Complex Enzymes in Microbial Natural Product Biosynthesis, Part A: Overview Articles and Peptides (eBook)

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2009 | 1. Auflage
350 Seiten
Elsevier Science (Verlag)
978-0-08-092335-2 (ISBN)

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.

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

Front Cover 1
Complex Enzymes in Microbial Natural Product Biosynthesis, Part A: Overview Articles and Peptides 2
Copyright 5
Contents 6
Contributors 14
Preface 20
Methods in Enzymology 24
Chapter 1: Approaches to Discovering Novel Antibacterial and Antifungal Agents 54
1. Introduction 55
2. Strains and Samples 58
3. Targets and Assays for Antibacterial and Antifungal Programs 64
4. Screening 67
5. Hit Follow-up 70
6. Databases, Operations, and Costs 74
7. Perspectives 76
Acknowledgment 76
References 76
Chapter 2: From Microbial Products to Novel Drugs that Target a Multitude of Disease Indications 80
1. Microbial Diversity and Biotechnological Products 81
2. Secondary Metabolites 81
3. Conclusions 104
Acknowledgments 105
References 105
Chapter 3: Discovering Natural Products from Myxobacteria with Emphasis on Rare Producer Strains in Combination with Improved Analytical Methods 110
1. Introduction 111
2. The Search for Novel Myxobacteria and Their Metabolites-Basic Considerations 114
3. Methods for Isolation, Purification, and Preservation of Novel Myxobacteria 122
4. Fermentation and Screening for Known and Novel Metabolites 129
Acknowledgment 139
References 139
Chapter 4: Analyzing the Regulation of Antibiotic Production in Streptomycetes 144
1. Introduction 145
2. The Regulation of Antibiotic Production in Streptomycetes 145
3. Identifying Regulatory Genes for Antibiotic Biosynthesis 148
4. Characterizing Regulatory Genes for Antibiotic Biosynthesis 155
Acknowledgments 164
References 164
Chapter 5: Applying the Genetics of Secondary Metabolism in Model Actinomycetes to the Discovery of New Antibiotics 168
1. Introduction 169
2. Actinomycetes as Antibiotic Factories 170
3. Effects of Culture Conditions and Metabolism 171
4. Molecular Genetic Factors that Regulate Antibiotic Production 176
5. Applications for New Antibiotic Screening Technologies 180
6. Future Prospects 184
Acknowledgments 185
References 185
Chapter 6: Regulation of Antibiotic Production by Bacterial Hormones 194
1. Introduction 195
2. Rapid Small-Scale gamma-Butyrolactone Purification 196
3. Antibiotic Bioassay 197
4. Kanamycin Bioassay 199
5. Identification of gamma-Butyrolactone Receptors 201
6. Identification of the gamma-Butyrolactone Receptor Targets 202
7. Gel Retardation Assay to Detect Target Sequences of the gamma-Butyrolactone Receptors 203
8. Conclusions 207
Acknowledgments 207
References 207
Chapter 7: Cloning and Analysis of Natural Product Pathways 210
1. Introduction 211
2. Cloning and Identification of Biosynthetic Gene Clusters 212
3. Analysis of Natural Product Pathways by PCR-Targeted Gene Replacement 214
4. In Vitro Transposon Mutagenesis 221
5. Heterologous Expression of Biosynthetic Gene Clusters 224
6. Reassembling Entire Gene Clusters by "Stitching" Overlapping Cosmid Clones 225
7. Conclusions 228
Acknowledgments 228
References 228
Chapter 8: Methods for In Silico Prediction of Microbial Polyketide and Nonribosomal Peptide Biosynthetic Pathways from DNA Sequence Data 232
1. Introduction 233
2. Converting Type I PKSs to Structural Elements 242
3. Converting NRPS Domain Strings to Structural Elements 256
4. Concluding Remarks 263
Acknowledgments 265
References 265
Chapter 9: Synthetic Probes for Polyketide and Nonribosomal Peptide Biosynthetic Enzymes 270
1. Introduction 271
2. Synthetic Probes of PKS and NRPS Mechanism 272
3. Synthetic Probes of PKS and NRPS Structure 284
4. Synthetic Probes for Proteomic Identification of PKS and NRPS Enzymes 292
5. Conclusions 301
References 301
Chapter 10: Using Phosphopantetheinyl Transferases for Enzyme Posttranslational Activation, Site Specific Protein Labeling and Identification of Natural Product Biosynthetic Gene Clusters from Bacterial Genomes 306
1. Introduction 307
2. Experimental Procedures 314
3. Conclusion 322
References 322
Chapter 11: Sugar Biosynthesis and Modification 328
1. Introduction 329
2. Deoxysugar Biosynthesis 330
3. Deoxysugar Transfer 334
4. Modification of the Glycosylation Pattern through Gene Inactivation 335
5. Modification of the Glycosylation Pattern through Heterologous Gene Expression 339
6. Modification of the Glycosylation Pattern through Combinatorial Biosynthesis 341
7. Gene Cassette Plasmids for Deoxysugar Biosynthesis 343
8. Generation of Glycosylated Compounds 350
