Complex Enzymes in Microbial Natural Product Biosynthesis, Part A: Overview Articles and Peptides (Volume 458) (Methods in Enzymology, Volume 458)

Microbial natural products have been an important traditional source of valuable antibiotics and other drugs but interest in them waned in the 1990s when big pharma decided that their discovery was no longer cost-effective and concentrated instead on synthetic chemistry as a source of novel compounds, often with disappointing results. Moreover understanding the biosynthesis of complex natural products was frustratingly difficult. With the development of molecular genetic methods to isolate and manipulate the complex microbial enzymes that make natural products, unexpected chemistry has been revealed and interest in the compounds has again flowered. This two-volume treatment of the subject will showcase the most important chemical classes of complex natural products: the peptides, made by the assembly of short chains of amino acid subunits, and the polyketides, assembled from the joining of small carboxylic acids such as acetate and malonate. In both classes, variation in sub-unit structure, number and chemical modification leads to an almost infinite variety of final structures, accounting for the huge importance of the compounds in nature and medicine. * Gathers tried and tested methods and techniques from top players in the field.* In depth coverage of ribosomally-synthesised and Non-ribosomally-synthesised peptides.* Provides an extremely useful reference for the experienced research scientist. Approaches to Discovering Novel Antibacterial and Antifungal Agents......Page 38 Objectives of a screening program......Page 39 Screening strategy and novelty of the program......Page 40 Strains and Samples......Page 42 Strain isolation, purification, and storage......Page 43 Procedure......Page 44 Procedure......Page 45 Sample preparation......Page 46 Procedure......Page 47 Target identification and validation......Page 48 Assay development......Page 49 Secondary assays......Page 50 Example: Screening for bacterial cell-wall inhibitors......Page 51 Procedure......Page 52 Hit Follow-up......Page 54 Novelty evaluation......Page 55 Profiling......Page 57 Databases, Operations, and Costs......Page 58 References......Page 60 From Microbial Products to Novel Drugs that Target a Multitude of Disease Indications......Page 64 Secondary Metabolites......Page 65 Source of new microbial drugs......Page 69 Overview of bioactivities and assays......Page 70 Successful drugs......Page 75 DNA-targeting antitumor antibiotics......Page 76 Tubulin-targeting anticancer drugs......Page 82 Discarded antifungals as agents for organ transplantation......Page 84 The best-selling drugs from natural products: Fungal statins......Page 86 Conclusions......Page 88 References......Page 89 Discovering Natural Products from Myxobacteria with Emphasis on Rare Producer Strains in Combination with Improved Analytical Methods......Page 94 Introduction......Page 95 Myxobacteria as proficient producers of bioactive compounds......Page 97 The Search for Novel Myxobacteria and Their Metabolites-Basic Considerations......Page 98 Prospects for the discovery of novel myxobacteria......Page 99 Choice of material to favor the discovery of novel myxobacteria......Page 100 A short survey of previous isolation efforts......Page 101 Genetic characterization of novel strains......Page 103 The issue of "unculturability"......Page 104 Methods for Isolation, Purification, and Preservation of Novel Myxobacteria......Page 106 Medium......Page 107 Light......Page 108 Extended incubation......Page 109 Swarming and fruiting body recognition......Page 110 A simple and effective method for the purification of myxobacteria......Page 111 Culture maintenance and preservation......Page 112 Fermentation and Screening for Known and Novel Metabolites......Page 113 Bioassays using crude extracts and prepurified fractions......Page 114 Analysis of myxobacterial metabolite profiles using high-resolution mass spectrometry......Page 116 High-resolution MS target screening