Complex Enzymes in Microbial Natural Product Biosynthesis, Part B: Polyketides, Aminocoumarins and Carbohydrates (eBook)

Front Cover 1
Methods in Enzymology Complex Enzymes inMicrobial Natural ProductBiosynthesis, Part B:Polyketides,Aminocoumarins andCarbohydrates 2
Copyright Page 5
Contents 6
Contributors 14
Preface 18
Methods in Enzymology 22
Chapter 1: Introduction to Polyketide Biosynthesis 52
1. Introduction 52
1.1. Types of PKS 52
1.2. Type II PKS 55
1.3. Type III PKS 55
1.4. Type I PKS 55
1.5. Combinatorial biosynthesis: Prospects and progress 55
Acknowledgments 62
References 62
Chapter 2: Structural Enzymology of polyketide Synthases 66
Chapter 3: Chapter Three: Fungal Type I Polyketide Synthases 97
1. Introduction 98
2. Partially Reducing PKSs: 6-Methylsalicylate Synthase 102
3. Nonreducing PKSs 105
3.1. Norsolorinic acid synthase 105
3.2. Tetrahydroxynaphthalene synthase 109
3.3. Bikaverin nonaketide synthase 112
4. Highly Reducing PKSs 114
4.1. Lovastatin (LNKS and LDKS) 114
4.2. HR PKS-NRPS: Fusarin and tenellin synthetases 117
5. NR/HR PKS Hybrid Systems: Zearalenone (ZAE1 and ZAE2) 120
6. Conclusions 122
References 122
Chapter 4: Tandem Acyl Carrier Protein Domains in Polyunsaturated Fatty Acid Synthases 127
1. Introduction 128
2. Methods 133
2.1. Production of PUFAs in E. coli by expressing the PUFAS genes 133
2.2. Mapping the active sites of PfaA-ACPs by site-directed mutagenesis 134
2.3. Overproduction of each of the PfaA-ACPs 137
2.4. Overproduction of PfaE and Svp PPTases 139
2.5. In vivo and in vitro preparation of the holo-form of PfaA-ACPs 140
2.6. Elucidation of the relationship between PUFA production and the number of active ACPs 141
3. Conclusion 141
Acknowledgments 143
References 144
Chapter 5: Iterative Type I Polyketide Synthases for Enediyne Core Biosynthesis 145
1. Introduction 146
2. Methods 149
2.1. PCR amplification of PKSE cassettes for predictive classification of new enediynes 149
2.2. Heterologous expression and overproduction of PKSE proteins 151
2.3. Production and isolation of the polyene intermediate from 9-membered PKSEs 153
2.4. Production of apo-ACPs from PKSE for in vitro functional analyses 155
2.5. In vitro preparation of holo-ACPs 156
3. Conclusion 157
Acknowledgments 159
References 159
Chapter 6: Chapter Six The DEBS Paradigm for Type I Modular Polyketide Synthases and Beyond 161
1. Introduction 162
2. DEBS and the Concept of a Module 162
2.1. Generalizability of the DEBS paradigm 166
3. Beyond the DEBS Paradigm 167
3.1. Specificity of the AT domains 170
3.2. Novel loading modules 171
3.3. Methylation domains 174
3.4. Trans PKS activities 175
3.5. Unusual modular organization 178
3.6. Unusual module functions 181
3.7. Intermodular interactions 183
4. Conclusion 184
References 184
Chapter 7: Formation and Characterization of Acyl Carrier Protein-Linked Polyketide Synthase Extender Units 191
1. Introduction 192
2. Overproduction and Purification of Recombinant Proteins 195
2.1. Principle 195
2.2. Materials 195
2.3. Heterologous overproduction of proteins 196
2.4. Purification of enzymes using batch-binding method with Nickel-NTA resin 197
3. Formation and Characterization of Hydroxymalonyl-ACP and Aminomalonyl-ACP 199
3.1. In vitro phosphopantetheinylation of the ACPs ZmaD and ZmaH 199
3.2. HPLC-based characterization of modified ACPs 200
3.3. Formation of (2R)-hydroxymalonyl-ACP 202