9. Tailoring Modifications of the Attached Deoxysugars 352
10. Detection of Glycosylated Compounds 354
Acknowledgments 354
References 354
Chapter 12: The Power of Glycosyltransferases to Generate Bioactive Natural Compounds 360
1. Introduction 361
2. Application of GTs in Producing Unnatural Bioactive Molecules 373
References 379
Chapter 13: Nonribosomal Peptide Synthetases: Mechanistic and Structural Aspects of Essential Domains 388
1. Introduction 389
2. Mechanistic and Structural Aspects of Essential NRPS Domains 390
3. Structural Insights into an Entire Termination Module 398
References 400
Chapter 14: Biosynthesis of Nonribosomal Peptide Precursors 404
1. Introduction 405
2. Precursors from Amino Acid Metabolism 406
3. Fatty Acid Precursor Biosynthesis 411
4. Polyketide Precursors 415
5. Glycosyl Building Blocks 422
6. Conclusion 423
References 424
Chapter 15: Plasmid-Borne Gene Cluster Assemblage and Heterologous Biosynthesis of Nonribosomal Peptides in Escherichia coli 430
1. Introduction 431
2. Biosynthetic Pathway of Nonribosomal Peptides 433
3. Echinomycin Biosynthetic Pathway 434
4. Construction of A Multigene Assembly on Expression Vectors 438
5. Heterologous Gene Expression and NRP Biosynthesis in E. coli 441
6. Self-Resistance Mechanism 442
7. Stability of Transformants Carrying Multiple Very Large Plasmids 443
8. Engineering of Heterologous NRP Biosynthetic Pathways in E. coli 444
9. Conclusion 446
References 447
Chapter 16: Enzymology of beta-Lactam Compounds with Cephem Structure Produced by Actinomycete 452
1. Introduction 453
2. Biosynthesis of Cephamycins: Enzymes and Genes 456
3. Early Steps Specific for Cephamycin Biosynthesis 456
4. Common Steps in Cephamycin-Producing Actinomycetes and Penicillin- or Cephalosporin-Producing Filamentous Fungi 460
5. Specific Steps for Tailoring the Cephem Nucleus in Actinomycetes 471
6. Regulation of Cephamycin C Production 473
Acknowledgments 474
References 474
Chapter 17: Siderophore Biosynthesis: A Substrate Specificity Assay for Nonribosomal Peptide Synthetase-Independent Siderophore Synthetases Involving Trapping of Acyl-Adenylate Intermediates with Hydroxylamine 482
1. Introduction 483
2. NRPS-Dependent Pathways for Siderophore Biosynthesis 485
3. NRPS-Independent Pathway for Siderophore Biosynthesis 493
4. Hybrid NRPS/NIS Pathway for Petrobactin Biosynthesis 499
5. Hydroxamate-Formation Assay for NIS Synthetases 501
References 506
Chapter 18: Molecular Genetic Approaches to Analyze Glycopeptide Biosynthesis 510
1. Structural Classification of Glycopeptide Antibiotics 511
2. Methods for Analyzing Glycopeptide Biosynthesis 513
3. Investigation of Glycopeptide Biosynthetic Steps 517
4. Regulation, Self-Resistance, and Excretion 528
5. Linking Primary and Secondary Metabolism 529
6. Approaches for the Generation of New Glycopeptides 530
Acknowledgments 531
References 531
Chapter 19: In Vitro Studies of Phenol Coupling Enzymes Involved in Vancomycin Biosynthesis 538
1. Introduction 539
2. Peptide Synthesis 542
3. Peptide Thioesters 550
4. In Vitro Assays with OxyB 553
5. Production and Purification of Enzymes 554
References 558
Chapter 20: Biosynthesis and Genetic Engineering of Lipopeptides in Streptomyces roseosporus 562
1. Introduction 563
2. Biosynthesis and Genetic Engineering of Daptomycin in S. rosesoporus 565
3. Sources of Genes for Combinatorial Biosynthesis 574
4. Genetic Engineering of Novel Lipopeptides 575
5. Concluding Remarks 578
Acknowledgments 579
References 579
Chapter 21: In Vitro Studies of Lantibiotic Biosynthesis 584
1. Introduction 585
2. Mining Microbial Genomes for Novel Lantibiotics 588
3. Expression and Purification of Lantibiotic Precursor Peptides (LanAs) 589
4. Expression, Purification, and Assay of LanM Enzymes 593
5. Expression, Purification, and Assays of LanC Cyclases 598
6. The Protease Domain of Class II Lantibiotic Transporters 603
7. Additional Posttranslational Modifications in Lantibiotics 604
References 605
Chapter 22: Whole-Cell Generation of Lantibiotic Variants 610
1. Introduction 610
2. Variant Generation 612
3. Conclusions 622
References 622
Chapter 23: Cyanobactin Ribosomally Synthesized Peptides-A Case of Deep Metagenome Mining 626
1. Introduction 627
2. Some Remaining Questions 634
3. Obtaining Prochloron Cells and DNA 634
4. Chemical Analysis 637
5. Cyanobactin Gene Cloning and Identification 638
6. Heterologous Expression in E. coli 641
7. Deep Metagenome Mining 642
8. Enzymatic Analysis of Cyanobactin Biosynthesis 644
9. Applying Deep Metagenome Mining: Pathway Engineering 644
Acknowledgments 646
References 646
Author Index 648
Subject Index 678
Colour Plates 688

