Novel Frontiers in the Production of Compounds for Biomedical Use  Volume 1

Novel Frontiers in the Production of Compounds for Biomedical Use Volume 1

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This work presents a selection of approaches for the production of novel biotechnological compounds for biomedical applications. In order to better focus on the challenges at hand, the editors have striven to adopt an integrative approach by selecting contributions linking genomics with bacterial resistance, antibiotics with improved production strategies, optimised production approaches with chemical development programs, chemical modifications with new antibody derivatives and biomaterials, the relation between bioartificial organs and xenotransplantation, apoptosis and process biotechnology, etc. The different chapters provide an overview of the state- of-the-art in the development of truly novel compounds for biomedical application using recombinant DNA approaches. This book should be of interest to academic and industrial scientists dealing with biomedical sciences and for teachers who want to be informed about developments in the field.

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Published 01 January 2001
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TABLEOFCONTENTS
EDITORS PREFACE............................................................................................... v
PART 1 – GENOMICS: THE NEW APPROACH TO THE DISCOVERY OF NEW COMPOUNDS........................................................ 13 The genomics approach: is it really the solution?...................................................... 15 Raul Goldschmidt and Karen Bush Summary................................................................................................................15 1.The Traditional Approach..................................................................................15 2. The Biochemical Genetics Approach................................................................ 16 3. The Genomics Approach................................................................................... 17 3.1 Target identification.....................................................................................17 3.2 Comparative genomics...................................................................................17 4. Perspectives for an Integrated Approach........................................................... 20 References............................................................................................................. 21 The Contribution of Genomics to the Discovery of new Antibiotics..................... 23 David J. Holmes, John P. Throup, Nicola G. Wallis, Martin K. R. Burnham, Magdalena Zalacain, Sanjoy Biswas, Alison F. Chalker, Karen A. Ingraham, Andrea Marra, Alex Bryant, Gary Woodnutt, Patrick V. Warren, James R Brown, Martin Rosenberg Abstract .................................................................................................................... 23 1. Introduction ........................................................................................................ 24 3. The Properties of an Antibacterial Target........................................................... 24 3.1 Novelty .......................................................................................................... 24 3.2 Spectrum/Selectivity................................................................................. 25  3.3 Expression during infection.................................................................. 25 3.4 Essential for cell viability.....................................................................26 3.5 Amenable to high-throughput screening ....................................................... 26 4. Aminoacyl tRNA synthetases.........................................................................27 5. Two Component Signal Transduction Systems..................................................28 6. Discussion.........................................................................................................29 References..............................................................................................................29 PART 2 – ANTIBIOTICS....................................................................................33 The antibiotic gallidermin-evolution of a production process .............................. 35 Markus Kempf, Uwe Theobald and Hans-Peter Fiedler 1. The antibiotic gallidermin........................................................................................35 2. The improvement of the production process..................................................... 36 3. Stabilisation of product-formation................................................................... 36 4. Stability test ......................................................................................................... 37 5. Hs-value.............................................................................................................. 37
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6. Economic improvement of the production process .......................................... 41 6.1 Nutrient sources......................................................................................... 42 6.2 Development of scale-up procedure ........................................................... 45 6.2.1 Development of a fed-batch process.................................................... 45 6.2.2 Investigation of batch processes.......................................................... 49 6.2.3 Small scale fermentations................................................................... 50 6.2.4 Variation of parameters in shake flask experiments............................ 50 6.2.5 Pilot scale fermentations ....................................................................... 51 6.2.6 An optimised scale-gallidermin .52up procedure for the production of References .............................................................................................................. 54 Resistance toβ-lactams, a self-regenerating problem................................................57 Jozsef Aszodi* and Andre Bryskier 1. Resistance to Beta-Lactams........................................................................ .... 57 2. Mode of Action ofβ-Lactams........................................................................... 57 3. Beta Lactamases............................................................................................... 59 3.1. Class Aβ-Lactamases............................................................................... 60 3.2. Class Cβ-Lactamases............................................................................... 61 3.3. Class Dβ-Lactamases.............................................................................. 61 3.4. Class Bβ-Lactamases.............................................................................. 62 4. PBPs................................................................................................................. 62 4.1. Naturally Resistant Pathogens.................................................................... 63 4.2. Intrinsic Resistance Through Acquisition of Resistant PBPs..................... 63 4.3. Mosaic Genes ............................................................................................ 65 4.4. Point Mutations......................................................................................... 66 5. Penetration Barrier ............................................................................................. 