Note: Front Cover -- Methods in Enzymology Complex Enzymes in Microbial Natural Product Biosynthesis, Part B: Polyketides, Aminocoumarins and Carbohydrates -- Copyright Page -- Contents -- Contributors -- Preface -- Methods in Enzymology -- Chapter 1: Introduction to Polyketide Biosynthesis -- 1. Introduction -- 1.1. Types of PKS -- 1.2. Type II PKS -- 1.3. Type III PKS -- 1.4. Type I PKS -- 1.5. Combinatorial biosynthesis: Prospects and progress -- Acknowledgments -- References -- Chapter 2: Structural Enzymology of polyketide Synthases -- Chapter 3: Chapter Three: Fungal Type I Polyketide Synthases -- 1. Introduction -- 2. Partially Reducing PKSs: 6-Methylsalicylate Synthase -- 3. Nonreducing PKSs -- 3.1. Norsolorinic acid synthase -- 3.2. Tetrahydroxynaphthalene synthase -- 3.3. Bikaverin nonaketide synthase -- 4. Highly Reducing PKSs -- 4.1. Lovastatin (LNKS and LDKS) -- 4.2. HR PKS-NRPS: Fusarin and tenellin synthetases -- 5. NR/HR PKS Hybrid Systems: Zearalenone (ZAE1 and ZAE2) -- 6. Conclusions -- References -- Chapter 4: Tandem Acyl Carrier Protein Domains in Polyunsaturated Fatty Acid Synthases -- 1. Introduction -- 2. Methods -- 2.1. Production of PUFAs in E. coli by expressing the PUFAS genes -- 2.2. Mapping the active sites of PfaA-ACPs by site-directed mutagenesis -- 2.3. Overproduction of each of the PfaA-ACPs -- 2.4. Overproduction of PfaE and Svp PPTases -- 2.5. In vivo and in vitro preparation of the holo-form of PfaA-ACPs -- 2.6. Elucidation of the relationship between PUFA production and the number of active ACPs -- 3. Conclusion -- Acknowledgments -- References -- Chapter 5: Iterative Type I Polyketide Synthases for Enediyne Core Biosynthesis -- 1. Introduction -- 2. Methods -- 2.1. PCR amplification of PKSE cassettes for predictive classification of new enediynes -- 2.2. Heterologous expression and overproduction of PKSE proteins. , 2.3. Production and isolation of the polyene intermediate from 9-membered PKSEs -- 2.4. Production of apo-ACPs from PKSE for in vitro functional analyses -- 2.5. In vitro preparation of holo-ACPs -- 3. Conclusion -- Acknowledgments -- References -- Chapter 6: Chapter Six The DEBS Paradigm for Type I Modular Polyketide Synthases and Beyond -- 1. Introduction -- 2. DEBS and the Concept of a Module -- 2.1. Generalizability of the DEBS paradigm -- 3. Beyond the DEBS Paradigm -- 3.1. Specificity of the AT domains -- 3.2. Novel loading modules -- 3.3. Methylation domains -- 3.4. Trans PKS activities -- 3.5. Unusual modular organization -- 3.6. Unusual module functions -- 3.7. Intermodular interactions -- 4. Conclusion -- References -- Chapter 7: Formation and Characterization of Acyl Carrier Protein-Linked Polyketide Synthase Extender Units -- 1. Introduction -- 2. Overproduction and Purification of Recombinant Proteins -- 2.1. Principle -- 2.2. Materials -- 2.3. Heterologous overproduction of proteins -- 2.4. Purification of enzymes using batch-binding method with Nickel-NTA resin -- 3. Formation and Characterization of Hydroxymalonyl-ACP and Aminomalonyl-ACP -- 3.1. In vitro phosphopantetheinylation of the ACPs ZmaD and ZmaH -- 3.2. HPLC-based characterization of modified ACPs -- 3.3. Formation of (2R)-hydroxymalonyl-ACP -- 3.4. Formation of (2S)-aminomalonyl-ACP -- 3.5. MALDI-TOF MS analysis of ACPs -- 3.6. Other methods for characterizing enzymes involved in (2S)-aminomalonyl-ACP formation -- 3.7. Other methods for characterizing enzymes involved in (2R)-hydroxymalonyl-ACP formation -- References -- Chapter 8: Type I Polyketide Synthases That Require Discrete Acyltransferases -- 1. Introduction -- 2. Methods -- 2.1. Heterologous expression and overproduction of apo-ACPs from AT-less PKS modules -- 2.2. In vitro preparation of holo-ACPs. , 2.3. Heterologous expression and overproduction of discrete ATs -- 2.4. In vitro assay for AT substrate specificity -- 2.5. In vitro assay of AT-catalyzed loading of acyl CoA extender substrate to holo-ACPs -- 3. Conclusion -- Acknowledgments -- References -- Chapter 9: The Enzymology of Polyether Biosynthesis -- 1. Introduction -- 2. Genetic Mining and Functional Analysis of Genes Specific to Polyether Biosynthetic Pathways -- 3. Premonensin, the Parent Unsaturated Monensin Polyketide -- 4. Epoxidases MonCI, NigCI, and NanO -- 5. Epoxide Hydrolases MonBI/BII, NigBI/BII, NanI, and Lsd19 -- 6. NanE, a Polyether-Specific Thioesterase -- 6.1. In vitro studies of NanE thioesterase -- 7. Assay of Polyether Thioesterase Activity -- 7.1. Site-directed mutagenesis of NanE -- 8. Transcriptional Analysis of the Nanchangmycin Biosynthetic Pathway Genes -- 9. Conclusions -- Acknowledgments -- References -- Chapter 10: Enzymology of the Polyenes Pimaricin and Candicidin Biosynthesis -- 1. Introduction -- 2. Pimaricin as a Prototype of Small Polyenes: Discovery and Properties -- 3. Pimaricin Biosynthesis in S. natalensis -- 3.1. The pimaricin gene cluster -- 3.2. Formation of pimaricinolide: The pimaricinolide synthase complex -- 3.3. Pimaricinolide tailoring and export -- 4. Regulation of Pimaricin Biosynthesis -- 4.1. Transcriptional regulators -- 4.2. Regulation by cholesterol oxidase -- 4.3. Inducers of pimaricin biosynthesis -- 4.4. Global regulatory mechanisms -- 5. Candicidin: A Prototype of ``Aromatic´´ Polyenes -- 6. The Candicidin/FR-008 Gene Cluster -- 7. Biosynthesis of PABA: The pabAB and pabC Genes -- 8. The Polyketide Synthases -- 9. Monooxygenase Genes: Modifications of the Polyketide Chain -- 10. Transporter Genes -- 11. Genes Related to Mycosamine Biosynthesis -- 12. Regulatory Genes -- 13. Phosphate Represses Expression of the pabAB Gene. , 14. Future Perspectives -- Acknowledgments -- References -- Chapter 11: Genetic Analysis of Nystatin and Amphotericin Biosynthesis -- 1. Introduction -- 2. Gene Inactivation and Replacement in the Nystatin Producer Streptomyces noursei -- 2.1. Conjugative transfer of a recombinant plasmid from E. coli ET12567 (pUZ8002) into S. noursei ATCC 11455 -- 2.2. Gene inactivation in S. noursei -- 2.3. Gene replacement in S. noursei -- 3. Gene Inactivation and Replacement in the Amphotericin Producer Streptomyces nodosus -- 3.1. Phage-mediated gene replacement in S. nodosus -- 4. Production, Purification, and Characterization of Novel Amphotericin- and Nystatin-Related Polyenes -- 4.1. Production and identification of nystatin-related polyenes -- 4.2. Scaled-up production of nystatin analogues -- 4.3. Preparative LC-MS purification of nystatin analogues -- 5. Conclusion -- Acknowledgments -- References -- Chapter 12: Polyketide Versatility in the Biosynthesis of Complex Mycobacterial Cell Wall Lipids -- 1. Introduction -- 2. Acetate and Propionate Feeding Studies -- 3. Genome Sequencing and Identification of Polyketide Synthases -- 4. Mycobacterial Polyketide Synthases -- 4.1. Biosynthesis of dimycocerosate esters (DIMs) by PKS15/1, PpsABCDE, and MAS -- 4.2. PKS2 is involved in biosynthesis of sulfolipids -- 4.3. PKS12 uses a novel ``modularly iterative´´ mechanism for biosynthesis of mannosyl-beta-1-phosphomycoketides -- 4.4. PKS13 catalyzes condensation of fatty-acyl chains during biosynthesis of mycolic acids -- 4.5. PKS3/4 is involved in the biosynthesis of phthenoic acids -- 4.6. PKS10, PKS7, PKS8, PKS17, PKS9, and PKS11 constitute an unusual PKS cluster -- 4.7. PKS18 is involved in biosynthesis of long-chain pyrones -- 4.8. MbtC and MbtD are involved in biosynthesis of iron-chelating siderophores from Mtb -- 4.9. PKS5 and PKS6. , 5.1. Product formation assay to study activity of PKS enzymes -- 5.2. Characterization of PKS derived saturated fatty acids -- References -- Chapter 13: Genetic Engineering to Produce Polyketide Analogues -- 1. Introduction -- 2. AT Domain Replacement to Alter α-Carbon Substitution -- 3. Procedure for Engineering AT Replacements in the Chromosome -- 4. Engineering beta-Carbon Processing -- 5. Engineering when only a Single Crossover Event is Possible -- 6. Heterologous Expression of Engineered PKS Genes -- 7. Chemobiosynthesis -- 8. Mutasynthesis -- 9. Gene Knockouts to Obtain Analogues -- References -- Chapter 14: Design and Synthesis of Pathway Genes for Polyketide Biosynthesis -- 1. Introduction -- 2. Redesign of PKS Genes to Accommodate Unique Restriction Sites Flanking Individual Components and for Efficient Expression in E -- 3. Validation of Synthetic PKS Gene Design -- 4. A Rapid Assay to Identify Productive Combinations of PKS Modules -- 5. Assembly of Larger Polyketide Synthases Using Information Gained with the Bimodular Assay -- 6. Design and Construction of Synthetic Operons for the Expression of Sugar Pathway Genes -- References -- Chapter 15: Heterologous Production of Polyketides in Bacteria -- 1. Introduction -- 2. General Considerations for the Heterologous Expression of Polyketide Pathways -- 3. S. coelicolor as a Model System for Heterologous Expression of Polyketides -- 4. Procedure for the Heterologous Production of Polyketides in S. coelicolor -- 5. System Improvements for the Heterologous Production of Polyketides in Streptomyces spp. -- 5.1. Handling large PKS genes -- 5.2. Improving polyketide titers -- 5.3. Optimization of conjugation protocols for industrial strains -- 5.4. Optimizing polyketide precursors supply -- 6. Recent Developments for the Production of Polyketides in Nonactinomycete Bacteria. , 6.1. E. coli as heterologous host.