3.4. Formation of (2S)-aminomalonyl-ACP 205
3.5. MALDI-TOF MS analysis of ACPs 206
3.6. Other methods for characterizing enzymes involved in (2S)-aminomalonyl-ACP formation 208
3.7. Other methods for characterizing enzymes involved in (2R)-hydroxymalonyl-ACP formation 209
References 209
Chapter 8: Type I Polyketide Synthases That Require Discrete Acyltransferases 213
1. Introduction 214
2. Methods 222
2.1. Heterologous expression and overproduction of apo-ACPs from AT-less PKS modules 222
2.2. In vitro preparation of holo-ACPs 223
2.3. Heterologous expression and overproduction of discrete ATs 225
2.4. In vitro assay for AT substrate specificity 225
2.5. In vitro assay of AT-catalyzed loading of acyl CoA extender substrate to holo-ACPs 227
3. Conclusion 230
Acknowledgments 231
References 231
Chapter 9: The Enzymology of Polyether Biosynthesis 235
1. Introduction 236
2. Genetic Mining and Functional Analysis of Genes Specific to Polyether Biosynthetic Pathways 239
3. Premonensin, the Parent Unsaturated Monensin Polyketide 243
4. Epoxidases MonCI, NigCI, and NanO 246
5. Epoxide Hydrolases MonBI/BII, NigBI/BII, NanI, and Lsd19 247
6. NanE, a Polyether-Specific Thioesterase 249
6.1. In vitro studies of NanE thioesterase 252
7. Assay of Polyether Thioesterase Activity 254
7.1. Site-directed mutagenesis of NanE 257
8. Transcriptional Analysis of the Nanchangmycin Biosynthetic Pathway Genes 257
9. Conclusions 258
Acknowledgments 258
References 259
Chapter 10: Enzymology of the Polyenes Pimaricin and Candicidin Biosynthesis 263
1. Introduction 264
2. Pimaricin as a Prototype of Small Polyenes: Discovery and Properties 265
3. Pimaricin Biosynthesis in S. natalensis 266
3.1. The pimaricin gene cluster 266
3.2. Formation of pimaricinolide: The pimaricinolide synthase complex 266
3.3. Pimaricinolide tailoring and export 271
4. Regulation of Pimaricin Biosynthesis 272
4.1. Transcriptional regulators 272
4.2. Regulation by cholesterol oxidase 273
4.3. Inducers of pimaricin biosynthesis 274
4.4. Global regulatory mechanisms 274
5. Candicidin: A Prototype of ``Aromatic´´ Polyenes 276
6. The Candicidin/FR-008 Gene Cluster 277
7. Biosynthesis of PABA: The pabAB and pabC Genes 278
8. The Polyketide Synthases 281
9. Monooxygenase Genes: Modifications of the Polyketide Chain 282
10. Transporter Genes 283
11. Genes Related to Mycosamine Biosynthesis 284
12. Regulatory Genes 284
13. Phosphate Represses Expression of the pabAB Gene 285
14. Future Perspectives 285
Acknowledgments 286
References 286
Chapter 11: Genetic Analysis of Nystatin and Amphotericin Biosynthesis 291
1. Introduction 292
2. Gene Inactivation and Replacement in the Nystatin Producer Streptomyces noursei 294
2.1. Conjugative transfer of a recombinant plasmid from E. coli ET12567 (pUZ8002) into S. noursei ATCC 11455 295
2.2. Gene inactivation in S. noursei 296
2.3. Gene replacement in S. noursei 296
3. Gene Inactivation and Replacement in the Amphotericin Producer Streptomyces nodosus 297
3.1. Phage-mediated gene replacement in S. nodosus 300
4. Production, Purification, and Characterization of Novel Amphotericin- and Nystatin-Related Polyenes 302
4.1. Production and identification of nystatin-related polyenes 302
4.2. Scaled-up production of nystatin analogues 303
4.3. Preparative LC-MS purification of nystatin analogues 303
5. Conclusion 304
Acknowledgments 304