66 6. Antibiotic Efflux ............................................................................................... 68 7. The future ofβ-Lactams.................................................................................... 69 7.1. New Families ............................................................................................. 70 7.2. New Generations...................................................................................... 70 7.3. Potentiators ofβ-Lactams....................................................................... 72 8. Conclusions....................................................................................................... 74 References ............................................................................................................... 75 Resistance to aminoglycoside antibiotics: Function meets structure................. 85 Gerard D. Wright and Albert M. Berghuis Abstract................................................................................................................. 85 1. Introduction....................................................................................................... 85 2. Aminoglycoside modifying enzymes .................................................................. 86 2.1. ANT............................................................................................................. 87 2.2. AAC.......................................................................................................... 91 2.3. APH........................................................................................................... 93 3. Conclusion........................................................................................................ 96 Acknowledgements............................................................................................... 96 References............................................................................................................. 97 The genetics and biochemistry of resistance to glycopeptide antibiotics ...........99 P. E. Reynolds
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Summary............................................................................................................ 99 1. Action of Glycopeptides: Vancomycin and Teicoplanin.............................. 99 2. Potential Mechanisms of Glycopeptide Resistance....................................... 100 3. Glycopeptide Resistance in Enterococci ....................................................... 101 3.1. The VanA phenotype.............................................................................. 101 3.1.1. Peptidoglycan Synthesis: a New Pathway........................................ 102 3.1.2. Peptidoglycan Synthesis: Control of Normal Host Pathway............ 102 3.2. Similarity and diversity of the resistance operons of VanA-, VanB- and VanD-type enterococci .................................................................................. 103 3.2.1. VanB-type resistance ..................................................................... 105 3.2.2. VanD - type ..................................................................................... 105 3.3. VanC-type resistance ........................................................................... 108 3.3.1. VanE-type Resistance.......................................................................109 4. Glycopeptide Resistance in Staphylococci.................................................... 109 5. The Future ................................................................................................... 110 Acknowledgement............................................................................................... 112 References................................................................................................................ 112 β117problem ........................................... old but ever renascent -Lactamases, an André Matagne, Moreno Galleni, Nezha Laraki, Gianfrance Amicosante, Gianmaria Rossolini and Jean-Marie Frère Abstract............................................................................................................... 117 1. Introduction....................................................................................................... 118 2. The target of penicillin and otherβ-Lactams........................................... 118 3. Resistance mechanisms................................................................................. 119 4.β-Lactamases.................................................................................................. 121 5. Carbapenems and carbapenem-hydrolysingβ-lactamases............................ 123 6. Hydrolysis of third- generation cephalosporins : the TEM and SHV variants124 7. Inhibitor- resistant enzymes............................................................................1258. Overproduction by deregulation of the induction system..............................126 9. Conclusion.......................................................................................................127 Acknowledgements..............................................................................................127 References............................................................................................................128 Metabolic flux analysis in streptomyces coelicolor: ..............................................131 Fereshteh Naeimpoor and Ferda Mavituna Abstract..................................................................................................................131 1. Introduction.....................................................................................................131 1.1 Streptomycetes.......................................................................................... 131 1.2 Streptomyces coelicolor ........................................................................133 1.3 Polyketides, peptide antibiotics and streptomycetes................................134 1.4 Metabolic engineering .............................................................................135 2. Metabolic Flux Analysis inS. coelicolor....................................................... . 135 2.1 Metabolite and Product Excretions........................................................ 136 3. Results and Discussion ............................................................................136 3.1 Model description...................................................................................136 3.2 Effect of Different Nitrogen Sources on Biomass Yield......................... 137