Note: Front Cover -- Complex Enzymes in Microbial Natural Product Biosynthesis, Part A: Overview Articles and Peptides -- Copyright -- Contents -- Contributors -- Preface -- Methods in Enzymology -- Chapter 1: Approaches to Discovering Novel Antibacterial and Antifungal Agents -- 1. Introduction -- 2. Strains and Samples -- 3. Targets and Assays for Antibacterial and Antifungal Programs -- 4. Screening -- 5. Hit Follow-up -- 6. Databases, Operations, and Costs -- 7. Perspectives -- Acknowledgment -- References -- Chapter 2: From Microbial Products to Novel Drugs that Target a Multitude of Disease Indications -- 1. Microbial Diversity and Biotechnological Products -- 2. Secondary Metabolites -- 3. Conclusions -- Acknowledgments -- References -- Chapter 3: Discovering Natural Products from Myxobacteria with Emphasis on Rare Producer Strains in Combination with Improved Analytical Methods -- 1. Introduction -- 2. The Search for Novel Myxobacteria and Their Metabolites-Basic Considerations -- 3. Methods for Isolation, Purification, and Preservation of Novel Myxobacteria -- 4. Fermentation and Screening for Known and Novel Metabolites -- Acknowledgment -- References -- Chapter 4: Analyzing the Regulation of Antibiotic Production in Streptomycetes -- 1. Introduction -- 2. The Regulation of Antibiotic Production in Streptomycetes -- 3. Identifying Regulatory Genes for Antibiotic Biosynthesis -- 4. Characterizing Regulatory Genes for Antibiotic Biosynthesis -- Acknowledgments -- References -- Chapter 5: Applying the Genetics of Secondary Metabolism in Model Actinomycetes to the Discovery of New Antibiotics -- 1. Introduction -- 2. Actinomycetes as Antibiotic Factories -- 3. Effects of Culture Conditions and Metabolism -- 4. Molecular Genetic Factors that Regulate Antibiotic Production -- 5. Applications for New Antibiotic Screening Technologies. , 6. Future Prospects -- Acknowledgments -- References -- Chapter 6: Regulation of Antibiotic Production by Bacterial Hormones -- 1. Introduction -- 2. Rapid Small-Scale gamma-Butyrolactone Purification -- 3. Antibiotic Bioassay -- 4. Kanamycin Bioassay -- 5. Identification of gamma-Butyrolactone Receptors -- 6. Identification of the gamma-Butyrolactone Receptor Targets -- 7. Gel Retardation Assay to Detect Target Sequences of the gamma-Butyrolactone Receptors -- 8. Conclusions -- Acknowledgments -- References -- Chapter 7: Cloning and Analysis of Natural Product Pathways -- 1. Introduction -- 2. Cloning and Identification of Biosynthetic Gene Clusters -- 3. Analysis of Natural Product Pathways by PCR-Targeted Gene Replacement -- 4. In Vitro Transposon Mutagenesis -- 5. Heterologous Expression of Biosynthetic Gene Clusters -- 6. Reassembling Entire Gene Clusters by "Stitching" Overlapping Cosmid Clones -- 7. Conclusions -- Acknowledgments -- References -- Chapter 8: Methods for In Silico Prediction of Microbial Polyketide and Nonribosomal Peptide Biosynthetic Pathways from DNA Sequence Data -- 1. Introduction -- 2. Converting Type I PKSs to Structural Elements -- 3. Converting NRPS Domain Strings to Structural Elements -- 4. Concluding Remarks -- Acknowledgments -- References -- Chapter 9: Synthetic Probes for Polyketide and Nonribosomal Peptide Biosynthetic Enzymes -- 1. Introduction -- 2. Synthetic Probes of PKS and NRPS Mechanism -- 3. Synthetic Probes of PKS and NRPS Structure -- 4. Synthetic Probes for Proteomic Identification of PKS and NRPS Enzymes -- 5. Conclusions -- References -- Chapter 10: Using Phosphopantetheinyl Transferases for Enzyme Posttranslational Activation, Site Specific Protein Labeling and Identification of Natural Product Biosynthetic Gene Clusters from Bacterial Genomes -- 1. Introduction -- 2. Experimental Procedures. , 3. Conclusion -- References -- Chapter 11: Sugar Biosynthesis and Modification -- 1. Introduction -- 2. Deoxysugar Biosynthesis -- 3. Deoxysugar Transfer -- 4. Modification of the Glycosylation Pattern through Gene Inactivation -- 5. Modification of the Glycosylation Pattern through Heterologous Gene Expression -- 6. Modification of the Glycosylation Pattern through Combinatorial Biosynthesis -- 7. Gene Cassette Plasmids for Deoxysugar Biosynthesis -- 8. Generation of Glycosylated Compounds -- 9. Tailoring Modifications of the Attached Deoxysugars -- 10. Detection of Glycosylated Compounds -- Acknowledgments -- References -- Chapter 12: The Power of Glycosyltransferases to Generate Bioactive Natural Compounds -- 1. Introduction -- 2. Application of GTs in Producing Unnatural Bioactive Molecules -- References -- Chapter 13: Nonribosomal Peptide Synthetases: Mechanistic and Structural Aspects of Essential Domains -- 1. Introduction -- 2. Mechanistic and Structural Aspects of Essential NRPS Domains -- 3. Structural Insights into an Entire Termination Module -- References -- Chapter 14: Biosynthesis of Nonribosomal Peptide Precursors -- 1. Introduction -- 2. Precursors from Amino Acid Metabolism -- 3. Fatty Acid Precursor Biosynthesis -- 4. Polyketide Precursors -- 5. Glycosyl Building Blocks -- 6. Conclusion -- References -- Chapter 15: Plasmid-Borne Gene Cluster Assemblage and Heterologous Biosynthesis of Nonribosomal Peptides in Escherichia coli -- 1. Introduction -- 2. Biosynthetic Pathway of Nonribosomal Peptides -- 3. Echinomycin Biosynthetic Pathway -- 4. Construction of A Multigene Assembly on Expression Vectors -- 5. Heterologous Gene Expression and NRP Biosynthesis in E. coli -- 6. Self-Resistance Mechanism -- 7. Stability of Transformants Carrying Multiple Very Large Plasmids. , 8. Engineering of Heterologous NRP Biosynthetic Pathways in E. coli -- 9. Conclusion -- References -- Chapter 16: Enzymology of beta-Lactam Compounds with Cephem Structure Produced by Actinomycete -- 1. Introduction -- 2. Biosynthesis of Cephamycins: Enzymes and Genes -- 3. Early Steps Specific for Cephamycin Biosynthesis -- 4. Common Steps in Cephamycin-Producing Actinomycetes and Penicillin- or Cephalosporin-Producing Filamentous Fungi -- 5. Specific Steps for Tailoring the Cephem Nucleus in Actinomycetes -- 6. Regulation of Cephamycin C Production -- Acknowledgments -- References -- Chapter 17: Siderophore Biosynthesis: A Substrate Specificity Assay for Nonribosomal Peptide Synthetase-Independent Siderophore Synthetases Involving Trapping of Acyl-Adenylate Intermediates with Hydroxylamine -- 1. Introduction -- 2. NRPS-Dependent Pathways for Siderophore Biosynthesis -- 3. NRPS-Independent Pathway for Siderophore Biosynthesis -- 4. Hybrid NRPS/NIS Pathway for Petrobactin Biosynthesis -- 5. Hydroxamate-Formation Assay for NIS Synthetases -- References -- Chapter 18: Molecular Genetic Approaches to Analyze Glycopeptide Biosynthesis -- 1. Structural Classification of Glycopeptide Antibiotics -- 2. Methods for Analyzing Glycopeptide Biosynthesis -- 3. Investigation of Glycopeptide Biosynthetic Steps -- 4. Regulation, Self-Resistance, and Excretion -- 5. Linking Primary and Secondary Metabolism -- 6. Approaches for the Generation of New Glycopeptides -- Acknowledgments -- References -- Chapter 19: In Vitro Studies of Phenol Coupling Enzymes Involved in Vancomycin Biosynthesis -- 1. Introduction -- 2. Peptide Synthesis -- 3. Peptide Thioesters -- 4. In Vitro Assays with OxyB -- 5. Production and Purification of Enzymes -- References -- Chapter 20: Biosynthesis and Genetic Engineering of Lipopeptides in Streptomyces roseosporus -- 1. Introduction. , 2. Biosynthesis and Genetic Engineering of Daptomycin in S. rosesoporus -- 3. Sources of Genes for Combinatorial Biosynthesis -- 4. Genetic Engineering of Novel Lipopeptides -- 5. Concluding Remarks -- Acknowledgments -- References -- Chapter 21: In Vitro Studies of Lantibiotic Biosynthesis -- 1. Introduction -- 2. Mining Microbial Genomes for Novel Lantibiotics -- 3. Expression and Purification of Lantibiotic Precursor Peptides (LanAs) -- 4. Expression, Purification, and Assay of LanM Enzymes -- 5. Expression, Purification, and Assays of LanC Cyclases -- 6. The Protease Domain of Class II Lantibiotic Transporters -- 7. Additional Posttranslational Modifications in Lantibiotics -- References -- Chapter 22: Whole-Cell Generation of Lantibiotic Variants -- 1. Introduction -- 2. Variant Generation -- 3. Conclusions -- References -- Chapter 23: Cyanobactin Ribosomally Synthesized Peptides-A Case of Deep Metagenome Mining -- 1. Introduction -- 2. Some Remaining Questions -- 3. Obtaining Prochloron Cells and DNA -- 4. Chemical Analysis -- 5. Cyanobactin Gene Cloning and Identification -- 6. Heterologous Expression in E. coli -- 7. Deep Metagenome Mining -- 8. Enzymatic Analysis of Cyanobactin Biosynthesis -- 9. Applying Deep Metagenome Mining: Pathway Engineering -- Acknowledgments -- References -- Author Index -- Subject Index -- Colour Plates.

