Handbook of Corynebacterium glutamicum

Front cover......Page 1
Foreword......Page 6
Editors......Page 8
Contributors......Page 10
Introduction......Page 12
Table of Contents......Page 14
Part I: History......Page 18
Introduction......Page 20
Role of Monosodium Glutamate......Page 21
References......Page 22
Part II: Taxonomy......Page 24
2.1 Position of Corynebacterium and Closely Related Genera within the Class Actinobacteria......Page 26
2.2 Taxonomy and Characteristics of the Genus Corynebacterium......Page 30
2.3 Methods for the Isolation, Identification, and Differentiation of Corynebacteria......Page 33
2.3.3 Mycolic Acids and Other Lipids......Page 37
2.4 Isolation, Classification, and Taxonomy of Industrially Relevant Corynebacteria......Page 38
2.4.1 Corynebacterium glutamicumAL......Page 39
2.4.3 Corynebacterium efficiensVP......Page 41
2.4.5 Corynebacterium ammoniagenesVP......Page 42
Acknowledgments......Page 43
References......Page 44
Part III: Genome, Plasmids, and Gene Expression......Page 52
3.1 Introduction......Page 54
3.2 Mapping and Sequencing of the C. glutamicum Genome......Page 55
3.4 Annotation of the C. glutamicum Genome......Page 56
3.5 The Overall Structure of the C. glutamicum Genome......Page 58
3.6 Prophages in the C. glutamicum Genome......Page 61
3.7 The Gene Inventory of C. glutamicum......Page 63
3.8 Comparative Corynebacterium Genome Analysis......Page 64
3.9 Conclusions......Page 68
Acknowledgments......Page 69
References......Page 70
4.1 Introduction......Page 74
4.2 Isolation of Plasmids from Amino Acid-Producing Corynebacteria......Page 78
4.3.1 The Archetype Plasmid pBL1 from B. lactofermentum ATCC 13869......Page 79
4.3.2 Other Members of the pBL1 Plasmid Family......Page 83
4.4.1 The Archetype Plasmid pCG1 from C. glutamicum ATCC 31808......Page 84
4.4.2 The Small Cryptic Plasmid pGA1 from C. glutamicum LP-6......Page 85
4.4.3 Large (Antibiotic Resistance) Plasmids of the pCG1 Family......Page 86
4.5 Structural Organization of Pxz10142 and Pxz10145 from C. glutamicum 1014......Page 89
4.7 Host Range of Plasmids from Amino Acid-Producing Corynebacteria......Page 90
4.8 Concluding Remarks and Perspectives......Page 91
References......Page 92
5.2 Promoters......Page 98
5.3 RNA Polymerase and Sigma Factors......Page 102
5.4 Regulation of Transcription Initiation......Page 104
5.6 Leaderless Transcripts......Page 107
5.7 Strategies of Modulation of Gene Expression......Page 109
Acknowledgments......Page 110
References......Page 111
6.1 Introduction......Page 116
6.2.1 Analysis of Protein Modifications......Page 117
6.2.2 Limitations of the Current Methods......Page 126
6.3 N-Terminal Processing of C. glutamicum Proteins......Page 128
6.4 Applications of Proteome Techniques......Page 130
6.5 Recent Developments and Outlook......Page 132
References......Page 133
Part IV: Transport......Page 136
7.1 Introduction......Page 138
7.2.1 Transmission Electron Microscopy......Page 139
7.2.2 Freeze-Etch Electron Microscopy......Page 140
7.3.1 The Plasma Membrane......Page 144
7.3.2.2 Arabinogalactan: Glycosyl Linkage Composition and Structural Features......Page 146
7.3.2.3 Mycolic Acids: Structure and Biosynthesis......Page 148
7.3.3 Cell Wall Proteins......Page 151
7.3.3.1 Mycoloyltransferases......Page 152
7.3.3.2 Porins......Page 154
7.3.5 Outer Layer......Page 155
7.5 Future Prospects......Page 156
Acknowledgments......Page 157
References......Page 158
8.1 Introduction......Page 166
8.4 Classes of Transporters Found in C. glutamicum and C. efficiens......Page 167