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3.3 Metabolite excretion with different nitrogen sources ............................... 138 4.Conclusion...................................................................................................140 Acknowledgements............................................................................................ 142 Nomenclature......................................................................................................143 References...........................................................................................................143 Metabolic engineering of the lysine pathway forβ-lactam overproduction in Penicillium chrysogenum........................................................................................ 147 Casqueiro, J., Bañuelos, O., Gutiérrez, S. and Martin, J.F 1. Lysine biosynthesis: synthesis ofα-aminoadipic acid a precursor ofβ-lactam antibiotics ............................................................................................................... 147 2. Relationships between lysine and penicillin biosynthesis .............................. 149 3. Metabolic engineering of the lysine pathway inP. chrysogenum................... 151 3.1 .-Metabolic engineering at theα-aminoadipate reductase level: Channelling of lysine metabolic flux towards penicillin biosynthesis. ........... 151 3.2.-Metabolic engineering at the homocitrate synthase level ..................... 152 4.-Futureperspectives......................................................................................... 156 Acknowledgements................................................................................................ 156 References ............................................................................................................ 156 Glycosylation of antibiotics and other agents from Actinomycetes ...................... 161 Wolfgang Piepersberg Summary ................................................................................................................ 161 1. Introduction..................................................................................................... 161 2. Sugars and Cyclitols as Building Blocks in Actinomycete Secondary Metabolites: Pathways for Modified Sugars and Cyclitols. ............................... 162 2.2. 6-Deoxyhexoses (6DOH) in Glycosylation of Antibiotics and Other Bioactive Secondary Metabolites.................................................................... 162 2.3. Cyclitols. ..................................................................................................... 164 2.4. Glycosyltransferases............................................................................... 164 3. Examples......................................................................................................... 165 3.1 The Streptomycin Pathway.....................................................................165 3.2 Macrolide Sugars..................................................................................... 165 3.3 Lincosamine. ...........................................................................................166 4. Conclusion .........................................................................................................166 References .......................................................................................................... 167 Enzymatic synthesis of amoxicillin....................................................................... 169 A.C. Spiess and V. Kasche 1. Introduction......................................................................................................... 169 2. Theory ............................................................................................................. 170 2.1. Solid phase and dissolution..................................................................... 170 2.2. Suspension to suspension conversion...................................................... 172 2.3. Limiting regimes.................................................................................... 173 3. Materials and Methods................................................................................... 174 3.1Enzymes and reagent . ...............................................................................174 3.2 Enzyme activity assay............................................................................. 174 3.3 HPLC analysis.......................................................................................... 175
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3.3 Solubility measurements.......................................................................... 175 3.4 Determination of kcatand Km............................................................. 175 3.5 Synthesis of amoxicillin in homogeneous reaction ............................ 175 3.6 Synthesis of amoxicillin in heterogeneous reaction. ................................ 176 3.7 Suspension pictures................................................................................. 176 3.8 Conjugation with fluorescent dyes. ........................................................... 176 3.9 CLSM (Confocal laser scanning microscopy) image acquisition. ......... 176 3.10 Reactor cycle:....................................................................................... 177 3.11 Image processing: ................................................................................... 177 3.12 pH calibration and pH measurement using CLSM............................ 177 3.13 Bipolar membrane module. Construction and operation....................... 177 3.14 Module characterisation........................................................................ 177 4. Results and Discussion .................................................................................. 178 4.1. Solubility and rate of dissolution ............................................................ 178 4.2. Kinetic parameters of amoxicillin synthesis and hydrolysis .................... 179 4.3. Evaluation of amoxicillin synthesis progress: pH, T,I-dependence ....... 180 4.4. Effect of concentration variation and pure substrate phase ......... 183 4.5. Comparison of soluble and immobilised enzyme ................................... 184 4.6. Activity and selectivity of immobilised enzymes................................... 185 4.7. pH profiles in immobilised biocatalysts under reaction........................ 186 4.8. Mass transfer limit and yield prediction ................................................. 187 4.9. Proposal for integrated reaction separation process............................. 187 5. Conclusion and prospects............................................................................. 189 References.......................................................................................................... 190 -PART 3 PRODUCTION OF THERAPEUTIC ANTIBODIES...... 193 New Recombinant bi-and trispecific antibody derivatives. .................................... 195 Nico Mertens, Reinilde Schoonjans, An Willems, Steve Schoonooghe, Jannick Leoen and Johan Grooten Summary ............................................................................................................ 195 1.Introduction..................................................................................................... 196 2. Material and Methods...................................................................................... 199 2.1 Cell lines.................................................................................................. 199 2.2 Plasmids and gene assembly.................................................................... 199 2.3 Production and purification of recombinant antibody fragments ............ 200 2.4 T-cell proliferation assay .......................................................................... 200 3. Results and discussion................................................................................... 200 3.1Heterodimerization by CL-CH1 interaction in eukaryotic cells depends on extension with VI and VH domains............................................................... 200 4.2 Fab-scFv fusion molecule as a model system for intermediate sized BsAb production...................................................................................................... 202 4.3 Fd:L mediated heterodimeration of two scFv molecules leads to efficient expression of trispecific molecules.............................................................. 203 4.4 Influence of linker length and composition on production and heterodimerization......................................................................................... 204 4. Discussion ...................................................................................................... 205
