Complex enzymes in microbial natural product biosynthesis: Overview articles and peptides

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\rEnzymes Involved in Vancomycin\rBiosynthesis......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\rLantibiotic 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\r......Page 597
Identification and processing of Prochloron-containing ascidians\r......Page 604
DNA purification from enriched Prochloron cells\r......Page 605
DNA purification from whole animals\r......Page 606
Chemical Analysis\r......Page 607
Cyanobactin Gene Cloning and Identification\r......Page 608
Genome mining and structure prediction\r......Page 609
Cyanobactin cloning from non-Prochloron cyanobacteria\r......Page 610
Heterologous Expression in E. coli\r......Page 611
Whole-cell PCR with Prochloron\r......Page 612
Example: Deep metagenome mining in Prochloron\r......Page 613
Applying Deep Metagenome Mining: Pathway Engineering\r......Page 614
Yeast recombination for pathway manipulation\r......Page 615
References\r......Page 616