checklist......Page 117 A diversity-oriented approach to mining myxobacterial secondary metabolomes......Page 119 Summary and outlook......Page 122 References......Page 123 Analyzing the Regulation of Antibiotic Production in Streptomycetes......Page 127 The Regulation of Antibiotic Production in Streptomycetes......Page 128 Random generation of antibiotic nonproducing or overproducing mutants by UV, NTG, transposon, and insertion mutagenesis......Page 131 Transposon mutants......Page 133 Genetic complementation......Page 134 Identifying antibiotic regulatory genes by overexpression......Page 135 Confirming the nature of antibiotic regulatory genes......Page 136 Insertion and deletion mutagenesis by homologous recombination......Page 137 Low-resolution S1 nuclease protection analysis......Page 138 Primer extension......Page 139 Reverse transcription PCR and quantitative real time RT-PCR......Page 140 Surface plasmon resonance......Page 141 Pleiotropic regulatory genes......Page 142 The effect of gene deletion......Page 144 Defining genome-wide DNA-protein interactions by chromatin immunoprecipitation and microarray analysis (ChIP-on-Chip)......Page 145 Systematic evolution of ligands by exponential enrichment (SELEX)......Page 146 References......Page 147 Applying the Genetics of Secondary Metabolism in Model Actinomycetes to the Discovery of New Antibiotics......Page 151 Introduction......Page 152 Actinomycetes as Antibiotic Factories......Page 153 Growth-dependent control mechanisms......Page 154 Phosphate-mediated control......Page 155 Interactions between metabolism and the DasR regulon......Page 156 Morphology as determinant of productivity......Page 157 Pathway-specific regulation......Page 159 Pleiotropic regulation......Page 161 Applications for New Antibiotic Screening Technologies......Page 163 Heterologous overexpression and mutant alleles......Page 164 Future Prospects......Page 167 References......Page 168 Regulation of Antibiotic Production by Bacterial Hormones......Page 176 Introduction......Page 177 Rapid Small-Scale gamma-Butyrolactone Purification......Page 178 Antibiotic Bioassay......Page 179 Kanamycin Bioassay......Page 181 Identification of gamma-Butyrolactone Receptors......Page 183 Identification of the gamma-Butyrolactone Receptor Targets......Page 184 Gel Retardation Assay to Detect Target Sequences of the gamma-Butyrolactone Receptors......Page 185 Radio-labeled probes......Page 186 Cy3-labeled probes......Page 188 References......Page 189 Cloning and Analysis of Natural Product Pathways......Page 191 Introduction......Page 192 Cloning and Identification of Biosynthetic Gene Clusters......Page 193 Analysis of Natural Product Pathways by PCR-Targeted Gene Replacement......Page 195 In Vitro Transposon Mutagenesis......Page 202 Heterologous Expression of Biosynthetic Gene Clusters......Page 205 Reassembling Entire Gene Clusters by "Stitching" Overlapping Cosmid Clones......Page 206 References......Page 209 Methods for In Silico Prediction of Microbial Polyketide and Nonribosomal Peptide Biosynthetic Pathways from DNA Sequence Data......Page 213 Introduction......Page 214 Software for PKS/NRPS domain analysis......Page 216 Manual PKS/NRPS parsing......Page 218 NRPS-PKS: A knowledge based resource for analyzing NRPSs and PKSs......Page 219 NRPSpredictor: A domain substrate prediction using transductive support vector machines (TSVMs)......Page 220 PKSsolNRPS analysis web site: An HMM implementation for domain parsing and a domain substrate prediction......Page 221 NORINE: A database of nonribosomal peptides......Page 222 The mechanistic approach......Page 223 Conserved active-site motifs......Page 225 Applicable systems......Page 226 Domain identification and ordering of proteins/modules......Page 228 Automated parsing......Page 229 Interprotein domain ordering......Page 231 Consensus motif analysis......Page 232 Ketoreductase (KR) domain analysis......Page 233 Heuristic approach......Page 234 Amphotericin-A classical type I modular PKS......Page 235 Dorrigocin/Migrastatin-An "AT-less" PKS......Page 236 Converting