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References ......................................................................................................... 206 Advantages of single-domain antigen-binding fragments derived from functional camel heavy-chain antibodies. ............................................................................... 209 Muyldermans Serge, Conrath Katja, Vu Khoa Bang, Serrao Teresa, Busch Magnus, Backmann Natasha, Silence Karen, Lauwereys Marc, Desmyter Aline 1. Introduction .................................................................................................... 209 2. Functional heavy-chain antibodies in sera of camelids ............................. 210 3. Single antigen-binding domain of camel heavy-210chain antibodies................ 4. Cloning and selecting the camel variable domains of heavy chain antibodies ............................................................................................................................ 211 5. Characteristicsm of the single domain antibody fragments....................... 212 5.1. Expression yield..................................................................................... 212 5.2. Solubility ................................................................................................. 212 5.3. Stability................................................................................................. 212 5.4. Specificity and affinity ........................................................................... 213 5.5. Enzyme inhibition................................................................................... 213 5.6. Multivalent constructs............................................................................ 214 5.7. Intrabodies.............................................................................................. 214 Acknowledgements............................................................................................ 214 References........................................................................................................... 214 -PART 4 HETEROLOGOUS PROTEIN PRODUCTION: NEW PRODUCTION STRATEGIES.........................................................217 Furin as a tool for the endoproteolytic maturation of susceptible recombinant biopharmaceuticals.. ............................................................................................. 219 M. Himmelspach, B. Plaimauer, F. Dorner and U. Schlokat 1. Introduction................................................................................................... 219 2. Sorting and processing of secretory proteins................................................ 220 2.1. The constitutive and regulated secretory pathways........................... 220 2.2. Endoproteolytic processing of precursor proteins................................. 222 3. The pro-protein convertases......................................................................... 222 3.1. Identification of eukaryotic pro-protein convertases. ............................. 222 3.2. Tissue distribution, sublocalisation and function .................................... 223 4. The endoprotease furin .................................................................................. 224 4.1. Structural organisation .......................................................................... 224 4.2. Subcellular localisation and trafficking............................................... 226 4.3. Substrate specificity................................................................................ 227 5. Improved biotechnological processes by the use of furin...................... 231 5.1. Development of recombinant coagulation factors.............................. 231 5.2. VonW232illebrand factor propeptide removal by full length furin ............. 5.3. Production of recombinant factor IX using a truncated soluble furin derivative........................................................................................................ 236 5.4. Processing of recombinant factor X precursors using furin derivatives in vitro ............................................................................................................... 236 5.5. Use of furin in transgenic animals....................................................... 238
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6. Perspectives .................................................................................................... 239 Acknowledgements............................................................................................ 241 References .............................................................................................................. 241 Development of bioprocesses for the generation of anti-inflammatory, anti-viral and anti-leukaemic agents...................................................................................... 249 Mahmoud Mahmoudian Abstract ..................................................................................................................... 249 1.Introduction...................................................................................................... 250 2. Process development for the generation of nucleoside 5'-carboxylic acids ... 250 TM 3. Abacavir (Ziagen )........................................................................................ 2554. Production of the anti-leukaemic agent 506U78............................................ 258 Acknowledgements ............................................................................................... 264 References............................................................................................................ 264 Apoptosis and bioprocess technology..................................................................... 267 R.P. Singh and M. Al-Rubeai 1. Introduction .......................................................................................................... 267 2. Apoptosis: basic features of cell death.......................................................... 268 Apoptosis and the mitochondria .................................................................... 269 3. Apoptosis and its control during industrial scale cell culture processes ........ 271 4. Conclusion...................................................................................................... 273 References .................................................................................................................... 273 Gram-positive Bacteria as host cells for Heterologous of production biopharmaceuticals ................................................................................................. 277 Lieve Van Mellaert and Jozef Anné Abstract .............................................................................................................. 277 1. Introduction ......................................................................................................... 278 2. The general secretion pathway.................................................................... 279 2.1. Early stage ................................................................................................... 279 2.2. Middle stage............................................................................................ 281 2.3. Late stage ................................................................................................... 281 2.4. Intrinsic features of secretory proteins.................................................. 