8.5 Classes of Substrates Transported......Page 168
8.7 Channels......Page 169
8.8 Secondary Carriers......Page 196
8.9 Primary Active Transporters......Page 198
8.14 Perspectives and Conclusions......Page 199
References......Page 202
9.1 Introduction......Page 204
9.2.1 Identification of the l-Lysine Exporter LysE......Page 206
9.2.2 Functional Residues within LysE......Page 207
9.2.4 Mechanism of l-Lysine Export......Page 209
9.2.5 Function of the Lysine Exporter......Page 210
9.2.7 Modulation of l-Lysine Export Activity......Page 211
9.3.1 The LysE and CadD Families......Page 212
9.4 Export of l-Threonine......Page 213
9.5 Export of Branched-Chain Amino Acids......Page 215
9.6 Export of l-Glutamate......Page 217
9.7 Contribution of Cell Wall to Amino Acid Export......Page 218
9.8 Further Exporters of C. glutamicum......Page 219
Acknowledgments......Page 220
References......Page 221
Part V: Physiology and Regulation......Page 230
Central Metabolism: Sugar Uptake and Conversion......Page 232
10.2 Sugar Uptake Systems......Page 233
10.2.1 Genome Analysis......Page 235
10.3 Glycolysis......Page 236
10.3.2.1 Glucose-6-Phosphate Isomerase......Page 238
10.3.2.4 Glyceraldehyde-3-Phosphate Dehydrogenase......Page 239
10.4 The Pentose Phosphate Pathway......Page 240
10.4.1 Genetic Organization......Page 242
10.4.2.1 Glucose-6-Phosphate Dehydrogenase......Page 245
10.5.1 Effect of Carbon Sources on the Operation of the Glycolytic and Pentose Phosphate Pathways......Page 246
10.5.2 Functional Operation of the Pentose Phosphate Pathway as Revealed by Mutant Analysis......Page 247
10.5.3.2 Flux Distribution in Lysine Producers......Page 248
10.6.1 Glycolysis......Page 249
10.7.1.1 Phosphoglucose Isomerase-Defective Mutant......Page 250
10.7.2.1 Aromatic Amino Acid Production by C. glutamicum......Page 251
10.7.2.2 Purine Nucleotide/Nucleoside Production by C. ammoniagenes......Page 252
References......Page 253
Central Metabolism: Tricarboxylic Acid Cycle and Anaplerotic Reactions......Page 258
11.1 Introduction......Page 259
11.2 Tricarboxylic Acid Cycle......Page 261
11.2.1.1 Pyruvate Dehydrogenase Complex......Page 262
11.2.1.2 Citrate Synthase......Page 266
11.2.1.4 Isocitrate Dehydrogenase......Page 267
11.2.1.5 2-Oxoglutarate Dehydrogenase Complex......Page 268
11.2.1.7 Succinate:Menaquinone Oxidoreductase......Page 269
11.2.1.9 Malate Dehydrogenase and Malate:Quinone Oxidoreductase......Page 270
11.2.2 Carbon Flux into and through the TCA Cycle......Page 271
11.2.3 Impact of TCA Cycle Reactions on Amino Acid Production......Page 273
11.3 Anaplerotic Reactions in Cells Growing on Carbohydrates......Page 274
11.3.1.2 Pyruvate Carboxylase......Page 275
11.3.1.4 Malic Enzyme......Page 276
11.3.2 Parallel and Bidirectional Carbon Fluxes at the PEP-Pyruvate-Oxaloacetate Node......Page 277
11.3.3 Impact of Anaplerotic Reactions on Amino Acid Production......Page 279
11.4.1.1 Isocitrate Lyase and Malate Synthase......Page 281
11.4.2 Carbon Fluxes through the Glyoxylate Cycle and Flux Control......Page 282
11.5 Global Control of the TCA Cycle and Anaplerosis......Page 283
11.6 Concluding Remarks and Perspectives......Page 284
References......Page 285
12.1 Introduction......Page 294
12.2.1 Prerequisites: Network Topology and Cellular Composition......Page 295
12.2.2 Metabolite Balancing......Page 296
12.2.3 Isotope Labeling......Page 301
12.3 Metabolic Fluxes in C. glutamicum......Page 303
12.3.1 Fluxes for the Generation of Reducing Power......Page 304