References 304
Chapter 12: Polyketide Versatility in the Biosynthesis of Complex Mycobacterial Cell Wall Lipids 307
1. Introduction 308
2. Acetate and Propionate Feeding Studies 313
3. Genome Sequencing and Identification of Polyketide Synthases 314
4. Mycobacterial Polyketide Synthases 315
4.1. Biosynthesis of dimycocerosate esters (DIMs) by PKS15/1, PpsABCDE, and MAS 315
4.2. PKS2 is involved in biosynthesis of sulfolipids 315
4.3. PKS12 uses a novel ``modularly iterative´´ mechanism for biosynthesis of mannosyl-beta-1-phosphomycoketides 320
4.4. PKS13 catalyzes condensation of fatty-acyl chains during biosynthesis of mycolic acids 321
4.5. PKS3/4 is involved in the biosynthesis of phthenoic acids 325
4.6. PKS10, PKS7, PKS8, PKS17, PKS9, and PKS11 constitute an unusual PKS cluster 326
4.7. PKS18 is involved in biosynthesis of long-chain pyrones 327
4.8. MbtC and MbtD are involved in biosynthesis of iron-chelating siderophores from Mtb 328
4.9. PKS5 and PKS6 329
5.1. Product formation assay to study activity of PKS enzymes 330
5.2. Characterization of PKS derived saturated fatty acids 330
References 332
Chapter 13: Genetic Engineering to Produce Polyketide Analogues 343
1. Introduction 344
2. AT Domain Replacement to Alter a-Carbon Substitution 348
3. Procedure for Engineering AT Replacements in the Chromosome 349
4. Engineering beta-Carbon Processing 353
5. Engineering when only a Single Crossover Event is Possible 356
6. Heterologous Expression of Engineered PKS Genes 357
7. Chemobiosynthesis 359
8. Mutasynthesis 361
9. Gene Knockouts to Obtain Analogues 362
References 363
Chapter 14: Design and Synthesis of Pathway Genes for Polyketide Biosynthesis 367
1. Introduction 368
2. Redesign of PKS Genes to Accommodate Unique Restriction Sites Flanking Individual Components and for Efficient Expression in E 370
3. Validation of Synthetic PKS Gene Design 372
4. A Rapid Assay to Identify Productive Combinations of PKS Modules 375
5. Assembly of Larger Polyketide Synthases Using Information Gained with the Bimodular Assay 378
6. Design and Construction of Synthetic Operons for the Expression of Sugar Pathway Genes 380
References 383
Chapter 15: Heterologous Production of Polyketides in Bacteria 387
1. Introduction 388
2. General Considerations for the Heterologous Expression of Polyketide Pathways 389
3. S. coelicolor as a Model System for Heterologous Expression of Polyketides 392
4. Procedure for the Heterologous Production of Polyketides in S. coelicolor 395
5. System Improvements for the Heterologous Production of Polyketides in Streptomyces spp. 397
5.1. Handling large PKS genes 397
5.2. Improving polyketide titers 399
5.3. Optimization of conjugation protocols for industrial strains 402
5.4. Optimizing polyketide precursors supply 403
6. Recent Developments for the Production of Polyketides in Nonactinomycete Bacteria 405
6.1. E. coli as heterologous host 405
6.2. Pseudomonas putida as heterologous host 407
References 408
Chapter 16: In Vitro Analysis of Type II Polyketide Synthase 415
1. Introduction 416
2. Expression and Purification of Type II PKSs 419
2.1. Escherichia coli as a host for protein expression 419
2.2. ACP expression and modification 420
2.3. Streptomyces as a host for PKS expression 421
3. In Vitro Activity Assays 424
3.1. Minimal PKS activity assays 424
3.2. Starter unit synthesis and incorporation assays 429
