Activation and Characterization of a Cryptic Polycyclic Tetramate Macrolactam Biosynthetic Gene Cluster

Polycyclic tetramate macrolactams (PTMs) are a widely distributed class of natural products with important biological activities. However, many of them have not been characterized. Here we apply a plug and play synthetic biology strategy to activate a cryptic PTM biosynthetic gene cluster SGR810-815 from Streptomyces griseus and discover three potential PTMs. This gene cluster is highly conserved in phylogenetically diverse bacterial strains and contains an unusual hybrid polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) which resembles iterative PKSs known in fungi. To further characterize this gene cluster, we use the same synthetic biology approach to create a series of gene deletion constructs and elucidate the biosynthetic steps for the formation of the polycyclic system. The strategy we employ bypasses the traditional laborious processes to elicit gene cluster expression and should be generally applicable to many other silent or cryptic gene clusters for discovery and characterization of new natural products.

[1]  K. Zaleta-Rivera,et al.  Biosynthesis of HSAF, a tetramic acid-containing macrolactam from Lysobacter enzymogenes. , 2011, Journal of the American Chemical Society.

[2]  Zengyi Shao,et al.  DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways , 2008, Nucleic acids research.

[3]  H. Kalinowski,et al.  Carbon 13 NMR Spectroscopy , 1988 .

[4]  M. Bibb,et al.  Cloning and analysis of the promoter region of the erythromycin resistance gene (ermE) of Streptomyces erythraeus. , 1985, Gene.

[5]  N. Kelleher,et al.  Heterologous production of fosfomycin and identification of the minimal biosynthetic gene cluster. , 2006, Chemistry & biology.

[6]  Y. Li,et al.  3-Hydroxylation of the polycyclic tetramate macrolactam in the biosynthesis of antifungal HSAF from Lysobacter enzymogenes C3 , 2012 .

[7]  Rolf Müller,et al.  Full-length RecE enhances linear-linear homologous recombination and facilitates direct cloning for bioprospecting , 2012, Nature Biotechnology.

[8]  Shu-Lin Chang,et al.  Illuminating the diversity of aromatic polyketide synthases in Aspergillus nidulans. , 2012, Journal of the American Chemical Society.

[9]  C. Hertweck,et al.  Triggering cryptic natural product biosynthesis in microorganisms. , 2009, Organic & biomolecular chemistry.

[10]  B. J. Royles.  NATURALLY OCCURRING TETRAMIC ACIDS : STRUCTURE, ISOLATION, AND SYNTHESIS , 1995 .

[11]  N. Kelleher,et al.  Unusual transformations in the biosynthesis of the antibiotic phosphinothricin tripeptide. , 2007, Nature chemical biology.

[12]  Christopher A. Voigt,et al.  Refactoring the nitrogen fixation gene cluster from Klebsiella oxytoca , 2012, Proceedings of the National Academy of Sciences.

[13]  I. Ial,et al.  Nature Communications , 2010, Nature Cell Biology.

[14]  R. Cox,et al.  First Heterologous Reconstruction of a Complete Functional Fungal Biosynthetic Multigene Cluster , 2010, Chembiochem : a European journal of chemical biology.

[15]  H. Bahl,et al.  Maltophilin: a new antifungal compound produced by Stenotrophomonas maltophilia R3089. , 1996, The Journal of antibiotics.

[16]  E. Chapin,et al.  Dihydromaltophilin: A Novel Fungicidal Tetramic Acid Containing Metabolite from Streptomyces sp. , 1998 .

[17]  R. Biener,et al.  Comparative analysis of transcriptional activities of heterologous promoters in the rare actinomycete Actinoplanes friuliensis. , 2009, Journal of biotechnology.

[18]  B. Wanner,et al.  One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[19]  A. Brakhage,et al.  Activation of fungal silent gene clusters: a new avenue to drug discovery. , 2008, Progress in drug research. Fortschritte der Arzneimittelforschung. Progres des recherches pharmaceutiques.

[20]  Eung-Soo Kim,et al.  Transcriptome Analysis of an Antibiotic Downregulator Mutant and Synergistic Actinorhodin Stimulation via Disruption of a Precursor Flux Regulator in Streptomyces coelicolor , 2011, Applied and Environmental Microbiology.

[21]  Yuemao Shen,et al.  Bioactive natural products from Lysobacter. , 2012, Natural product reports.

[22]  K. Zaleta-Rivera,et al.  Structure and Biosynthesis of Heat-Stable Antifungal Factor (HSAF), a Broad-Spectrum Antimycotic with a Novel Mode of Action , 2006, Antimicrobial Agents and Chemotherapy.

[23]  Takuma Sasaki,et al.  Alteramide A, a new tetracyclic alkaloid from a bacterium Alteromonas sp. associated with the marine sponge Halichondria okadai , 1992 .

[24]  R. Schobert,et al.  Tetramic and tetronic acids: an update on new derivatives and biological aspects. , 2008, Bioorganic & medicinal chemistry.

[25]  Huimin Zhao,et al.  Rapid characterization and engineering of natural product biosynthetic pathways via DNA assembler. , 2011, Molecular bioSystems.

[26]  Kay Nieselt,et al.  The dynamic architecture of the metabolic switch in Streptomyces coelicolor , 2010, BMC Genomics.

[27]  Huimin Zhao,et al.  Direct cloning of large genomic sequences , 2012, Nature Biotechnology.

[28]  Jun Ishikawa,et al.  Genome Sequence of the Streptomycin-Producing Microorganism Streptomyces griseus IFO 13350 , 2008, Journal of bacteriology.

[29]  Y. Kuroda,et al.  A new antibiotic, ikarugamycin. , 1972, The Journal of antibiotics.

[30]  N. Kelleher,et al.  Cloning, expression, and biochemical characterization of Streptomyces rubellomurinus genes required for biosynthesis of antimalarial compound FR900098. , 2008, Chemistry & biology.

[31]  Corinna Lange,et al.  Genomics-driven discovery of PKS-NRPS hybrid metabolites from Aspergillus nidulans. , 2007, Nature chemical biology.

[32]  A. Pineda-Lucena,et al.  Amino Acid Precursor Supply in the Biosynthesis of the RNA Polymerase Inhibitor Streptolydigin by Streptomyces lydicus , 2011, Journal of bacteriology.

[33]  Richard H. Baltz,et al.  Streptomyces and Saccharopolyspora hosts for heterologous expression of secondary metabolite gene clusters , 2010, Journal of Industrial Microbiology & Biotechnology.

[34]  Clay C C Wang,et al.  Total biosynthesis of antitumor nonribosomal peptides in Escherichia coli , 2006, Nature chemical biology.

[35]  E. Chapin,et al.  Dihydromaltophilin; a novel fungicidal tetramic acid containing metabolite from Streptomyces sp. , 1997, The Journal of antibiotics.

[36]  Satoshi Omura,et al.  Genome-minimized Streptomyces host for the heterologous expression of secondary metabolism , 2010, Proceedings of the National Academy of Sciences.

[37]  R. Kolter,et al.  Common biosynthetic origins for polycyclic tetramate macrolactams from phylogenetically diverse bacteria , 2010, Proceedings of the National Academy of Sciences.

[38]  J. Engels,et al.  Promoter constructions for efficient secretion expression in Streptomyces lividans , 2004, Applied Microbiology and Biotechnology.