12.3.2 Anaplerotic Fluxes......Page 306
12.3.3 Fluxes in a Genealogy of Strains......Page 308
12.3.4 Fluxes on Different Carbon Sources......Page 310
12.3.5 Response of Fluxes to Different Cellular Demands......Page 313
12.3.6 Nitrogen Fluxes......Page 315
References......Page 317
13.1 Introduction......Page 322
13.2.1 NADH Dehydrogenase......Page 323
13.2.2 Succinate Dehydrogenase......Page 328
13.2.3 Malate:Quinone Oxidoreductase......Page 329
13.2.5 Lactate Dehydrogenases......Page 330
13.2.6 Glycerol-3-Phosphate Dehydrogenase......Page 331
13.2.8 Electron-Transferring Flavoprotein......Page 332
13.3 Electron Transfer from Menaquinol to Oxygen......Page 333
13.3.1 Cytochrome bc1 Complex......Page 334
13.3.2 Cytochrome aa3 Oxidase......Page 335
13.3.4 Cytochrome bd Menaquinol Oxidase......Page 338
13.4 Electron Transfer from Menaquinol to Nitrate......Page 339
13.5 Heme Biosynthesis and Cytochrome c Maturation......Page 340
13.6 Impact of F1F0-ATP Synthase on Metabolism......Page 341
13.7 Influence of Respiratory Chain Composition on the ATP Yield......Page 342
13.8 Biotechnological Aspects......Page 343
References......Page 344
14.1 Introduction......Page 350
14.2 Uptake of Nitrogen Sources......Page 351
14.2.2 Uptake of Urea and Urease Activity......Page 352
14.2.3 Transport of Other Nitrogen Sources......Page 353
14.3 Assimilation of Ammonium......Page 354
14.3.2 The Glutamine Synthetase/Glutamate Synthase Pathway......Page 356
14.4.1 Regulation of GS Activity......Page 357
14.4.2 The GlnK/UTase Pathway......Page 358
14.5.1 The Global Regulator Protein AmtR......Page 359
14.5.2 Influence of Two-Component Signal Transduction Systems......Page 361
14.7 Open Questions......Page 362
References......Page 363
15.1 Introduction......Page 368
15.2 Assimilation of Sulfur......Page 369
15.3 Transport of Sulfate......Page 374
15.4.1 Cysteine......Page 375
15.4.2 Methionine......Page 377
15.5.2 Regulation of Methionine Biosynthesis......Page 383
15.6 Constructing Methionine-Producing Strains......Page 386
15.7 Conclusions and Perspectives......Page 387
References......Page 388
16.1 Introduction......Page 394
16.2.1 Phosphorus Uptake......Page 395
16.2.2 Extracytoplasmic Phosphorus Mobilization......Page 401
16.2.3 Alternative Phosphorus Sources......Page 402
16.2.4 Phosphorus Assimilation and Polyphosphate Metabolism......Page 403
16.3 The Phosphate Starvation Response......Page 405
16.5 Comparison of Phosphorus Metabolism and Its Regulation in C. glutamicum, E. coli, B. subtilis, and M. tuberculosis......Page 409
References......Page 411
17.1 Carotenoids in Corynebacteria......Page 414
17.1.1 Carotenoid Synthesis in C. glutamicum......Page 416
17.1.2 Carotenogenic Genes of Corynebacteria......Page 419
17.2.1 Synthesis of Biotin......Page 422
17.2.3 The bio Loci in Corynebacterium......Page 424
17.3.1 Pantothenate Synthesis in C. glutamicum......Page 425
Acknowledgments......Page 428
References......Page 429
18.1 The Impact of Osmotic Stress on Bacterial Physiology......Page 434
18.2 Response of C. glutamicum to Hypoosmotic Stress......Page 435
18.3.1 Potassium and Glutamate Response......Page 437
18.3.3 Accumulation of Compatible Solutes under N-Limitation......Page 438
18.3.4 Accumulation of Compatible Solutes from the Environment......Page 440
18.4.1 Regulation of Proline Biosynthesis......Page 441
18.4.2 Regulation of Trehalose Biosynthesis......Page 442
18.5.1 The Betaine Uptake System BetP......Page 443
18.5.4 The Betaine/Ectoine Permease LcoP......Page 447