Note: Front Cover -- Natural Product Biosynthesis by Microorganisms and Plants, Part B -- Copyright -- Contents -- Contributors -- Preface -- Methods in Enzymology -- Section 1: Peptides -- Chapter 1: In Vivo Production of Thiopeptide Variants -- 1. Introduction -- 2. Generation of Thiopeptide Variants -- 2.1. Thiostrepton analogs -- 2.1.1. Overview -- 2.1.2. Thiostrepton variants obtained by precursor peptide mutagenesis -- 2.1.2.1. Deletion of tsrA from the S. laurentii chromosome -- 2.1.2.2. Fosmid engineering to generate complementation vector int-3A10 -- 2.1.2.3. Generation of an intermediate fosmid int-3A100 -- 2.1.2.4. Site-directed mutagenesis of tsrA -- 2.1.2.6. Evaluation of thiostrepton analog production in S. laurentii -- 2.1.3. Gene inactivation to produce thiostrepton analogs -- 2.1.3.1. Deletion of tsrU and tsrT from S. laurentii -- 2.1.3.2. Evaluation of thiostrepton analog production in S. laurentii -- 2.2. Thiocillin analogs -- 2.2.1. Overview -- 2.2.2. Thiocillin analogs obtained by precursor peptide mutagenesis -- 2.2.2.1. Construction of B. cereus DeltatclE-H -- 2.2.2.2. Generation of complementation vector pMGA-tclE-KI -- 2.2.2.3. Evaluation of thiocillin analog production in B. cereus -- 2.3. Nosiheptide analogs -- 2.3.1. Overview -- 2.3.2. Gene inactivation to produce nosiheptide analogs -- 2.3.2.1. Deletion of nosN and nosA in S. actuosus -- 2.3.2.3. Evaluation of nosiheptide analog production in S. actuosus -- References -- Chapter 2: Microviridin Biosynthesis -- 1. Introduction -- 2. Reconstitution In Vitro of the Cyclization Reactions of the Microviridin Biosynthetic Pathway from P. agardhii -- 2.1. Expression of GRASP-like ligase genes mvdC and mvdD -- 2.2. Expression of mvdB -- 2.3. Expression of His6-mvdE -- 2.4. LCMS analysis -- 2.5. Investigation of the lactonization/lactamization reaction in vitro. , 3. Influence of Leader Peptide on the Cyclization Reactions -- 4. Summary -- Acknowledgments -- References -- Chapter 3: Cyclotide Isolation and Characterization -- 1. Introduction -- 2. Isolation of Cyclotides from Plant Tissues -- 3. Isolation of Nucleic Acids Encoding Cyclotide Precursors -- 3.1. Isolating RNA -- 3.2. Primer design for PCR amplification of cyclotide-encoding genes -- 4. Mass Spectrometric Detection and Characterization of Cyclotides -- 4.1. Detection by reduction and alkylation -- 4.2. Sequencing following reduction and digestion -- 5. Structural Analysis of Cyclotides -- 6. Membranolytic Assays of Cyclotides -- 6.1. Preparation of lipid vesicles -- 6.2. Membrane binding by SPR -- 6.3. Vesicle leakage studies -- 7. Summary -- Acknowledgments -- References -- Chapter 4: Ribosomally Encoded Cyclic Peptide Toxins from Mushrooms -- 1. Introduction -- 2. Detection and Purification of Amanita Cyclic Peptide Toxins -- 2.1. Purification of Amanita cyclic peptides -- 2.1.1. Purification from fruiting bodies (mushrooms) -- 2.1.2. Purification from liquid culture of G. marginata -- 2.1.3. Purification of α-amanitin from G. marginata cultured on petri plates -- 3. Identification of Cyclic Peptide Genes in Amanita and Other Fungi -- 4. Purification and Assay of Prolyl Oligopeptidase (POP) from Mushrooms -- 4.1. Isolation of POP from fruiting bodies of C. apala -- 4.1.1. Preparation of crude extract -- 4.1.2. HPLC purification of POP -- 4.1.2.1. Hydrophobic interaction chromatography -- 4.1.2.2. Hydroxyapatite chromatography -- 4.1.2.3. Anion exchange chromatography -- 4.2. Assays for POP -- 4.2.1. Assay with chromogenic substrate -- 4.2.2. Assay with synthetic peptides -- 5. Immunodetection of POP in Mushroom Tissues -- 5.1. Preparation of antibodies -- 5.2. Immunological and microscopic methods -- 6. Summary -- Acknowledgments -- References. , Chapter 5: The Pictet-Spengler Mechanism Involved in the Biosynthesis of Tetrahydroisoquinoline Antitumor Antibiotics: A Novel Function for a Nonribosomal Peptide Synthetase -- 1. Introduction -- 2. Biosynthetic Analysis of THIQ Core Scaffold Assembly -- 2.1. Previous biosynthetic studies on saframycin family members -- 2.2. Biogenesis of saframycin A based on bioinformatic analysis -- 3. Reaction Mechanism for Constructing a Pentacyclic THIQ Core Skeleton Catalyzed by a Novel Function for a Single NRPS Modul -- 4. Comparison Between Typical Peptide Synthesis and the Pictet-Spengler Reaction Catalyzed by NRPS Machinery -- 5. Reaction Profiles Affected by the Chain Length of the Acyl Moiety -- 6. Multiple Roles for the Fatty Acyl Chain During the Maturation Process in Late-Stage Biosynthesis -- 7. Methods for Substrate Synthesis, Enzyme Preparation, and Assay Conditions -- 7.1. Synthesis of the substrates -- 7.1.1. Synthesis of the fatty acyl-peptidyl CoA esters -- 7.1.2. Synthesis of the fatty acyl-peptidyl aldehydes -- 7.2. Overexpression, purification, and phosphopantetheinylation of SfmC -- 7.2.1. Construction of SfmC expression vectors -- 7.2.2. Overexpression