Preface ....................................................... xix
Part I. Genetic Technology
1. Practical Molecular Taxonomy of Fungi
David M. Geiser
1 Introduction .............................................. 3
2 Identifying a Fungus to Species—What does it Mean? ........ 3
2.1 Useful Species Definitions ........................... 3
2.2 Genealogical Concordance as a Means to Recognize
Fungal Species ....................................... 4
2.3 Molecular Taxonomy in Practice ....................... 6
3 How do I Identify an Unknown Fungus? ...................... 6
3.1 The Molecular Toolbox ................................ 7
3.1.1 DNA Sequence Tools ............................ 7
3.1.2 Genotyping Methods: Comparing and Identifying
Isolates within a Species ..................... 9
3.2 Using the Toolbox ................................... 11
3.2.1 Tools for Identifying any Fungus ............. 11
3.2.2 Tools for Identifying Fungi in a Particular
Taxonomic Group of Intensive Study ........... 12
4 What is Next? ............................................ 12
References .................................................. 13
2. Genomics of Filamentous Fungi
Ulrich Schulte
1 Introduction ............................................. 15
2 Genomic Projects Focusing on Fungi ....................... 16
3 Genome Structure ......................................... 18
4 Gene Identification and Annotation ....................... 19
5 Gene Complement of a Filamentous Fungus .................. 21
6 Novel Aspects of Fungal Biology .......................... 26
7 Summary .................................................. 27
References .................................................. 27
3. A Molecular Tool Kit for Fungal Biotechnology
John E. Hamer
1 Introduction ............................................. 31
2 Vectors and Transformation ............................... 32
1 Gene Cloning Tools for Genomic Approaches ................ 32
4 Fungal Transposons as Tools .............................. 33
5 Tools for Identifying Essential Genes .................... 34
5.1 Generating Conditional Lethal Mutants ............... 34
5.2 Inference ........................................... 34
5.3 Using Controllable Promoters ........................ 34
5.4 Post-Transcriptional Gene Silencing (PTGS) .......... 35
6 Genome-Based Tools ....................................... 35
6.1 Genome-Wide Insertional Mutagenesis ................. 36
6.2 Genome-Shuffling .................................... 36
7 Summary .................................................. 37
References .................................................. 37
4. Transformation Mediated by Agrobacterlum tumefaclens
Paul J.J. Hooykaas
1 Introduction ............................................. 41
2 Agrobacterium ............................................ 42
3 Host Range ............................................... 44
4 T-DNA Transfer Resembles Bacterial Conjugation ........... 44
5 Accessory Functions Enabling Trans-Kingdom DNA Transfer .. 49
6 Protein Translocation from Agrobacterium into Host Cells . 50
7 T-DNA Integration ........................................ 51
8 Agrobacterium-Bastd Vector Systems ....................... 52
9 Transformation of Yeasts and Filamentous Fungi ........... 54
10 Concluding Remarks ....................................... 57
References .................................................. 58
Part II. Special (Secondary) Metabolism
5. Fungal Polyketide Synthases in the Information Age
Russell J. Cox and Frank Glod
1 Introduction ............................................. 69
1.1 Secondary Metabolites ............................... 69
1.2 Polyketides ......................................... 71
1.3 Types of Polyketide Synthase ........................ 72
2 Non-Fungal PKS ........................................... 73
3 Fungal PKS ............................................... 75
3.1 6-Methylsalicyclic Acid Synthase .................... 76
3.2 Fungal PKS Involved in Biosynthesis of Conidial
Pigment and Melanin ................................. 77
3.3 Fungal Polyketide Mycotoxins—Norsolorinic Acid
Synthase (NAS) ...................................... 78
3.4 Polyketide Synthase in T-Toxin Production ........... 79
3.5 Polyketide Synthase in Fumonisin Production ......... 79
3.6 Lovastatin Synthases ................................ 80
4 Novel Methods for Accessing PKS Genes .................... 81
4.1 Problems Associated with Cloning Fungal PKS Genes ... 81
4.2 Early Efforts to Develop Fungal PKS Probes .......... 82
4.3 Assessing Biosynthetic Potential .................... 85
4.3.1 Prokaryotes .................................. 85
4.3.2 Lichens ...................................... 85
4.3.3 Insect and Nematode Associated Fungi ......... 86
4.3.4 Endophytic Fungi ............................. 88