18.6 Relevance of Osmostress for Fermentation Processes......Page 448
References......Page 449
Part VI: Synthesis and Production......Page 454
19.1 Introduction......Page 456
19.2.1 Carbon Source......Page 457
19.2.2 Fermentation Process......Page 458
19.2.3 Crystallization......Page 459
19.2.4 Waste Reduction......Page 460
19.3 Induction of l-Glutamate Overproduction......Page 461
19.4.1 Reactions Leading to 2-Oxoglutarate......Page 462
19.4.2 Ammonia Incorporation......Page 464
19.4.4 Cloning and Analysis of the odhA Gene......Page 465
19.4.5 Cloning of dtsR Genes......Page 468
19.4.6 Characterization of dtsR Mutants......Page 469
19.4.7 Regulation of dtsR1 Expression......Page 470
19.5 Next Generation Glutamate Producer: Corynebacterium efficiens......Page 471
19.5.1 Comparative Analysis of C. efficiens......Page 473
References......Page 474
20.1 Introduction......Page 482
20.2.1 Conventional Production Strains Generated by Random Mutation and Selection......Page 484
20.2.2 Strains with Defined Improvements in Biosynthetic Pathways......Page 487
20.2.3 Strains with Altered Regulatory Networks......Page 489
20.3 Manufacturing Process......Page 490
20.3.1 Media Components and Sterile Media Preparation......Page 491
20.3.3 Production Step Cultivation......Page 493
20.4 Downstream Processing......Page 497
20.5 Scale-up of l-Lysine Production with C. glutamicum......Page 498
References......Page 499
21.1 Introduction......Page 506
21.2.1 Common Aromatic Pathway......Page 507
21.2.2 Tryptophan-Specific Pathway......Page 511
21.2.3.1 The aro Genes......Page 512
21.3 Aromatic Amino Acid Transport......Page 513
21.4.1 Fermentation Operations......Page 514
21.5 Recent Progress in Strain Development......Page 515
21.5.1.1 C. glutamicum Strains......Page 516
21.5.1.2 Escherichia and Bacillus Strains......Page 517
21.5.2.1 C. glutamicum Strains......Page 518
21.5.3 Transport Engineering......Page 519
21.6 Conclusions and Perspectives......Page 520
References......Page 521
22.1 Introduction......Page 528
22.2 Threonine Synthesis......Page 529
22.3 Threonine Overproduction......Page 532
22.4 Isoleucine and Valine Synthesis......Page 534
22.5 Isoleucine Overproduction......Page 537
22.6 Valine Overproduction......Page 539
22.7 Leucine Synthesis......Page 540
22.8 Leucine Overproduction......Page 542
References......Page 543
Part VII: Experiments......Page 550
23.1 Introduction......Page 552
Step 1 (Day 1): Preculture and Medium Preparation......Page 553
Step 3 (Day 3): Glutamate Accumulation......Page 554
Note on growth......Page 555
Step 2 (Day 2): Inoculation and Ethambutol Treatment......Page 556
Step 2 (Day 2): Precultivation......Page 557
Step 4: Electroporation and Plating......Page 558
Step 2 (Day 2): Cultivation of Donor and Recipient......Page 559
Step 4 (Day 4): Selection for Conjugants......Page 560
23.6 Plasmid Vectors for Corynebacterium glutamicum......Page 561
23.7 Chromosomal Integration......Page 568
23.8 Deletion of Chromosomal Sequences and Allelic Exchange......Page 574
Step 2: Selection for Vector Integration......Page 575
23.9 Transposon Mutagenesis......Page 576
Step 3: Competent Cells of C. glutamicum ATCC14752......Page 577
Step 4: Transposon Mutagenesis......Page 578
References......Page 579
A......Page 584
B......Page 587
C......Page 589
D......Page 596
E......Page 598
F......Page 600
G......Page 601
H......Page 605
I......Page 606
L......Page 608
M......Page 610
N......Page 614
O......Page 616
P......Page 617
R......Page 624
S......Page 625
T......Page 628
W......Page 632
Z......Page 633