NRPS Domain Strings to Structural Elements......Page 237 Modifications and stereochemistry......Page 238 Caveats......Page 239 Interprotein domain ordering......Page 240 Heuristic structure prediction......Page 241 Ramoplanin......Page 242 Concluding Remarks......Page 244 References......Page 246 Synthetic Probes for Polyketide and Nonribosomal Peptide Biosynthetic Enzymes......Page 250 Introduction......Page 251 Carrier protein posttranslational modification and PPTase promiscuity......Page 252 Utility of CoA analogues to study PKS and NRPS mechanism......Page 254 One-pot chemoenzymatic synthesis of CoA and carrier protein analogues......Page 256 Synthesis of PMB-pantothenic acid (9)......Page 260 Synthesis of pantetheine azide (11)......Page 261 General materials and protocols......Page 262 One-pot chemoenzymatic loading of CPs by pantetheine analogues......Page 263 Challenges in the structural analysis of modular biosynthetic enzymes......Page 264 Synthetic probes of PKS and NRPS protein-substrate interactions......Page 265 Chemoenzymatic Crosslinking of ACP and Partner Enzymes......Page 267 Reaction protocol......Page 271 Background......Page 272 Proteomic preparation......Page 274 Background......Page 275 Labeling and visualization......Page 277 Background......Page 278 In vivo labeling of endogenous CPs......Page 279 Visualization of azide-labeled CPs via Cu-catalyzed [3 + 2] cycloaddition with fluorophore or biotin alky......Page 280 References......Page 281 Using Phosphopantetheinyl Transferases for Enzyme Posttranslational Activation, Site Specific Protein Labeling and Identification of Natural Product biosynthetic.........Page 286 Introduction......Page 287 Expression of Sfp from E. coli......Page 294 Coexpression of NRPS or PKS modules with Sfp in E. coli......Page 295 Preparation of small molecule-CoA conjugates......Page 296 Preparation of Qdot-CoA conjugates......Page 297 Sfp catalyzed protein labeling with small molecule-CoA conjugates......Page 298 Sfp-catalyzed protein labeling on the cell surface......Page 299 Construction of the genomic library for phage selection of NRPS and PKS fragments......Page 300 Phage selection by Sfp catalyzed carrier protein modification with biotin-SS-CoA 2......Page 301 References......Page 302 Sugar Biosynthesis and Modification......Page 307 Introduction......Page 308 Deoxysugar Biosynthesis......Page 309 Deoxysugar Transfer......Page 313 Modification of the Glycosylation Pattern through Gene Inactivation......Page 314 Generation of a mithramycin derivative by inactivating the mtmU gene......Page 315 Modification of the Glycosylation Pattern through Heterologous Gene Expression......Page 318 Selection of suitable expression vectors......Page 319 Modification of the Glycosylation Pattern through Combinatorial Biosynthesis......Page 320 Gene Cassette Plasmids for Deoxysugar Biosynthesis......Page 322 Construction of pLN2......Page 326 Generation of Glycosylated Compounds......Page 329 Generation of S. albus GB16......Page 330 Tailoring Modifications of the Attached Deoxysugars......Page 331 References......Page 333 The Power of Glycosyltransferases to Generate Bioactive Natural Compounds......Page 339 Introduction......Page 340 Pharmaceutical relevance......Page 341 Characteristics of the GT-B superfamily......Page 342 Classical sugar transfer......Page 343 Flexible GTs......Page 345 Bi-functional GTs......Page 351 Detection of new GTs......Page 352 Gene inactivation......Page 354 Expression of parts of an entire gene cluster......Page 355 Altering the substrate specificity of a GT......Page 356 Mutagenesis via error prone PCR......Page 357 References......Page 358 Nonribosomal Peptide Synthetases: Mechanistic and Structural Aspects of Essential Domains......Page 364 Introduction......Page 365 Mechanistic and Structural Aspects of Essential NRPS Domains......Page 366 Structural Insights into an Entire Termination Module......Page 374 References......Page 376 Biosynthesis of Nonribosomal Peptide Precursors......Page 379 Introduction......Page 380 Precursors from Amino Acid Metabolism......Page 