4.4 Biosynthetically Informed Approaches for Accessing
Fungal PKS Genes .................................... 88
4.4.1 KS-Specific Primers .......................... 88
4.4.2 KR-Specific Primers .......................... 90
4.4.3 CmeT-Specific Primers ........................ 90
4.4.4 Lessons and Outlook .......................... 92
5 The Genomic Era ............................................. 92
References .................................................. 93
6 More Functions for Multifunctional Polyketide Synthases
Isao Fujii, Akira Watanabe, and Yutaka Ebizuka
1 Introduction ............................................. 97
2 Architecture and Functions of Fungal Polyketide
Synthases ............................................... 100
2.1 MS AS/OAS Polyketide Synthases ..................... 100
2.2 Polyketide Synthases for Aromatic Multi-Ring
Products (AR-PKSs) ................................. 100
2.2.1 Pentaketide 1,3,6,8-Tetrahydroxynaphthalene
Synthases ................................... 101
2.2.2 Heptaketide Naphthopyrone Synthases ......... 102
2.2.3 PKSs Involved in Aflatoxin Biosynthesis ..... 103
2.3 Polyketide Synthases for Reduced Products
(RD-PKSs) .......................................... 103
2.3.1 T-toxin PKS ................................. 104
2.3.2 PKSs Involved in Lovastatin Biosynthesis .... 105
2.3.3 Fumonisin PKS ............................... 105
2.3.4 RD-PKS from Altemaria solani ................ 106
3 More Functions for Fungal Polyketide Synthases .......... 106
3.1 Claisen Cyclase Domain in AR-PKSs .................. 107
3.2 More Functions for AR-PKSs ......................... 110
3.2.1 Starter Units ............................... 110
3.2.2 N-Termini ................................... 111
3.2.3 InterdomainRegions .......................... 111
3.2.4 ACPDomains .................................. 111
3.3 C-Methyltransferase Domains in RD-PKSs ............. 113
3.4 PSED (Peptide Synthetase Elongation Domain)-Like
Domains in RD-PKSs ................................. 114
3.5 "Diels-Alderase" in RD-PKSs ........................ 115
4 Concluding Remarks ...................................... 118
Acknowledgments ............................................ 120
References ................................................. 120
7 Peptide Synthesis Without Ribosomes
Jonathan D. Walton, Daniel G. Panaccione, and Heather
E. Hallen
1 Introduction ............................................ 127
2 Overview of Non-Ribosomal Peptide Synthetases ........... 129
3 The "Non-Ribosomal Code" for Fungal NRP Synthetases ..... 132
4 Parsing Fungal NRP Synthetases .......................... 136
4.1 Guideline 1 ........................................ 136
4.2 Guideline 2 ........................................ 137
4.3 Guideline 3 ........................................ 137
5 Strategies to Identify NRP Synthetases and Genes ........ 138
6 Tailoring Enzymes and Auxiliary Domains ................. 139
6.1 N-Methylation ...................................... 139
6.2 Epimerization ...................................... 140
6.3 Other Tailoring Reactions of Fungal NRP
Synthetases ........................................ 140
6.4 Pantetheinylation .................................. 141
7 Regulation .............................................. 142
8 Status of Research on Selected Fungal Systems ........... 143
8.1 AM-Toxin ........................................... 143
8.2 Cyclosporin ........................................ 144
8.3 Destruxins ......................................... 145
8.4 Enniatins .......................................... 145
8.5 Ergopeptines ....................................... 146
8.6 НС-Toxin ........................................... 148
8.7 Penicillin and Cephalosporin ....................... 149
8.8 Peptaibols ......................................... 150
9 Evolution of NRPs and NRP Synthetases ................... 151
9.1 Clustering ......................................... 151
9.2 Evolution of Secondary Metabolite Pathways ......... 152
10 NRP Synthetases in the Genomics Age ..................... 154
Acknowledgments ............................................ 154
References ................................................. 155
8. Isoprenoids: Gene Clusters and Chemical Puzzles
D. Barry Scott, Geoffrey B. Jameson, and Emily J. Parker
1 Introduction ............................................ 163
2 Sesquiterpenes .......................................... 165
2.1 Trichothecenes ..................................... 165
2.1.1 Chemical Diversity .......................... 165
2.1.2 Gene Clusters ............................... 166
2.1.3 Biosynthesis of T2-Toxin .................... 167
2.1.4 Regulation .................................. 172
2.2 Aristolochenes ..................................... 173
3 Diterpenes .............................................. 174