283 3. Improvement of secretion............................................................................... 284 3.1. early stage secretion improvement.......................................................... 284 3.2. middle stage secretion Improvement .................................................... 285 3.3. late stage secretion Improvement ......................................................... 285 3.3.1. SPase activity.................................................................................... 286 3.3.2. Proteolytic breakdown...................................................................... 286 3.3.3. Protein folding.................................................................................. 287 4. Examples of Gram-positive bacteria as host cells for the production of heterologous proteins......................................................................................... 287 4.1. High-of biopharmaceutical compounds ...................... level production 288 B.brevis.......................................................................................................... 289 4.2. Gram-positive bacteria as live vaccine delivery systems................. 291 4.2.1. Gram-291positive bacteria used as antigen delivery systems ............... 4.2.2.Approaches for antigen presenatation............................................... 293
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4.2.3. Heterologous antigens presented by Gram-294positive bacteria .......... Clostridium tetani........................................................................................... 295 4.3. Other application areas.............................................................................. 296 5. Conclusions and perspectives........................................................................ 296 Referenc..e..s............................................................................................................ 297 Multiple Pathways of Exoprotein Secretion in Gram-................. 301negative bacteria Anthony P Pugsley 1. Introduction.............................................................................................................. 301 2. The General Secretory Pathway...................................................................... 303 3. The Type I or ABC secretion pathway................................................................ 306 4. The type III or Contact Secretion Pathway...................................................... 306 5. Progress and technical problems ......................................................................... 308 6. A specific example: theKlebsiella oxytocapullulanase secreton...................308 Referenc..e..s.............................................................................................................309Alterations of Metabolic Flux Distributions in RecombinantEscherichia coliin Response to Heterologous Protein Production ........................................................ 313 Jan Weber and Ursula Rinas Summary............................................................................................................. 313 1. Introduction.................................................................................................... 313 2.Methodology.................................................................................................... 315 2.1 Metabolic Flux Analysis......................................................................... 315 2.2 Underdetermined Networks ............................................................................... 316 2.3 Why Linear Programming?........................................................................ 317 2.4 Properties of the Metabolic Network ....................................................... 318 2.5 Optimal amino acid drain for protein production..................................... 319 3.Applications...................................................................................................... 320 3.1 Production of hfgf-2 by temperature shift..................................................... 320 3.2 Temperature-...................................326induced production of human insulin 3.3 Effect of a stable and unstable recombinant protein on the host metabolism ......................................................................................................................... 328 4. Summary and Concluding Remarks............................................................ 329 Appendix............................................................................................................... 332 Bioreaction Network ofE. coli....................................................................... 332 Phosphotransferase System........................................................................ 332 Embden-Meyerhof-332Parnas Pathway............................................................ PEP Carboxykinase and PEP Carboxlyase ............................................... 332 By-products................................................................................................. 332 TCA Cycle ............................................................................................... 332 Glyoxylate shunt ........................................................................................ 332 Transhydrogenase....................................................................................... 332 Oxidative Phosphorylation......................................................................... 333 Pentose Phosphate Pathway........................................................................ 333 Methylglyoxal Pathway .............................................................................. 333 Ammonium, Glutamate and Glutamine ...................................................... 333
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Amino Acids............................................................................................. 333 Pretein........................................................................................................ 334 Nucleotides................................................................................................. 334 RNA .............................................................................................................. 334 DNA........................................................................................................... 334 Lipids ............................................................................................................. 334 Lipopolysaccharide.................................................................................... 334 Peptidoglucane ......................................................................................... 334 Glycogen ............................................................................................................ 334 One-.............................................................................. 334carbon unit and Polyamine Biomass...................................................................................................... 335 Miscellaneous .................................................................................................... 335 Fibroblast Growth Factor hFGF-2............................................................. 335 Objective .................................................................................................................. 335 References.......................................................................................................... 