381 Biosynthesis of (2S,3R)-methylglutamic acid: In vivo studies......Page 385 Fatty Acid Precursor Biosynthesis......Page 386 Biosynthesis of the 2,3-epoxyhexanoyl side chain of CDA......Page 389 Polyketide Precursors......Page 390 3,5-Dihydroxyphenylglycine biosynthesis......Page 391 In vivo studies of DHPG biosynthesis......Page 392 In vitro studies of DHPG biosynthesis......Page 393 Variation of the balhimycin DHPG moiety by mutasynthesis......Page 394 Bmt biosynthesis (cyclosporin precursor)......Page 395 3-Methoxy-5-methylnapthoic acid biosynthesis......Page 396 Glycosyl Building Blocks......Page 397 Conclusion......Page 398 References......Page 399 Plasmid-Borne Gene Cluster Assemblage and Heterologous Biosynthesis of Nonribosomal Peptides in Escherichia Coli......Page 405 Introduction......Page 406 Biosynthetic Pathway of Nonribosomal Peptides......Page 408 Echinomycin Biosynthetic Pathway......Page 409 Construction of A Multigene Assembly on Expression Vectors......Page 413 Heterologous Gene Expression and NRP Biosynthesis in E. coli......Page 416 Self-Resistance Mechanism......Page 417 Stability of Transformants Carrying Multiple Very Large Plasmids......Page 418 Engineering of Heterologous NRP Biosynthetic Pathways in E. coli......Page 419 Conclusion......Page 421 References......Page 422 Enzymology of......Page 426 Introduction......Page 427 Early Steps Specific for Cephamycin Biosynthesis......Page 430 Common Steps in Cephamycin-Producing Actinomycetes and Penicillin- or Cephalosporin-Producing Filamentous......Page 434 Isopenicillin N synthase......Page 437 Genes......Page 438 Mutagenesis and crystallization......Page 439 Isopenicillin N epimerase......Page 440 Deacetoxycephalosporin C synthase......Page 442 Deacetoxycephalosporin C hydroxylase......Page 444 Specific Steps for Tailoring the Cephem Nucleus in Actinomycetes......Page 445 Regulation of Cephamycin C Production......Page 447 References......Page 448 Siderophore Biosynthesis: A Substrate Specificity Assay for Nonribosomal Peptide Synthetase-Independent Side......Page 455 Introduction......Page 456 NRPS-Dependent Pathways for Siderophore Biosynthesis......Page 458 NRPS-Independent Pathway for Siderophore Biosynthesis......Page 466 Hybrid NRPS/NIS Pathway for Petrobactin Biosynthesis......Page 472 Hydroxamate-Formation Assay for NIS Synthetases......Page 474 Assay principle......Page 475 Buffers, reagents, and others materials......Page 476 Technical notes......Page 477 References......Page 479 Molecular Genetic Approaches to Analyze Glycopeptide Biosynthesis......Page 482 Structural Classification of Glycopeptide Antibiotics......Page 483 Identification of glycopeptide biosynthetic gene clusters......Page 485 Systems for the genetic manipulation of glycopeptide-producing bacteria......Page 486 Heterologous over-expression of glycopeptide biosynthetic genes......Page 487 The biosynthesis of amino acid building blocks......Page 489 Biosynthesis of beta-hydroxytyrosine (AA2 and AA6)......Page 491 Biosynthesis of 3,5-dihydroxyphenylglycine (AA7)......Page 492 Biosynthesis of carbohydrate building blocks......Page 493 Peptide assembly by NRPSs......Page 494 Vancomycin-type glycopeptides......Page 496 Time point of halogenation and side chain cyclization in glycopeptide biosynthesis......Page 498 Methylation of glycopeptide antibiotics......Page 499 Regulation, Self-Resistance, and Excretion......Page 500 Linking Primary and Secondary Metabolism......Page 501 Approaches for the Generation of New Glycopeptides......Page 502 References......Page 503 In Vitro Studies of Phenol Coupling Enzymes Involved in Vancomycin Biosynthesis......Page 510 Introduction......Page 511 Peptide Synthesis......Page 514 Synthesis of hexapeptide S-phenyl thioester (1-SPh)......Page 522 Synthesis of hexapeptide-SCoA thioester (1-SCoA)......Page 523 Synthesis of hexapeptide-PCP conjugate (1-S-PCP)......Page 524 In Vitro Assays with OxyB......Page 525 Production and purification of OxyB......Page 526 Production and purification of spinach ferredoxin......Page 527 Production and purification of