and purification -- 7.2.3. Phosphopantetheinylation of SfmC -- 7.3. Enzymatic reactions with saframycin NRPS SfmC -- 7.3.1. SfmC-catalyzed reaction using the fatty-acyl-peptidyl-CoA thioester or the fatty acyl-peptidyl aldehyde -- 7.3.2. Large-scale enzymatic reaction for preparation of presaframycins -- 8. Conclusion -- Acknowledgments -- References -- Section 2: Other Compounds -- Chapter 6: Discovery and Biosynthesis of Phosphonate and Phosphinate Natural Products -- 1. Introduction -- 2. Spectrum of Phosphonate Natural Products: Structures and Bioactivities -- 3. Biosynthetic Pathways -- 4. Screening Bacteria for Phosphonate Producing Gene Clusters. , 4.1. Protocol to identify pepM or ppD genes -- 5. Purification of Phosphonates/Phosphinates -- 5.1. Screening by NMR spectroscopy -- 5.2. Protocol to distinguish between phosphonates and cyclic phosphate esters -- 5.3. Phosphonate purification strategies -- 5.4. Protocol to remove salts -- 6. Structural Elucidation of Phosphonates -- 6.1. 31P NMR spectroscopy -- 6.2. Liquid chromatography-Fourier transform mass spectrometry -- 7. A Wealth of Unusual Biosynthetic Enzymes -- 8. Conclusion and Outlook -- Acknowledgments -- References -- Chapter 7: RlmN and AtsB as Models for the Overproduction and Characterization of Radical SAM Proteins -- 1. Introduction -- 2. RlmN as a Model for Gene Expression and Overproduction of RS Proteins -- 2.1. General cloning strategy -- 2.2. Gene expression and protein overproduction -- 2.3. Protocol for expression of the rlmN gene -- 3. RlmN as a Model for Purification of RS Proteins -- 3.1. Excluding O2 -- 3.2. Protocol for purifying RlmN -- 3.3. Chemical reconstitution of as-isolated RlmN -- 3.4. Protocol for chemical reconstitution of as-isolated RlmN -- 3.5. Removing adventitiously bound iron and sulfide from chemically reconstituted RS proteins -- 4. Overproduction and Purification of E. coli Flavodoxin and Flavodoxin Reductase -- 4.1. Protocol for overexpressing E. coli genes for Flv and Flx -- 4.2. Protocol for purification of Flv or Flx -- 5. AtsB as a Model for Determination of Configuration and Stoichiometry of Iron-Sulfur Clusters in RS Enzymes -- 5.1. Strategy for determining iron-sulfur cluster configuration and stoichiometry -- 5.2. Protocol for preparation of iron-sulfur proteins for amino acid analysis -- 5.3. Protocol for preparation of samples for Mössbauer and EPR spectroscopies -- 6. Activity Determination of RS Enzymes -- 6.1. Protocol for analysis of SAM-related products in RS reactions. , 6.2. Protocol for standard curve preparation of SAM-related products -- 6.3. Protocol for HPLC quantification of SAM-related products -- Acknowledgments -- References -- Chapter 8: Fe(II)-Dependent, Uridine-5-Monophosphate α-Ketoglutarate Dioxygenases in the Synthesis of 5-Modified Nucleosides -- 1. Introduction -- 2. Methods -- 2.1. Bioinformatic analysis of LipL and homologous dioxygenases -- 2.2. Cloning and heterologous expression -- 2.3. SEC-RI-MALLS-ICP-MS to determine metalloprotein stoichiometry -- 2.4. Activity assays -- 2.4.1. Detection of succinate using an enzyme-coupled reaction -- 2.4.2. HPLC analysis -- 2.4.3. Malachite-green binding assay -- 3. Summary -- Acknowledgments -- References -- Section 3: Tailoring Reactions -- Chapter 9: Tailoring Reactions Catalyzed by Heme-Dependent Enzymes: Spectroscopic Characterization of the L-Tryptophan-Nitrating Cytochrom P450 TxtE -- 1. Introduction -- 1.1. Cytochromes P450 -- 1.2. The catalytic mechanisms of CYPs -- 1.3. CYP tailoring enzymes in thaxtomin phytotoxin biosynthesis -- 2. Overexpression, Purification, and Characterization of the Novel Nitrating CYP TxtE -- 2.1. His6-TxtE purification -- 3. Protein Characterization Using UV-vis Spectroscopy -- 3.1. Recording the spectrum of the Fe(III) resting state -- 3.2. Reduction of the ferric heme to ferrous heme using sodium dithionite -- 3.3. Recording the soret spectrum of the ferrous-CO complex -- 4. Ligand and Substrate Binding -- 4.1. Qualitative binding assays: Type I difference spectra -- 4.1.1. Dual beam spectrophotometer -- 4.1.2. Single beam spectrophotometer -- 4.2. Qualitative binding assays: Type II spectra -- 4.2.1. Generation of amine Fe(III) complexes (tryptamine, imidazole, clotrimazole, bifonazole) -- 4.2.2. Generation of an Fe(III)-NO complex -- 5. Summary -- Acknowledgment -- References. , Chapter 10: Oxidative Tailoring Reactions Catalyzed by Nonheme Iron-Dependent Enzymes: Streptorubin B Biosynthesis as an Example.