3.1 GibbereUins ........................................ 174
3.1.1 Chemical Diversity .......................... 174
3.1.2 Gene Cluster ................................ 175
3.1.3 Biosynthesis of GA3 ......................... 175
3.1.4 Regulation .................................. 179
3.2 Indole-Diterpenes .................................. 179
3.2.1 Chemical Diversity .......................... 180
3.2.2 Gene Cluster ................................ 180
4. Tetraterpenes ........................................... 184
4.1 Carotenoids ........................................ 184
4.1.1 Chemical Diversity .......................... 185
4.1.2 Biosynthetic Pathway ........................ 185
5 Proteins of Isoprenoid Biosynthetic Pathways ............ 189
5.1 Initiation of Prenyl Transfer ...................... 189
5.2 Prenyl Transferase Structure and Classification .... 190
5.3 Trichodiene Synthase ............................... 191
5.4 Aristolochene Synthase ............................. 192
6 Final Remarks ........................................... 192
Acknowledgments ......................................... 193
References .............................................. 193
Part III. Enzymes and Green Chemistry
9 Heterologous Expression and Protein Secretion in
Filamentous Fungi
Wendy Thompson Yoder and Jan Lehmbeck
1 Introduction ............................................ 201
2 The Past Decade ......................................... 202
3 Development of a New Fungal Expression Host: Fusarium
venenatum Nirenberg ..................................... 208
3.1 Selection Criteria ................................. 208
3.2 Heterologous Expression ............................ 209
3.3 Improved Morphological Mutants ..................... 209
3.4 Selectable Markers ................................. 209
3.5 Targeted Gene Deletions ............................ 210
3.6 GRAS Status for the First Heterologous Enzyme
Produced in F. venenatum ........................... 210
3.7 The First Commercial Recombinant F. venenatum
Product ............................................ 210
3.8 Fusarium venenatum Genomics ........................ 210
4 "To Infinity and Beyond" ................................ 211
5 Conclusions ............................................. 212
References ................................................. 213
10 Artificial Evolution of Fungal Proteins
Jesper Vind
1 Introduction ............................................ 221
2 Artificial Evolution in General ......................... 221
2.1 Idea Generation .................................... 221
2.2 In Vitro Generation of Gene Variants ............... 222
3 In Vitro Mutagenesis and Expression of Fungal Proteins .. 222
3.1 Characterization of Protein Variants Expressed in
Yeast .............................................. 222
3.2 Characterization of Protein Variants Expressed
in Filamentous Fungi ............................... 224
3.3 Library Generation in Filamentous Fungi ............ 225
4 In Vivo Mutagenesis in Fungi ............................ 226
4.1 In Vivo Shuffling in Yeast ......................... 226
4.2 In Vivo Shuffling in Neurospora .................... 226
4.3 In Vivo Mutagenesis with the RIP System ............ 228
4.4 In Vivo Mutagenesis with the Mismatch Repair
System ............................................. 228
5 Future in Artificial Evolution of Fungal Proteins ....... 231
References ................................................. 232
11.Biocatalysis and Biotransformation
Frieder Schauer and Rainer Borriss
1 Preface ................................................. 237
2 Fungal Enzymes and Biotransformations—An Introduction ... 237
3 Glycosyl Hydrolases ..................................... 242
3.1 Starch Hydrolysis: Amylases and Glucoamylases ...... 244
3.2 Cellulose and Cellulases ........................... 245
3.2.1 Cellulases in Textile and Laundry
Biotechnology ............................... 246
3.3 Hydrolysis of Hemicellulose: Xylanases and
Mixed-Linked β-Glucanases .......................... 246
3.3.1 Xylanases ................................... 246
3.3.2 Application: Delignification of Kraft
Pulps by Trichoderma Xylanases .............. 247
3.3.3 Mixed Linked β-Glucan Hydrolyzing Enzymes ... 247
3.3.4 Application of Cellulases and
Hemicellulases in Animal Feed
Biotechnology ............................... 249
3.4 Cell Wall Lytic Enzymes ............................ 249
3.4.1 Macerating Enzymes in Fruit and Vegetable
Processing .................................. 249
4 Phosphorous Mobilization: Phytases ...................... 249
4.1 Engineering of Improved Functionality in
Aspergillus Phytase ................................ 252
5 Lipases (Triacylglycerol Hydrolases, EC 3.1.1.3) ........ 253
6 Proteases ............................................... 254
7 Degradation of Lignocellulose: Ligninolytic Enzymes ..... 255
