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Umfang: 1 online resource (428 pages) : , illustrations (some color).

ISBN: 9780128046166 , 0128046163 , 9780128045848 , 0128045841

Serie: Methods in Enzymology, Volume 575

Anmerkung: Front Cover -- Synthetic Biology and Metabolic Engineering in Plants and Microbes Part A: Metabolism in Microbes -- Copyright -- Contents -- Contributors -- Preface -- Chapter One: Directing Biosynthesis: Practical Supply of Natural and Unnatural Cyanobactins -- 1. Introduction -- 2. Discovery of Cyanobactin Pathways -- 3. Elucidating Natural Rules of Engineering in Cyanobactin Pathways -- 4. Heterologous Expression of Cyanobactin Pathways in E. coli -- 5. Optimization for Increased Yield of Cyanobactins in E. coli -- 6. Synthesis of Cyanobactins In Vitro -- 7. Conclusions -- 8. Outlook -- Acknowledgments -- References -- Chapter Two: Synthetic Biology Approaches to New Bisindoles -- 1. Introduction -- 2. Identification of New Bisindole Gene Clusters -- 3. Heterologous Expression -- 3.1. Host Strains Commonly Used -- 3.2. Introduction of the Bisindole Gene Cluster into a Chassis Host -- 4. Mutational Biosynthesis to Generate New Bisindoles -- 5. Mixing Genes from Phylogenetically Related Clusters to Generate New Bisindoles -- 6. Chemical Isolation and Structural Characterization -- 6.1. Strain Growth and Product Extraction -- 6.2. Analysis and Purification -- 6.3. Metabolite Characterization -- 7. Conclusions -- References -- Chapter Three: Enzymatic [4+2] Cycloadditions in the Biosynthesis of Spirotetramates and Spirotetronates -- 1. Introduction -- 2. Strategy -- 2.1. Prediction of the Presence of [4+2] Cycloaddition Reactions in the PYR Biosynthetic Pathway -- 2.2. Identification of the Candidates Coding for Cascade [4+2] Cycloadditions from the PYR Biosynthetic Gene Cluster -- 3. Methods -- 3.1. In Vivo Validation of the Involvement of pyrE3 and pyrI4 in PYR Biosynthesis -- 3.1.1. Inactivation of pyrE3 or pyrI4 and Associated Homologous Complementation in S. rugosporus. , 3.1.2. Characterization of the Intermediate Isolated from the ΔpyrE3 S. rugosporus Mutant Strain -- 3.2. In Vitro Determination of the Functions of PyrE3 and PyrI4 for Pentacyclic Core Formation -- 3.2.1. Expression and Purification of PyrE3 and PyrI4 from E. coli -- 3.2.2. Assay of the Activities of PyrE3, PyrI4, and Their Combination Along with Characterization of the Products -- 3.3. In Vivo and In Vitro Mechanistic Evaluation of the Generality of Pentacyclic Core Formation in the CHL Biosynthetic ... -- 3.3.1. Inactivation of chlE3 or chlL in S. antibioticus -- 3.3.2. Exchanges Between pyrE3 and chlE3 as well as pyrI4 and chlL by Heterologous Complementation -- 3.3.3. Expression and Purification of ChlE3 and ChlL from E. coli -- 3.3.4. Assay of the Activities of ChlE3, ChlL, and Their Combination -- 3.4. Examination of the Protein Natures of PyrE3 and ChlE3 -- 4. Discussion and Perspectives -- References -- Chapter Four: Application and Modification of Flavin-Dependent Halogenases -- 1. Introduction -- 2. Inactivation of Halogenases Under Reaction Conditions -- 2.1. Improvement of Halogenase Stability by Error-Prone PCR -- 2.2. Stabilization of Halogenases by Formation of Cross-Linked Enzyme Aggregates (CLEAs) -- 2.2.1. Preparation of CLEAs -- 2.2.2. Halogenation Using CLEAs -- 2.2.3. Gram-Scale Halogenation Reaction Using CLEAs -- 3. The Biocatalytic Scope of Flavin-Dependent Tryptophan Halogenases -- 3.1. Substrate Specificity of Tryptophan Halogenases -- 3.1.1. A High-Throughput Assay for the Detection of Halogenase Activity -- 3.2. Modification of Biosynthetic Pathways Using Tryptophan Halogenases -- 3.2.1. Introduction of thal/thdH into P. chlororaphis and Isolation of 3-(2'-Amino-4'-Chlorophenyl)Pyrrole -- 3.2.2. In Vivo Modification of Biosynthetic Pathways Using Halogenase Genes. , 3.3. Chemical Substitution of Enzymatically Introduced Halogen Atoms -- 3.4. Modification of the Regioselectivity of Tryptophan Halogenases -- 4. Synthesis of PCP-Bound Halogenase Substrates -- 4.1. Enzymatic Synthesis of Pyrrolyl-S-PCPs -- 4.2. Chemoenzymatic Synthesis of Pyrrolyl-S-PCPs -- 4.2.1. Synthesis of Pyrrolyl-S-CoA via Acid Chloride -- 4.2.2. Synthesis of Pyrrolyl-S-CoA via S-Phenyl Thioates -- 4.3. Transfer of Pyrrolyl-S-CoA Thioesters to Carrier Proteins and Halogenation of the Substrate -- 4.4. Release of Halogenated Pyrrole-2-Carboxylic Acid from PCPs -- 5. Conclusions -- Acknowledgments -- References -- Chapter Five: Engineering Flavin-Dependent Halogenases -- 1. Introduction -- 2. Improving the Stability of FDHs -- 2.1. Improving FDH Stability via Directed Evolution -- 2.1.1. Procedure for Evolving FDH Thermostability -- 2.1.2. Procedure for Evolving