Note: Front Cover -- Natural Product Biosynthesis by Microorganisms and Plants, Part A -- Copyright -- Contents -- Contributors -- Preface -- Methods in Enzymology -- Section1:Terpenoids -- Chapter 1:Steady-State Kinetic Characterization of Sesquiterpene Synthases by Gas Chromatography-Mass Spectroscopy -- 1. Introduction -- 1.1. The vial assay for enzyme kinetics -- 1.2. Steady-state kinetics and the Michaelis-Menten model -- 2. Experimental Components and Considerations -- 2.1. Enzyme purification and quantification -- 2.2. Substrates -- 2.3. Buffer and pH -- 2.4. Metal ions -- 2.5. Internal standard -- 2.6. Vial assay method -- 2.7. GC-MS instrument and run parameters -- 2.8. Additional points -- 3. Pilot Experiments -- 3.1. Analyte detection and quantification -- 3.2. Instrument calibration -- 3.3. Linear range of protein concentration: Measuring kcat apparent -- 4. Steady-State Kinetic Experiments -- 4.1. Reaction velocity versus substrate concentration -- 4.2. Steady-state kinetics experiment -- 5. Data Handling/Processing -- 6. Summary -- References -- Chapter 2: Automating Gene Library Synthesis by Structure-Based Combinatorial Protein Engineering: Examples from Plant Sesquiterpene Synthses -- 1. Overview -- 2. Plasmid Library Synthesis -- 2.1. Deconstructing the gene -- 2.2. Cloning of the three-plasmid system -- 3. Optimization of SCOPE PCR Conditions -- 4. Generic SCOPE Method -- 4.1. Primer design -- 4.2. Fragment amplification -- 4.3. SCOPE synthesis: Recombination -- 4.4. SCOPE synthesis: Amplification -- 5. Applications of SCOPE Synthesis -- 5.1. Synthesis of complex mixtures of diverse mutants -- 5.1.1. Deconstructing the gene -- 5.1.2. Plasmid library synthesis -- 5.1.3. SCOPE synthesis -- 5.2. Synthesis of arrays of individual mutants -- 5.2.1. Deconstructing the gene -- 5.2.2. Plasmid library synthesis and fragment amplification. , 5.2.3. SCOPE synthesis -- 6. Troubleshooting -- 6.1. Presence of alternative amplification products -- 6.2. Low levels of full-length gene product -- 7. Conclusions -- References -- Chapter 3:In Planta Transient Expression Analysis of Monoterpene Synthases -- 1. Introduction -- 2. Plant Transformation Vector Construction -- 2.1. Materials -- 2.2. Preparing attB-PCR products using attB adaptor PCR -- 2.3. Protocol for destination vector cloning -- 3. Agrobacterium-Mediated Transient Expression -- 3.1. Materials -- 3.2. Preparation of competent A. tumefaciens -- 3.3. Transformation of A. tumefaciens GV3101 -- 3.4. Preparation of transformed Agrobacterium for transient expression -- 3.5. Transient transformation of N. benthamiana leaves -- 4. Agrobacterium Preparation for High-Throughput Analysis -- 5. Product Identification -- 5.1. Headspace volatile analysis -- 6. Transient Expression of a Kiwifruit Linalool Synthase -- 6.1. Vectors and cloning -- 6.2. Infiltration setup -- 6.3. Solvent extraction and analysis -- 6.4. Headspace trapping and analysis -- 6.5. Results and discussion -- 7. Conclusions -- Acknowledgments -- References -- Chapter 4:Natural Rubber Biosynthesis in Plants: Rubber Transferase -- 1. Introduction -- 2. Preparing Samples for Assaying Rubber Transferase Activity -- 2.1. Rubber particle-bound activity -- 2.1.1. Purification of enzymatically active rubber particles -- 2.1.2. Enzyme stability -- 2.1.3. Assay system purity -- 2.1.4. Sources of competing enzymes and the separate roles of cis and trans prenyl transferases in rubber biosynthesis -- 3. The Rubber Transferase Assay -- 3.1. Assay method -- 3.2. Kinetic analyses -- 3.3. Regulation of molecular weight -- 4. Identification and Purification of Rubber Transferase -- 4.1. Rubber transferase is a distinct cis prenyl transferase -- 4.2. Photoaffinity labeling. , 4.2.1. A photoaffinity labeling protocol -- 4.2.2. Purification of labeled proteins -- 4.3. Antibodies -- 4.3.1. Immunoinhibition and immunoprecipitation -- 4.3.2. Latex-free animals -- 5. Qualitative Protein Analysis -- 6. Protein Quantification -- 7. Summary -- Acknowledgments -- References -- Chapter 5:Discovery and Characterization of Terpenoid Biosynthetic Pathways of Fungi -- 1. Introduction -- 2. Identification, Cloning, and Characterization of Terpene Synthases -- 2.1. Identification and cloning of terpene synthase genes without sequence information -- 2.2. Identification and cloning of terpene synthase genes using sequence information -- 2.3. Characterization of terpene synthase gene function -- 3. Structural Characterization and Engineering of Fungal Terpene Synthase Activities -- 3.1. Site-directed mutagenesis guided by crystal structures -- 3.2. Exploring terpene synthase structure through molecular models -- 4. Identification and Characterization of Biosynthetic Gene