7.1 Lignin Peroxidase and Manganese Peroxidase ......... 256
7.2 Laccase ............................................ 256
7.2.1 Distribution ................................ 257
7.2.2 Biological Function of Laccase .............. 257
7.2.3 Isoenzymes .................................. 258
7.2.4 Characterization and Some Biochemical
Properties .................................. 258
7.2.5 Regulation of Laccase Production ............ 259
7.2.6 Laccase Mediator Systems .................... 259
7.2.7 Delignification of Ligninocellulosics by
Laccase ..................................... 260
7.2.8 Purification of Colored Waste Waters ........ 260
7.2.9 Textile Dye Decolorization .................. 260
7.2.10 Transformation and Inactivation of Toxic
Environmental Pollutants .................... 260
7.2.11 Beverage and Food Treatment ................. 261
7.2.12 Laccase-Based Biosensors .................... 261
7.2.13 Synthesis of New Chemicals by Laccase ....... 261
7.2.14 Desulfurization and Solubilization of Coal .. 262
8 Utilization of Aromatic and Aliphatic Compounds and
Hydrocarbons ............................................ 262
9 Inactivation of Fungal Biocontrol Agents ................ 263
9.1 Creosote ........................................... 263
9.2 Pentachlorophenol .................................. 264
9.3 Inorganic Wood Preservatives ....................... 264
9.4 Disinfectants and Deodorants ....................... 265
9.5 Fungicides in Agriculture and Medicine ............. 265
9.6. Food Preservatives ................................. 265
10 Biotransformation of Biphenyls by Fungi ................. 266
10.1 Biphenyl ........................................... 266
10.2 Polychlorinated Biphenyls .......................... 268
11 Oxidation of Dibenzofurans and Dibenzodioxins ........... 269
12 Biotransformation of Diphenyl Ethers and Phenoxy
Herbicides .............................................. 272
13 Dehalogenation of Aromatic Xenobiotics .................. 275
14 Trends and Future Developments .......................... 276
14.1 Novel Fungal Enzymes: Screening, Development, and
Specific Features .................................. 277
14.2 Screening of Fungi Producing Improved Phytases ..... 278
14.3 Diversity of Microbial Enzymes Catalyzing
Stereoselective Reactions .......................... 279
14.4 Lactonase in D-Pantothenic Acid Production ......... 279
14.5 Aldehyde Reductase in the Production of Chiral
Alcohols ........................................... 279
14.6 Laccase-Catalyzed Heteromolecular Coupling of
Molecules .......................................... 279
14.7 Heterologous Expression of Fungal Ligninolytic
Enzymes ............................................ 280
14.8 Impact of DNA Recombinant Techniques ............... 280
14.9 Expression of Aspergillus Phytase in Transgenic
Plants ............................................. 281
14.10 Gene Libraries .................................... 282
14.11 Biomolecular Engineering .......................... 282
14.12 Concept of Directed Evolution ..................... 283
References ................................................. 283
12 Organic Acid Production by Filamentous Fungi
Jon K. Magnuson and Linda L. Lasure
1 Introduction ............................................ 307
2 Commercial Successes: Organic Acids from Filamentous
Fungi ................................................... 308
2.1 Citric Acid ........................................ 309
2.2 Gluconic Acid ...................................... 310
2.3 Itaconic Acid ...................................... 310
2.4 L-Lactic Acid ...................................... 311
2.5 Market Prospects ................................... 311
3 Biochemistry and Genetics of Organic Acid Production
by Filamentous Fungi .................................... 312
3.1 Aspergillus and Organic Acid Production ............ 312
3.1.1 Citric Acid ................................. 312
3.1.2 Oxalic Acid ................................. 319
3.1.3 Gluconic Acid ............................... 322
3.1.4 Itaconic Acid ............................... 323
3.2 Rhizopus and Organic Acid Production ............... 325
3.2.1 L-Lactic Acid ............................... 325
3.2.2 Fumaric Acid ................................ 328
3.2.3 L-Malic Acid ................................ 329
3.2.4 Succinic Acid ............................... 330
3.2.5 (—)-trans-2,3-Epoxysuccinic Acid and
meso-Tartaric Acid .......................... 330
4 Final Perspective ....................................... 332
References ................................................. 333
13 Flavors and Fragrances
Ralf G. Berger and Holger Zorn
1 Introduction ............................................ 341
2 Biotransformation of Terpenoids by Fungi ................ 343
3 Biosynthesis of Terpenyl Esters ......................... 348