Chapter 1

Approaches to Discovering Novel Antibacterial and Antifungal Agents

Stefano Donadio*,†; Paolo Monciardini*; Margherita Sosio* * KtedoGen, Via Fantoli, Milano, Italy
† NAICONS, Via Fantoli, Milano, Italy

Abstract

The need for novel antibiotics to fight multidrug-resistant pathogens calls for a return to natural product screening, but novel approaches must be implemented to increase the chances of discovering novel compounds. This chapter illustrates strategic considerations and the required ingredients for screening programs: microbial diversity, samples for screening, targets and assays, assay development and implementation, hit identification and follow-up. When appropriate, we highlight the impact that chemical diversity consisting of mixtures of different compounds, amid a large background of known antibiotics, has on the screening process. Examples of detailed procedures are described for strain isolation and preservation, sample preparation, primary and secondary assays, and extract fractionation. While these limited examples are not sufficient to organize a complete screening program, they provide a basis for understanding the details of microbial product screening in the anti-infective field.

1 Introduction

There is a need for novel and more effective antibiotics to combat multidrug resistant pathogens. Due to aging, immunosuppression and invasive surgical procedures, an ever-larger population is at risk of contracting severe infections, while bacterial pathogens are becoming increasingly resistant to currently available antibiotics. As a consequence, infections caused by drug-resistant bacteria are associated with increased morbidity, mortality, and health care costs.

It is now widely accepted that, despite the plethora of novel targets provided by the sequenced genomes of bacterial pathogens, combinatorial chemistry and high throughput screening (HTS) have failed to provide novel drug candidates in the anti-infective field, resulting in a virtually empty pipeline of compounds under development (Donadio et al., 2005a; Payne et al., 2007). This has prompted many players in the field to advocate a return to screening natural products, which constitute most of the clinically used antibiotics and the bulk of compounds under development (Butler, 2008).

During the golden era of antibiotic discovery in the 1950s and 1960s, the then-untapped diversity of soil actinomycetes provided most of the antibiotics known today. Over several decades, tens of millions of soil microorganisms were screened for anti-infective activity (Baltz, 2005). Thus, most low-hanging fruits have probably been picked and the discovery of a new antibiotic is nowadays a rare event. Furthermore, newly discovered compounds must possess advantages over the many antibiotics in clinical use, which implies the early recognition of potentially valuable compounds.

1.1 Objectives of a screening program

The objective of a screening program for anti-infectives is the discovery of a new, patentable chemical entity possessing desired properties, such as antimicrobial spectrum, molecular weight range, solubility, and preferred route of administration. Obviously, the higher the bar is set in terms of desired properties, the lower the probability of identifying the desired compound(s). Thus, a compromise is reached by defining the minimal properties a compound should have to be pursued and characterized. To reduce costs, it is also important that these properties can be established using a small amount of compound. For some classes of antibiotics (e.g., β-lactams, macrolides, glycopeptides, aminoglycosides, and polyenes) many variants are known, either as the product of different strains or because they have undergone extensive chemical analoguing. There is thus a small probability of discovering improved variants through screening in these classes. Consequently, screening programs usually aim at discovering either a novel class or an improved variant of a poorly explored class.

