Synthetic Biology and Metabolic Engineering in Plants and Microbes Part A: Metabolism in Microbes

 
 
Academic Press
  • 1. Auflage
  • |
  • erschienen am 11. Juli 2016
  • |
  • 396 Seiten
 
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978-0-12-804616-6 (ISBN)
 

Synthetic Biology and Metabolic Engineering in Plants and Microbes: Part A, the new volume in the Methods in Enzymology series, 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 microbes
  • Presents 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
0076-6879
  • Englisch
  • San Diego
  • |
  • USA
Elsevier Science
  • 31,20 MB
978-0-12-804616-6 (9780128046166)
0128046163 (0128046163)
weitere Ausgaben werden ermittelt
  • 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 ?pyrE3 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
  • 3.3.3.2. Protocol: Conditions for Detection of BIA Assay Intermediates and Products Using Mass Spectrometry
  • 4. Pathway Reconstitution and Optimization
  • 4.1. Pathway Assembly
  • 4.2. Debottlenecking Strategies
  • 4.2.1. Genetic Tuning
  • 4.2.2. Directed Enzyme Evolution
  • 4.2.3. Bioprospecting of Enzyme Variants
  • 4.2.4. Cultivation Conditions
  • 5. Challenges and Future Directions
  • References
  • Chapter Ten: Engineering Microbes to Synthesize Plant Isoprenoids
  • 1. Introduction
  • 2. Improving Production of C20 ISMs by Modular Genetic Engineering of Escherichia coli
  • 2.1. Constructing Expression Vectors for IsoSs
  • 2.1.1. IsoS-Encoding Genes
  • 2.1.2. Primer Design
  • 2.1.3. PCR Reaction
  • 2.1.4. DNA Electrophoresis and Recovery
  • 2.1.5. Iodine Treatment and Ethanol Precipitation
  • 2.1.6. DNA Assembly
  • 2.1.7. Cell Transformation
  • 2.2. Testing the Expression Vectors
  • 2.2.1. Competent Cell Preparation and Cell Transformation
  • 2.2.2. Preparation of E. coli Seed Culture
  • 2.2.3. Preparation of 1000x K3 Trace Elements Stock Solution
  • 2.2.4. Preparation of K3 Basal Medium
  • 2.2.5. Preparation of K3 Master mix
  • 2.2.6. Testing C20 Isoprenoid Production in Test Tube
  • 2.2.7. Extraction of C20 Isoprenoids for GCMS Analysis
  • 2.2.8. GCMS Analysis
  • 3. Improving Production of C20 ISMs by Optimizing Bioreactor Operation
  • 3.1. Bioreactor Specifications
  • 3.2. Protocol for Operating the Bioreactor
  • 3.2.1. Preparation of the Bioreactor
  • 3.2.2. Initiating Bioreactor Run and Bioreactor Setting
  • 3.2.3. Feeding the Bioreactor
  • 3.2.4. Sampling Bioreactor and HPLC Analysis
  • 3.2.5. Purification of Isoprenoids
  • 4. Oxygenating C20 ISMs by Using E. coli-Saccharomyces cerevisiae Coculture
  • 4.1. Expression of a CYP in S. cerevisiae
  • 4.1.1. Constructing Yeast Expression Vector for CYP
  • 4.1.2. Yeast Transformation
  • 4.1.3. YNB CSM-URA Glucose Agar Plate
  • 4.1.4. YNB CSM-URA Glucose Medium
  • 4.2. Characterization of the S. cerevisiae
  • 4.2.1. Preparation of Isoprenoid-DMSO Stock Solution
  • 4.2.2. Preparation of Seed Culture
  • 4.2.3. Feeding Isoprenoid into Yeast Culture
  • 4.3. Coculture of the S. cerevisiae with an Isoprenoid-Overproducing E. coli
  • 4.3.1. Preparation of Yeast Preculture
  • 4.3.2. Bioreactor Run
  • 5. Discussion
  • References
  • Chapter Eleven: Natural Product Biosynthesis in Escherichia coli: Mentha Monoterpenoids
  • 1. Introduction
  • 2. Operon Construction
  • 2.1. Vector and Gene Selections
  • 2.2. Assembly of Mentha-Like Biosynthetic Pathways in E. coli
  • 2.2.1. General Assembly Protocols
  • 2.2.2. In-Fusion Reactions with Repeating Sequences
  • 3. Multigene Protein Expression and Validation
  • 4. Biotransformations
  • 4.1. General Monoterpenoid Reaction and Detection
  • 4.2. Alternative Protocols
  • 5. Further Studies
  • 5.1. Expression Constructs
  • 5.2. Strain and Expression Optimization