335 Dynamics of proteolysis and its influence on the accumulation of intracellular recombinant protein .............................................................................................. 339 Rozkov A., Yang S. and Enfors S.-O Summary .................................................................................................................... 339 1.Introduction..................................................................................................... 339 2.Materials and Methods..................................................................................... 341 2.1 Microorganism ....................................................................................... 341 2 .2 Media and Cultivation: ........................................................................... 341 2.3 Product concentrations ........................................................................... 342 2.4 Determination of the proteolysis rate constant .................................. 342 3. Results and Discussion .................................................................................. 342 4. Conclusions.................................................................................................... 346 References........................................................................................................... 346 PART 5 – ARTIFICIAL ORGANS AND XENOGRAFTING...........349 The impact of transgenesis and cloning on cell and organ xenotransplantation to humans ................................................................................................................... 351 Louis-Marie Houdebine , Bernard Weill Summary ............................................................................................................ 351 1. Why xenografting ? ...................................................................................... 352 3. The mechanisms of xenograft rejection........................................................... 354 4. The preventive treatments of xenograft rejection........................................... 355 5. The biosafety of xenografting......................................................................... 358 6. The acceptability of xenografting ....................................................................... 359 7.Conclusion and perspectives........................................................................ 361 References.............................................................................................................. 361 Reinforced Bioartificial Skin In The Form Of Collagen Sponge And Threads....... 365 Eun Kyung Yang, Young Kwon Seo and Jung Keug Park Abstract ...................................................................................................................... 365 1. Introduction ................................................................................................... 365 2. Materials and Methods.................................................................................. 367
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2.1 Collagen scaffolds ...................................................................................... 367 2.1.1 Extraction of Type I Collagen Solution ............................................... 367 2.1.2 Fabrication of Macroporous Collagen Sponge..................................... 367 2.1.3 Fabrication of Collagen Threads and Mesh ........................................ 368 2.1.4 Reinforcement by Cross-linking Treatments ......................................... 369 2.1.5 Reinforcement by Incorporating with Collagen Mesh........................ 370 2.1.6 Measurement of Mechanical Strength............................................... 370 2.1.7 Morphological Analysis...................................................................... 370 2.2 Bioartificial skin .............................................................................................. 371 2.2.1 Primary Cell Culture ............................................................................... 371 2.2.2 Culture of Bioartificial Skin............................................................... 371 2.2.3 Morphology Examination .................................................................. 372 3. Results and Discussion .......................................................................................... 372 3.1 Collagen scaffolds...................................................................................... 372 3.1.1 Preparation of Collagen Threads........................................................ 372 3.1.2 Ultimate Tensile Strength of Collagen Sponges.................................... 374 3.2Bioartificial skin ........................................................................................... 377 4. Conclusion and Future work............................................................................... 379 Acknowledgement................................................................................................. 379 References.............................................................................................................. 380 -PART 6 ANTITUMOUR COMPOUNDS................................................381 Towards the generation of novel antitumour agents from actinomycetes by combinatorial biosynthesis........................................................................................... 383 Jose A. Salas, Gloria Blanco, Alfredo F. Braña, Ernestina Fernandez, a M Jose Fernandez, Jose Garcia Bernardo, Ana Gonzalez, Felipe Lombo, Laura Prado, Luis M. Quiros, Cesar Sanchez and Carmen Mendez 1. Introduction ..................................................................................................... 383 2. Anticancer biosynthetic gene clusters.............................................................. 385 3. The aureolic acid group........................................................................................ 386 3.1. Genes involved in the biosynthesis of the polyketide moiety. ................... 387 3.2. Genes encoding enzymes modifying the polyketide skeleton.................... 388 3.3. Genes encoding an activated methyl cycle................................................ 388 3.4. Genes encoding sugar biosynthetic enzymes ............................................. 388 3.5. Genes encoding glycosyltransferases.......................................................... 389 3.6. Genes responsible for resistance and secretion................................... 389 4. Generation of novel compounds....................................................................... 390 4.1. Insertional inactivation.............................................................................. 390 4.2. Tailoring modification ............................................................................. 392 4.3. Combinatorial biosynthesis ............................................................................ 394 5. Concluding remarks ............................................................................................... 397 Acknowledgements .............................................................................................. 397 References .................................................................................................................. 397 Cell immobilisation ofTaxus media.............................................................................. 401 Chi Wai Tang and Ferda Mavituna Summary .................................................................................................................... 401
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