FDH Organic Solvent Tolerance -- 2.2. Alternative Screening Methods for FDH Evolution -- 2.3. Improving Enzyme Stability Through Immobilization -- 3. Altering the Regioselectivity of FDHs -- 3.1. Altered Regioselectivity via Iterative Mutagenesis and Screening -- 3.1.1. Procedure for Evolving FDH Regioselectivity via Random Mutations -- 3.1.2. Procedure for Altering FDH Regioselectivity via Targeted Mutations -- 4. Expanding the Substrate Scope of FDHs -- 4.1. Expanding FDH Substrate Scope via Targeted Mutations -- 4.2. Expanding FDH Substrate Scope via Random Mutations -- 4.2.1. Procedure for Evolving FDH Substrate Scope via Random Mutations -- 5. Conclusions -- References -- Chapter Six: Heterologous Expression of Fungal Secondary Metabolite Pathways in the Aspergillus nidulans Host System -- 1. Introduction -- 2. Identification of Secondary Metabolite Genes in Fungal Genomes -- 3. Design Primers for Fusion PCR -- 4. Obtain Genomic DNA for PCR Template -- 4.1. Materials. , 4.2. DNA Extraction from Hyphae -- 4.3. Materials -- 4.4. DNA Extraction from Spores -- 5. Fusion PCR Construction -- 5.1. Materials -- 5.2. Genomic PCR -- 5.3. One Pot Fusion Reaction -- 5.4. Two Pot Fusion Reaction -- 6. Transformation -- 6.1. Materials -- 6.2. Protoplasting -- 7. Diagnostic PCR -- 8. Liquid Culturing of Mutant Strains -- 8.1. Materials -- 8.2. Culturing Strains in Glucose Minimal Media -- 9. Recipes -- 10. Conclusions -- Acknowledgments -- References -- Chapter Seven: Plug-and-Play Benzylisoquinoline Alkaloid Biosynthetic Gene Discovery in Engineered Yeast -- 1. Introduction -- 1.1. Benzylisoquinoline Alkaloid Metabolic Biochemistry -- 1.1.1. BIA Biosynthesis in Plants -- 1.1.2. Localization of BIA Biosynthetic Pathways in Plants -- 1.2. Functional Genomics in BIA-Producing Plants -- 1.3. Engineering BIA Pathways in Yeast -- 2. Transcriptome Resources and Mining Candidate Genes -- 2.1. Selection of Plants and Tissues -- 2.2. RNA Extraction and DNA Sequencing -- 2.3. Selection of Candidate Genes -- 3. Building Yeast Platform Strains -- 3.1. Overview of USER Cloning -- 3.2. Preparing USER-Compatible Vectors -- 3.3. Simultaneous Cloning of Multiple PCR Fragments -- 3.4. Yeast Transformation -- 3.5. Recombinant Protein Analysis -- 3.6. Ura Marker excision -- 4. Functional Testing of Candidate Genes -- 4.1. Transient Expression Constructs -- 4.2. Yeast Transformation, Culture, and Substrate Feeding -- 5. Liquid Chromatography-Tandem Mass Spectrometry -- 5.1. Analytical Strategy Overview -- 5.2. List of Instrumentation and Software -- 5.3. List of Consumables, Solvents, and Reagents -- 5.4. Analytical Methods -- 5.5. Technical Notes -- 6. Summary and Future Prospects -- Acknowledgments -- References -- Chapter Eight: Optimizing Metabolic Pathways for the Improved Production of Natural Products -- 1. Introduction. , 2. Genetic Optimization -- 2.1. Gene Homolog Sourcing -- 2.2. Selection of Expression Plasmid Backbone(s) -- 2.3. DNA Copy Number Balancing -- 2.4. Transcriptional Balancing -- 2.5. Translational Balancing -- 2.6. Posttranslational Balancing -- 2.7. Dynamic Balancing -- 2.8. Coculture Optimization -- 3. Fermentation Optimization -- 3.1. Media Optimization -- 3.2. Temperature Optimization -- 3.3. Induction Optimization -- 3.3.1. Induction Point -- 3.3.2. Inducer Concentration -- 3.3.3. Substrate Delay -- 4. Conclusion -- Acknowledgments -- References -- Chapter Nine: Reconstituting Plant Secondary Metabolism in Saccharomyces cerevisiae for Production of High-Value Benzylis ... -- 1. Introduction -- 2. BIA Biosynthetic Gene Sources and Selection of Candidate Genes -- 3. Yeast Functional Expression Strategies -- 3.1. Genetic Considerations -- 3.2. Enzyme Expression and Localization -- 3.2.1. Flow Cytometry -- 3.2.1.1. Protocol: Quantitative Determination of Mean Culture Fluorescence Using GFP-Tagged Proteins and Flow Cytometry -- 3.2.1.2. Protocol: Rapid Identification of Fluorescent Colonies Expressing GFP-Tagged Proteins Using Flow Cytometry -- 3.2.2. Fluorescence Microscopy -- 3.2.2.1. Protocol: Analysis of BIA Enzyme Expression and Localization in Yeast Using Fluorescence Microscopy -- 3.2.3. Immunoblotting -- 3.3. Enzyme Activity -- 3.3.1. In Vitro Enzyme Assays (Purified Proteins, Microsomal Preparations, or Crude Cell Lysates) -- 3.3.1.1. Protocol: Preparation of Yeast Crude Cell Lysates, Microsomal Preparations, and Purified Proteins -- 3.3.1.2. Protocol: In Vitro Assay of BIA Biosynthetic Enzymes -- 3.3.2. In Vivo Cell-Feeding Assays -- 3.3.2.1. Protocol: In Vivo Cell-Feeding Assays -- 3.3.3. LC-MS Analysis of In Vitro and In Vivo Assay Products. , 3.3.3.1. Protocol: Conditions for Separation of BIA Assay Intermediates and Products Using Liquid Chromatography.