4 Generation of Aromatic Flavor Compounds ................. 349
5 Flavor Compounds from Other Chemical Classes ............ 353
6 Bioprocess Technology ................................... 353
7 Conclusion .............................................. 355
References ................................................. 355
Part IV. Host-Fungal Interactions
14 Human Mycoses: The Role of Molecular Biology
Donald C. Sheppard, Ashraf S. Ibrahim, and John
E. Edwards Jr.
1 Introduction ............................................ 361
2 Goals in the Study of Pathogenic Filamentous Fungi ...... 362
2.1 Identification of Virulence Factors ................ 362
2.2 Identification of Other Drug Targets ............... 362
3 The Genus Aspergillus ................................... 362
3.1 Aspergillosis: Spectrum of Disease ................. 363
3.2 Aspergilloma ....................................... 363
3.3 Invasive Aspergillosis (IA) ........................ 363
3.3.1 Epidemiology and Significance ............... 363
3.3.2 Pathophysiology ............................. 363
3.3.3 Virulence Factors of A. fumigatus ........... 364
3.3.4 Clinical Presentation of IA ................. 364
3.3.5 Therapy of IA ............................... 364
3.4 Molecular Techniques for the Study of Aspergillus
sp ................................................. 366
3.4.1 Selection Markers for A. fumigatus .......... 366
3.4.2 Transformation Techniques ................... 369
3.4.3 Parasexual Genetics ......................... 370
3.4.4 Signature-Tagged Mutagenesis ................ 371
3.4.5 Reporter Gene Systems ....................... 372
3.4.6 Transposable Elements in Aspergilli ......... 373
3.4.7 Complementation and Heterologous
Expression in Aspergilli .................... 373
3.4.8 Genome Sequencing ........................... 374
4 The Agents of Mucormycosis .............................. 375
4.1 Molecular Techniques for the Study of
Mucormycosis ....................................... 376
4.1.1 Transformation Techniques ................... 377
4.1.2 Sexual Cycle ................................ 378
4.1.3 Heterologous Expression ..................... 378
4.1.4 Summary ..................................... 379
5 Other Pathogenic Filamentous Fungi ...................... 379
6 Future Directions ....................................... 379
References ................................................. 379
15 Molecular Interactions of Phytopathogens and Hosts
Joanna M. Jenkinson and Nicholas J. Talbot
1 Introduction ............................................ 385
1.1 The Life Cycles of Magnaporthe grisea and
Ustilago maydis .................................... 386
2 Pathogenicity Factors ................................... 387
2.1 Regulators of Infection ............................ 387
2.1.1 The cAMP Response Pathway ................... 387
2.1.2 PMK1 and MAP Kinase Pathways in Fungal
Pathogens ................................... 389
2.1.3 РАШ-Related MAP Kinases in Other
Phytopathogenic Fungi ....................... 392
2.1.4 Alternative МАРК Pathways in M. grisea ...... 392
2.1.5 Nutritional Regulatory Genes ................ 393
2.2 Pathogen-Specific Molecules ........................ 394
2.2.1 Toxins and Host-Specific Toxins ............. 394
2.3 Plant Recognition Evasion .......................... 395
2.3.1 Saponin Detoxification ...................... 395
2.3.2 Phytoalexin Detoxification .................. 395
2.4 Proteins of Unknown Function ....................... 396
2.5 Pathogen Associated Molecular Patterns ............. 397
2.5.1 Plant Resistance Mechanisms ................. 397
2.5.2 R Gene and Avr Gene Signaling ............... 398
3 Genomics of Phytopathogens .............................. 399
4 Future Prospects ........................................ 400
References ................................................. 400
16 Structural and Functional Genomics of Symbiotic Arbuscular
Mycorrhizal Fungi
V. Gianinazzi-Pearson, C. Azcon-Aguilar, G. Becard,
P. Bonfante, N. Ferrol, P. Franken, A. Gollotte,
L.A. Harrier, L. Lanfranco, and D. van Tuinen
1 Introduction ............................................ 405
2 Genome Structure and Organization ....................... 407
3 Fungal Genes in the Symbiotic Context ................... 408
3.1 Targeted Analyses of Gene Expression ............... 408
3.2 Transcriptome Profiling ............................ 412
4 Manipulating the Symbiotic Genome ....................... 414
5 Endobacterial Genes ..................................... 417
6 Conclusions ............................................. 418
Acknowledgments ......................................... 419
References .............................................. 419
Index ......................................................... 425
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