  • 5.3. High-Throughput Optimization Studies
  • 6. Conclusions
  • Acknowledgments
  • References
  • Chapter Twelve: High-Efficiency Genome Editing of Streptomyces Species by an Engineered CRISPR/Cas System
  • 1. Introduction
  • 2. Design of pCRISPomyces for Genome Editing in Streptomyces Species
  • 3. pCRISPomyces for Single Gene Disruption in S. lividans
  • 4. pCRISPomyces for Multiplex Gene Deletion in S. lividans
  • 5. Conjugation, Genotype Screening, and pCRISPomyces Clearance
  • 6. Evaluation of pCRISPomyces in Other Streptomyces Species
  • 7. Conclusion
  • Acknowledgment
  • References
  • Chapter Thirteen: Rapid Optimization of Engineered Metabolic Pathways with Serine Integrase Recombinational Assembly (SIRA)
  • 1. Introduction
  • 2. Mechanism of Serine Integrase Recombination
  • 3. How SIRA Works
  • 3.1. attPxattB Recombination
  • 3.2. attLxattR Recombination
  • 4. Applications of SIRA
  • 4.1. Inserting a Single Piece of DNA into a Plasmid
  • 4.2. Assembly of Multiple Genes in Predefined Orders for Compound Biosynthesis
  • 4.3. Combinatorial Construct Libraries
  • 4.3.1. Degenerate RBSs for Combinatorial Optimization of Expression Levels of Biosynthetic Pathway Genes
  • 4.3.2. Varying Biosynthetic Pathway Gene Order for Phenotypic Diversity
  • 4.4. Targeted Postassembly Modification for Enhanced Productivity
  • 5. Design Principles for att Sites
  • 5.1. Naming Convention for att Sites
  • 5.2. Central Dinucleotides of att Sites Determine the Positions of DNA Parts in a Construct
  • 5.3. Central Dinucleotides of att Sites Determine Polarity of Recombination
  • 5.4. Elimination of Intramolecular Recombination
  • 5.5. Assembled Products Contain Only attL Sites
  • 6. Materials Preparation
  • 6.1. DNA Components
  • 6.1.1. DNA Parts
  • 6.1.1.1. Primers for DNA Parts to be Assembled in Predefined Gene Orders
  • 6.1.1.2. Primers for Degenerate RBS Libraries
  • 6.1.1.3. Primers for Combinatorial Construct Libraries
  • 6.1.1.4. Secondary SIRA Insertion Sites
  • 6.1.1.5. PCR Conditions for Making DNA Parts
  • 6.1.2. SIRA Substrate Plasmids
  • 6.1.3. DNA Purification by Ethanol Precipitation
  • 6.2. Purification of FC31 Integrase and gp3
  • 6.2.1. Purification of FC31 Integrase
  • 6.2.2. Purification of gp3
  • 6.3. SIRA Reaction Buffer
  • 6.4. Competent E. coli Strains
  • 6.4.1. Chemically Competent E. coli
  • 6.4.2. Electrocompetent E. coli
  • 7. Example SIRA Protocols
  • 7.1. SIRA Example 1: Inserting a Single Gene Using SIRA
  • 7.2. SIRA Example 2: Assembly of Multiple DNA Parts in a Predefined Order
  • 7.3. SIRA Example 3: Combinatorial Assembly of DNA Parts for a Library of Constructs with Different Gene Orders
  • 7.4. SIRA Example 4: Targeted Postassembly Modification of a SIRA Construct
  • 8. The Future of SIRA
  • Acknowledgments
  • References
  • Chapter Fourteen: Rewiring Riboswitches to Create New Genetic Circuits in Bacteria
  • 1. Introduction
  • 2. Overview
  • 3. Methods
  • 3.1. Construction of Inducible-Riboswitch Controlled Gene Expression Vectors for E. coli
  • 3.2. CheZ Motility Assays in E. coli
  • 3.3. Construction of Repressible-Riboswitch Controlled Constructs for Targeted Chromosomal Integration in B. subtilis
  • 3.3.1. lacZ Reporter Gene Constructs
  • 3.3.2. Native Gene Expression Constructs
  • 3.4. ß-Galactosidase Assays to Validate Synthetic Riboswitch Function
  • 3.5. Western Blot Analysis to Quantify Target Gene Expression Levels
  • 3.6. MreB Morphology Assays in B. subtilis
  • 3.7. Construction of Chimeric Riboswitches to Couple Orthogonal Aptamers to Host-Specific Expression Platforms
  • 4. Concluding Remarks
  • Acknowledgments
  • References
  • Author Index
  • Subject Index
  • Color Plate
  • Back Cover

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