Sprache: Englisch

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Umfang: 74 S , Ill

Serie: Spomenica preminulim akademicima. Jugoslavenska Akademija znanosti i umjetnosti 6

Sprache: Unbestimmte Sprache

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Umfang: 1 Online-Ressource (xvi, 379 Seiten, [30 Tafeln])

Ausgabe: First edition

ISBN: 9780128046166

Serie: Methods in Enzymology volume 575

Inhalt: Synthetic Biology and Metabolic Engineering in Plants and Microbes: Part A, the new volume in the Methods in Enzymologyseries,continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers research methods, synthetic biology, and metabolic engineering in plants and microbes, and includes sections on such topics as the uses of integrases in microbial engineering, biosynthesis, and engineering of tryptophan derived metabolites, regulation and discovery of fungal natural products, and elucidation and localization of plant pathways.Continues the legacy of this premier serial with quality chapters authored by leaders in the field Contains two volumes covering research methods in synthetic biology and metabolic engineering in plants and microbesPresents sections on such topics as the uses of integrases in microbial engineering, biosynthesis, and engineering of tryptophan derived metabolites, regulation and discovery of fungal natural products, and elucidation and localization of plant pathways

Anmerkung: Description based upon print version of record

Weitere Ausg.: ISBN 9780128045848

Weitere Ausg.: Erscheint auch als Druck-Ausgabe O'Connor, Sarah E Synthetic Biology and Metabolic Engineering in Plants and Microbes Part A: Metabolism in Microbes Saint Louis : Elsevier Science,c2016 ISBN 9780128045848

Sprache: Englisch

Schlagwort(e): Synthetische Biologie ; Mikroorganismus ; Pflanzen ; Stoffwechsel ; Electronic books

URL: Volltext