PCP domain......Page 528 Production and purification of E. coli NADPH-flavodoxin reductase......Page 529 References......Page 530 Biosynthesis and Genetic Engineering of Lipopeptides in......Page 533 Introduction......Page 534 Cloning of the daptomycin biosynthetic genes and deductions on biosynthesis......Page 536 Construction of deletion mutants of S. roseosporus and expression vectors......Page 539 Functions of dptGHIJ genes......Page 542 Reconstitution of the daptomycin biosynthetic pathway by ectopic expression of individual genes or groups of genes......Page 543 The A54145 biosynthetic genes from Streptomyces fradiae and the CDA genes from Streptomyces coelicolor......Page 545 Gene replacement......Page 546 Module and multidomain replacements......Page 547 In vitro antibacterial activities of novel lipopeptides......Page 548 Concluding Remarks......Page 549 References......Page 550 In Vitro Studies of Lantibiotic Biosynthesis......Page 554 Introduction......Page 555 Overview......Page 558 Overview......Page 559 Procedure......Page 560 Mutagenesis and construction of synthetic analogs of LanA peptides......Page 562 Overview......Page 563 Procedure......Page 564 Procedure......Page 565 Order of dehydration and cyclization......Page 567 Overview......Page 568 Procedure......Page 570 Procedure......Page 573 Additional Posttranslational Modifications in Lantibiotics......Page 574 References......Page 575 Introduction......Page 580 Cis complementation system......Page 582 Mutacin II......Page 583 Nisin......Page 584 Mersacidin......Page 585 In cis expression of cinnamycin......Page 586 Comparison between cis and trans complementation for the production of mersacidin analogues......Page 587 Variants from targeted or saturation mutagenesis libraries......Page 589 Analytical strategy......Page 591 References......Page 592 Cyanobactin Ribosomally Synthesized Peptides-A Case of Deep Metagenome Mining......Page 596 Introduction ......Page 597 Identification and processing of Prochloron-containing ascidians ......Page 604 DNA purification from enriched Prochloron cells ......Page 605 DNA purification from whole animals ......Page 606 Chemical Analysis ......Page 607 Cyanobactin Gene Cloning and Identification ......Page 608 Genome mining and structure prediction ......Page 609 Cyanobactin cloning from non-Prochloron cyanobacteria ......Page 610 Heterologous Expression in E. coli ......Page 611 Whole-cell PCR with Prochloron ......Page 612 Example: Deep metagenome mining in Prochloron ......Page 613 Applying Deep Metagenome Mining: Pathway Engineering ......Page 614 Yeast recombination for pathway manipulation ......Page 615 References ......Page 616 Microbial natural products have been an important traditional source of valuable antibiotics and other drugs but interest in them waned in the 1990s when big pharma decided that their discovery was no longer cost-effective and concentrated instead on synthetic chemistry as a source of novel compounds, often with disappointing results. Moreover understanding the biosynthesis of complex natural products was frustratingly difficult. With the development of molecular genetic methods to isolate and manipulate the complex microbial enzymes that make natural products, unexpected chemistry has been revealed and interest in the compounds has again flowered. This two-volume treatment of the subject will showcase the most important chemical classes of complex natural products: the peptides, made by the assembly of short chains of amino acid subunits, and the polyketides, assembled from the joining of small carboxylic acids such as acetate and malonate. In both classes, variation in sub-unit structure, number and chemical modification leads to an almost infinite variety of final structures, accounting for the huge importance of the compounds in nature and medicine.

* Gathers tried and tested methods and techniques from top players in the field.
* In depth coverage of ribosomally-synthesised and Non-ribosomally-synthesised peptides.
* Provides an extremely useful reference for the experienced research scientist.