Clusters -- 4.1. Characteristics of fungal biosynthetic gene clusters -- 4.2. Obtaining a biosynthetic cluster sequence -- 4.2.1. Identifying an anchor gene sequence -- 4.2.2. Obtaining a sequencing dataset -- 4.2.2.1. Small-scale sequencing approaches -- 4.2.2.2. Whole-genome sequencing -- 4.3. Cluster annotation -- 4.4. Characterization of biosynthetic clusters -- 4.4.1. Gene knockout and complementation studies -- 4.4.2. Pathway construction in a heterologous host -- 5. Genome Mining for Sesquiterpene Synthases -- 5.1. Phylogeny-guided analysis of terpene synthase gene families -- 6. Conclusions -- Acknowledgments -- References -- Chapter 6:Menaquinone Biosyntheses in Microorganisms -- 1. Introduction -- 2. Futalosine Pathway -- 2.1. Tracer experiments clearly showed the presence of an alternative pathway -- 2.2. Genes participating in the alternative pathway. , 2.3. Intermediates in the alternative pathway -- 2.3.1. Isolation of an intermediate accumulated by the SCO4327-disruptant -- 2.3.2. Identification of the intermediate next to FL -- 2.3.3. Identification of the intermediate following DHFL -- 2.3.4. Identification of the intermediate following cyclic DHFL -- 2.4. The early biosynthetic steps -- Acknowledgments -- References -- Chapter 7:Diversity and Analysis of Bacterial Terpene Synthases -- 1. Terpenoid Metabolites from Bacterial Cultures -- 2. Bacterial Terpene Synthases -- 2.1. Monoterpene synthases -- 2.2. Sesquiterpene synthases -- 2.3. Diterpene synthases -- 3. Methods for the Study of Bacterial Terpene Synthases -- 3.1. Bioinformatic analysis of bacterial terpene synthases -- 3.1.1. Properties of terpene synthases and database search procedures -- 3.1.2. Genome mining of bacterial terpene synthases -- 3.2. Expression of genes encoding bacterial terpene synthases in heterologous hosts -- Protocol 1. Construction of expression cassette on integrating vector for terpene synthase gene of interest -- Protocol 2. Introduction of recombinant integrating plasmid into S. avermitilis SUKA17 -- Protocol 3. Cultivation for the production of terpene compounds and analysis of products -- References -- Chapter 8: Platensimycin and Platencin Biosynthesis in Streptomyces platensis, Showcasing Discovery and Characterization of Novel Bacteria Diterpene Synthases -- 1. Introduction -- 2. Methods -- 2.1. In vivo confirmation of PtmT1 and PtmT3 as DTSs in PTM and PTN biosynthesis -- 2.1.1. Bioinformatic analyses to identify candidate DTSs -- 2.1.2. Construction of a gene replacement or deletion on an isolated cosmid -- 2.1.3. Replacement of the DTS gene in vivo with the antibiotic resistance cassette -- 2.1.4. Southern analysis to verify the mutant strain genotype. , 2.1.5. HPLC analysis to determine the mutant strain chemotype -- 2.2. In vivo confirmation of ptmT3 encoding a DTS by heterologous expression -- 2.2.1. Construction of heterologous expression strain -- 2.2.2. Structural validation of the diterpenoids produced in heterologous hosts -- 2.3. In vitro characterization of PtmT2 and PtmT3 as DTSs in PTM biosynthesis -- 2.3.1. Expression, overproduction, and purification of PtmT2 and PtmT3 from E. coli -- 2.3.2. Synthesis of GGDP from geranylgeraniol -- 2.3.3. Functional characterization of PtmT2 as an ent-CDP synthase -- 2.3.4. Functional characterization of PtmT3 as an ent-kauran-16-ol synthase -- 3. Conclusions -- Acknowledgment -- References -- Section 2:Alkaloids andGlucosinolates -- Section 2: Alkaloids and Glucosinolates -- Chapter 9:Strategies for Engineering Plant Natural Products: The Iridoid-Derived Monoterpene Indole Alkaloids of Catharanthus roseus -- 1. Introduction -- 2. Metabolic Engineering Strategies -- 2.1. Precursor-directed biosynthesis -- 2.1.1. Example of directed biosynthesis of monoterpene indole alkaloids -- 2.1.2. Methods for precursor-directed biosynthesis in C. roseus seedlings and hairy root cultures -- 2.2. Mutasynthesis -- 2.2.1. Example of mutasynthesis for monoterpene indole alkaloids -- 2.2.2. Methods for mutasynthesis in C. roseus -- 2.3. Incorporation of engineered pathway enzymes in biosynthesis -- 2.3.1. Example of incorporation of an engineered pathway enzyme in C. roseus -- 2.3.2. Methods for incorporation of an engineered pathway enzyme in C. roseus -- 3. Conclusions and Future Directions -- References -- Chapter 10:Discovery and Functional Analysis of Monoterpenoid Indole Alkaloid Pathways in Plants -- 1. Introduction -- 2. Enzyme Assays for MIA Pathway Steps -- 2.1. Tabersonine-16-hydroxylase assay coupled to 16-OMT -- 2.2. Tabersonine 6,7-epoxidase assay. , 2.3. Tabersonine 19 hydroxylase assay.