Nonribosomal Peptide and Polyketide Biosynthesis: Methods and Protocols (Methods in Molecular Biology)

This volume presents an overview of nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) structure and function.  It then continues with methods for the analysis of these pathways including conventional enzymological assays, contemporary mass spectrometric analysis techniques, specialized molecular biological approaches applicable to NRPSs and PKSs, and small molecule analysis tools tailored to this very special class of natural products, and concludes by examining bioinformatics tools for the analysis of these enzymes, pathways, and molecules. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Nonribosomal Peptide and Polyketide Biosynthesis: Methods and Protocols serves as a valuable reference for those experienced in studying NRPS and PKS enzymes, pathways, and natural products as well as a gateway for those just entering the field. Part I: Background and Overview   1. Structural Biology of Nonribosomal Peptide Synthetases             Bradley R. Miller and Andrew M. Gulick   2. The Assembly Line Enzymology of Polyketide Biosynthesis             Marisa Till and Paul R. Race   Part II: In Vitro Methods   3. Measurement of Nonribosomal Peptide Synthetase Adenylation Domain Activity Using a Continuous Hydroxylamine Release Assay             Benjamin P. Duckworth, Daniel J. Wilson, and Courtney C. Aldrich   4. Affinity Purification Method for the Identification of Nonribosomal Peptide Biosynthetic Enzymes Using a Synthetic Probe for Adenylation Domains             Fumihiro Ishikawa and Hideaki Kakeya   5. Colorimetric Detection of the Adenylation Activity in Nonribosomal Peptide Synthetases             Chitose Maruyama, Haruka Niikura, Masahiro Takakuwa, Hajime Katano, and Yoshimitsu Hamano   6. Facile Synthetic Access to Glycopeptide Antibiotic Precursor Peptides for the Investigation of Cytochrome P450 Action in Glycopeptide Antibiotic Biosynthesis             Clara Brieke, Veronika Kratzig, Madeleine Peschke, and Max J. Cryle   7. Reconstitution of Fungal Nonribosomal Peptide Synthetases in Yeast and In Vitro             Ralph A. Cacho and Yi Tang   8. The Continuing Development of E. coli as a Heterologous Host for Complex Natural Product Biosynthesis             Haoran Zhang, Lei Fang, Marcia S. Osburne, and Blaine A. Pfeifer   9. Screening for Expressed Nonribosomal Peptide Synthetases and Polyketide Synthases Using LCMS/MS-Based Proteomics             Yunqiu Chen, Ryan A. McClure, and Neil L. Kelleher   10. Enhancing Nonribosomal Peptide Biosynthesis in Filamentous Fungi             Alexandra A. Soukup, Nancy P. Keller, and Philipp Wiemann   11. In Situ Analysis of Bacterial Lipopeptide Antibiotics by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging             Delphine Debois, Marc Ongena, Hlne Cawoy, and Edwin De Pauw   12. Secondary Metabolic Pathway-Targeted Metabolomics             Maria I. Vizcaino and Jason M. Crawford   Part III: Bioinformatic Methods   13. Annotating and Interpreting Linear and Cyclic Peptide Tandem Mass Spectra