression of heterologous genes in both expression hosts. We found that the phylogenetic content of the DNA library was extremely diverse, representing mostly microorganisms that have not been described previously. The library was screened by PCR for sequences similar to parts of type I polyketide synthase genes and tested for the expression of new molecules by screening of live colonies and cell extracts. The results revealed new polyketide synthase genes in at least eight clones. In addition, at least five additional clones were confirmed by high-pressure liquid chromatography analysis and/or biological activity to produce heterologous molecules. These data reinforce the idea that exploiting previously unknown or uncultivated microorganisms for the discovery of novel natural products has potential value and, most importantly, suggest a strategy for developing this technology into a realistic and effective drug discovery tool.

[1]  J. Short.  Molecular Evolution: Recombinant approaches for accessing biodiversity , 1997, Nature Biotechnology.

[2]  Eugene W. Myers,et al.  Basic local alignment search tool. Journal of Molecular Biology , 1990 .

[3]  J. Pernodet,et al.  Characterization of a Glycosyl Transferase Inactivating Macrolides, Encoded by gimA from Streptomyces ambofaciens , 1998, Antimicrobial Agents and Chemotherapy.

[4]  R Amann,et al.  Analysis of broad-scale differences in microbial community composition of two pristine forest soils. , 1998, Systematic and applied microbiology.

[5]  T. Kieser.  Practical streptomyces genetics , 2000 .

[6]  D. Hopwood.  Genetic manipulation of Streptomyces : a laboratory manual , 1985 .

[7]  C. Thompson,et al.  DNA cloning in Streptomyces: resistance genes from antibiotic-producing species , 1980, Nature.

[8]  C. Thompson,et al.  Construction of a series of pSAM2-based integrative vectors for use in actinomycetes. , 1990, Gene.

[9]  J Davies,et al.  A study of iterative type II polyketide synthases, using bacterial genes cloned from soil DNA: a means to access and use genes from uncultured microorganisms , 1997, Journal of bacteriology.

[10]  K. Schleifer,et al.  Phylogenetic identification and in situ detection of individual microbial cells without cultivation. , 1995, Microbiological reviews.

[11]  G. Wahl,et al.  Cosmid vectors for rapid genomic walking, restriction mapping, and gene transfer. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[12]  E. Myers,et al.  Basic local alignment search tool. , 1990, Journal of molecular biology.

[13]  P. Branny,et al.  Antibiotic resistance gene cassettes derived from the omega interposon for use in E. coli and Streptomyces. , 1997, Gene.

[14]  P. Simonet,et al.  Quantification of bacterial subgroups in soil: comparison of DNA extracted directly from soil or from cells previously released by density gradient centrifugation. , 2001, Environmental microbiology.

[15]  P Green,et al.  Base-calling of automated sequencer traces using phred. II. Error probabilities. , 1998, Genome research.

[16]  N. Pace,et al.  Novel Division Level Bacterial Diversity in a Yellowstone Hot Spring , 1998, Journal of bacteriology.

[17]  J. Handelsman,et al.  The Earth's bounty: assessing and accessing soil microbial diversity. , 1999, Trends in biotechnology.

[18]  J. Short.  Recombinant approaches for accessing biodiversity. , 1997, Nature biotechnology.

[19]  M. Gilman,et al.  Expression and isolation of antimicrobial small molecules from soil DNA libraries. , 2001, Journal of Molecular Microbiology and Biotechnology.

[20]  N. Pace.  A molecular view of microbial diversity and the biosphere. , 1997, Science.

[21]  W. Wade,et al.  Design and Evaluation of Useful Bacterium-Specific PCR Primers That Amplify Genes Coding for Bacterial 16S rRNA , 1998, Applied and Environmental Microbiology.

[22]  L. Bakken,et al.  Recovery of Bacterial Cells from Soil , 1995 .

[23]  J Davies,et al.  Novel natural products from soil DNA libraries in a streptomycete host. , 2000, Organic letters.

[24]  P. Green,et al.  Consed: a graphical tool for sequence finishing. , 1998, Genome research.

[25]  P. Green,et al.  Base-calling of automated sequencer traces using phred. I. Accuracy assessment. , 1998, Genome research.

[26]  J. Handelsman,et al.  Cloning the Soil Metagenome: a Strategy for Accessing the Genetic and Functional Diversity of Uncultured Microorganisms , 2000, Applied and Environmental Microbiology.

[27]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[28]  J. Ishikawa,et al.  FramePlot: a new implementation of the frame analysis for predicting protein-coding regions in bacterial DNA with a high G + C content. , 1999, FEMS microbiology letters.