3.3. Assays of tailoring enzymes responsible for aromatic polyketide scaffold formation: KR, ARO, and CYC 432
3.4. Assays of downstream tailoring enzymes 434
Acknowledgments 437
References 438
Chapter 17: Bacterial Fatty Acid Synthesis and its Relationships with Polyketide Synthetic Pathways 443
1. Introduction 444
2. Bacterial Fatty Acids 444
3. Acyl Carrier Protein, the Key Component of Bacterial Fatty Acid Synthesis 445
4. Overview of the Reactions of Fatty Acid Biosynthesis 448
5. The Initiation Steps of Fatty Acid Synthesis 450
6. The Enzymes of the Fatty Acid Elongation Cycle 452
6.1. The 3-ketoacyl-ACP synthase reaction (FabB, FabF, FabH) 452
6.2. 3-ketoacyl-ACP reductase (FabG) 454
6.3. The 3-hydroxyacyl-ACP dehydratase (FabZ) 454
6.4. The enoyl-ACP reductase (FabI) 455
7. Unsaturated Fatty Acid Synthesis in E. coli 455
7.1. The 3-hydroxydecanoyl-ACP dehydratase (FabA) 455
8. The Abundant Exceptions to the E. coli Fatty Acid Synthesis Paradigm 458
8.1. Branched-chain fatty acids 458
8.2. Anaerobic synthesis of unsaturated fatty acids 458
8.3. Diversity of enoyl-ACP reductases 458
8.4. Type I megasynthase fatty acid synthesis 459
9. Relationships between Fatty Acid Synthesis and Polyketide Synthesis 460
10. Methods for Study of Bacterial Fatty Acid Synthesis 463
10.1. Preparation of the holo and apo forms of E. coli ACP 463
10.2. Strains and plasmids used 464
10.3. Holo-ACP 464
10.4. Apo-ACP 466
10.5. Synthesis of Acyl-ACP substrates 466
10.6. Purification of fatty acid synthetic enzymes 468
10.7. Direct assays of fatty acid synthetic enzyme activities 468
10.8. Reconstituted fatty acid synthesis systems 470
10.9. Resolution of ACP species by conformationally sensitive gel electrophoresis 471
10.10. ACPs behave abnormally in SDS-polyacrylamide gel electrophoresis and gel filtration 473
References 474
Chapter 18: Aminocoumarins: Mutasynthesis, Chemoenzymatic Synthesis, and Metabolic Engineering 485
1. Introduction 486
2. Generation of Integrative Cosmids and Heterologous Expression of Novobiocin, Clorobiocin, and Coumermycin A1 Gene Clusters 489
3. Generation of Single and or Multiple Deletions in the Integrative Cosmids 490
4. Mutasynthetic Generation of New Aminocoumarin Antibiotics 492
5. In Vitro Amide Synthetase Assays for the Identification of Suitable Ring A Analogues for Mutasynthesis 494
6. Use of Various Amide Synthetase Genes for Expanding Mutasynthesis Product Range 495
7. Generation of Substrates for Chemoenzymatic Synthesis 495
8. Chemoenzymatic Synthesis of New Clorobiocin Analogues 497
9. Generation of New Aminocoumarin Antibiotics by Metabolic Engineering 499
10. Conclusion 501
Acknowledgments 501
References 502
Chapter 19: Chapter Nineteen Enzymology of Aminoglycoside Biosynthesis-Deduction from Gene Clusters 507
1. Introduction 508
1.1. Basic concepts of CAG biosynthesis 512
1.2. Key to the biosynthetic classes of CAGs 515
2. Some Key Enzymes in the Biosyntheses of Aminoglycoside Antibiotics 517
2.1. Investigation of a dTDP-6-deoxyhexose pathway in STR-producing Streptomyces griseus subsp. griseus DSM 517
2.2. dTDP-D-glucose synthase (StrD, RfbA) 518
2.3. dTDP-D-glucose 4,6-dehydratase (StrE, RfbB) 519
2.4. dTDP-4-6-glucose 3,5-epimerase (StrM, RfbC) 519
2.5. dTDP-L-rhamnose synthase (StrL, RfbD) 519
2.6. L-glutamine:scyllo-inosose aminotransferase StsC (EC 2.6.1.50) from Streptomyces griseus subsp. griseus DSM 4023 520