Operationally, screening microbial products requires: (1) a library of microbial strains that produce a diverse and biologically relevant set of compounds; (2) appropriate tests to detect compounds of potential value; (3) samples derived from the strains; (4) instrumentation and data capture tools adequate to the size of the screening program; (5) identification of the molecules responsible for the activity and recognition of known compounds; (6) additional tests to profile the molecules; and (7) ability to supply increasing amounts of active compounds. These ingredients, schematized in Fig. 1.1, are strongly intertwined as, for example, the tests must take into account the known compounds produced by the screened strains. Screening also uses its own jargon and relevant terminology is summarized in Table 1.1.

Figure 1.1 Elements of a screening process.

Table 1.1

Common terms used in microbial product screening

Term

Meaning

Primary assay

A test used to screen all samples in the library

Secondary assay

One or more tests used only on the positives

Screening algorithm

The sequence of primary and secondary assays with the corresponding threshold values for a sample being positive or negative

Positive

A sample giving a signal above the established threshold in a primary assay

Hit	A sample passing the selectivity criteria of the screening algorithm

Dereplication

The procedure of selecting one representative only among identical strains or among strains producing an identical compound

Extract

A sample used in screening obtained by processing a fermentation broth or a cell culture

1.2 Screening strategy and novelty of the program

Once the objective is set, the screening strategy must consider chemical diversity (how many different strains are available? how frequently do they produce bioactive compounds? do these belong to different chemical classes?), the test system (selectivity and sensitivity of the assay, hit rate), and the known natural products detected by the assay. This knowledge helps delineate the screening program in terms of size, screening algorithm, and hit dereplication (Table 1.1).

A key factor for success is to introduce elements of novelty with respect to previous screening campaigns. Although many details are not known, a reasonable assumption is that easy-to-isolate strains (e.g., streptomycetes and other easily retrieved actinomycetes) were screened for antibacterial and antifungal agents by simple growth inhibition tests, prioritizing the many positives by potency, selective effect on a macromolecular synthesis and/or lack of toxicity. Consequently, most discovered compounds are produced by relatively abundant species within the sampled genera, act on canonical targets (e.g., cell-wall synthesis, transcription, and translation), and are rather potent and not obviously toxic (or coproduced with toxic compounds). The novelty of a program can thus result from a relatively unexplored source of microbial diversity, from a sensitive assay that detects a subset of growth inhibition events, and from a screening algorithm (Table 1.1) that effectively filters out most known compounds. These factors greatly contribute to the probability that a screening campaign will identify novel compounds at a reasonable cost.

The following sections describe the elements of a screening process, providing also selected experimental details. They are mostly based on the authors' experience at previous companies (Biosearch Italia and Vicuron Pharmaceuticals), with modifications implemented at their current affiliations.

2 Strains and Samples

Chemical diversity is obtained by the simplified steps illustrated in Fig. 1.2. Several methods allow culturing of microorganisms from environmental samples, which are then purified as single colonies and grown in liquid media for inclusion in the strain library (Fig. 1.2A). A sample bank can be obtained by culturing the strains in appropriate media, followed by sample processing (Fig. 1.2B). Although samples can be immediately screened, it is preferable to store them as separate aliquots for comparing results obtained with different tests.

Figure 1.2 Strains and samples. (A) activities needed to generate a strain library. (B) activities for generation of a sample bank from the strain library.

The key element in generating a sample bank is diversification, which results from the diversity of the strains and their growth under appropriate conditions. This ideally requires the ad hoc development of methods for strain cultivation. However, since newly isolated strains are, by definition, uncharacterized, new fermentation or processing methods are usually developed through a pilot study on a limited number of strains, and then systematically applied to the entire library.

The results from the screening establish the true value of the samples, in terms of diversity of isolated strains,...

Erscheint lt. Verlag	10.4.2009
Sprache	englisch
Themenwelt	Medizin / Pharmazie ► Gesundheitsfachberufe
	Medizin / Pharmazie ► Medizinische Fachgebiete ► Pharmakologie / Pharmakotherapie
	Naturwissenschaften ► Biologie ► Biochemie
	Naturwissenschaften ► Physik / Astronomie ► Angewandte Physik
	Technik
ISBN-10	0-08-092335-6 / 0080923356
ISBN-13	978-0-08-092335-2 / 9780080923352

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