2.7. L-arginine:scyllo-inosamine-phosphate amidinotransferase StrB1 from Streptomyces griseus subsp. griseus D 521
2.8. Streptomycin-phosphate phosphatase StrK (EC 3.1.3.39) from Streptomyces griseus subsp. griseus DSM 40236 (ATCC 10137) 521
2.9. Myo-inositol-2-dedydrogenase (IDH scyllo-inosose synthase) ForG (EC 1.1.1.18) from Micromonospora523
2.10. Glycosyltransferases involved in CAG pathways 524
2.11. KanM1, KanM2, and KanN of S. kanamyceticus DSM 40500 525
3. Acarbose and Related Metabolites 526
3.1. The synthesis and modification of C7-cyclitols 527
3.2. The biochemistry of carbophors: A unique system for the acquisition of glucose from starch in actinomycetes 529
References 535
Chapter 20: chapter twenty Biosynthetic Enzymes for the Aminoglycosides Butirosin and Neomycin 540
1. Introduction 541
2. General Method to Investigate Functions of the Biosynthetic Enzymes for Aminoglycosides 546
3. Neamine Biosynthetic Enzymes (Enzymes 17, and 20) 547
3.1. 2-Deoxy-scyllo-inosose (2DOI) synthase 547
3.2. L-Glutamine:DOI aminotransferase-a dual-function aminotransferase in 2DOS biosynthesis 548
3.3. 2-N-Acetylparomamine synthase (UDP-GlcNAc:2DOS N-acetylglucosaminyltransferase) 551
3.4. 2-N-Acetylparomamine deacetylase 552
3.5. FAD-dependent paromamine 6-dehydrogenase 552
3.6. 6-Dehydro-6-oxoparomamine aminotransferase 553
4. Ribostamycin Biosynthetic Enzymes (Enzymes 8 and 9) 554
4.1. Phosphoribostamycin synthase (PRPP: neamine phosphoribosyltransferase) 554
4.2. Phosphoribostamycin phosphatase 555
5. Neomycin Biosynthetic Enzymes (Enzymes 57, 10, and 11) (Addition of Neosamine B to Ribostamycin) 555
5.1. UDP-GlcNAc:ribostamycin N-acetylglucosaminyltransferase 555
5.2. 2-N-Acetyl-6-deamino-6& hyphe
5.3. 6-Deamino-6-hydroxyneomycin C dehydrogenase (probable repetitiv 556
5.4. 6-Deamino-6-dehydro-6-oxoneomycin C aminot 557
6. (S)-4-Amino-2-Hydroxybutyrate Biosynthetic Enzymes (Enzymes 1219) (Addition of AHBA to Ribost 557
6.1. gamma-L-glutamyl-ACP ligase and the pathway-specific acyl carrier protein 558
6.2. gamma-L-glutamyl ACP decarboxylase 559
6.3. gamma-L-glutamyl-4-aminobutyryl ACP mono-oxygenase and NAD(P)H:FMN oxidoreductase 560
6.4. gamma-L-glutamyl-4-amino-2-hydroxybutyryl ACP:ribostamycin gamma-L& hyphe
6.5. gamma-L-glutamyl-butirosin B gamma-L-glutamyl cyclotransferase 561
7. Other Related Enzymes in the Biosynthesis of 2DOS-Containing Aminoglycoside Antibiotics 562
8. Concluding Remarks and Future Perspectives 563
References 563
Chapter 21: Enzymatic Synthesis of TDP-Deoxysugars 568
1. Introduction 569
2. Enzymatic Synthesis of TDP-a-d-glucose 571
2.1. Preparation of enzymes required for in vitro synthesis of TDP-a-d-glucose (5) 572
2.2. Enzymatic synthesis of TDP-a-d-glucose (5) 573
2.3. Purification of TDP-a-d-glucose (5) 574
3. Generation of TDP-4-keto-6-deoxy-a-d-glucose (6) 576
3.1. Generation of TDP-2,6-dideoxysugars 578
4. In vitro Reconstitution of Entire Deoxysugar Biosynthetic Pathways 580
4.1. One-pot synthesis of TDP-a-d-mycaminose (12) 581
4.2. Two-stage one-pot synthesis of TDP-beta-l-mycarose (13) 582
4.3. Multistep enzymatic synthesis of TDP-a-d-forosamine (14) 583
4.4. TDP-a-d-desosamine (11) 585
5. Synthesis of Deoxysugars In Vivo by Metabolic Pathway Engineering 586
6. Summary 588
Acknowledgments 589
References 589
Author Index 592
Subject Index 618
Color Plates 629