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

 
 
Academic Press
  • 1. Auflage
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  • erschienen am 29. Juli 2016
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  • 446 Seiten
 
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978-0-12-811123-9 (ISBN)
 

Synthetic Biology and Metabolic Engineering in Plants and Microbes, Part B, the latest 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 usage 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 of enzymology
  • Contains two volumes covering research methods in synthetic biology and metabolic engineering in plants and microbes
  • 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
0076-6879
  • Englisch
  • San Diego
  • |
  • USA
Elsevier Science
  • 20,68 MB
978-0-12-811123-9 (9780128111239)
0128111232 (0128111232)
weitere Ausgaben werden ermittelt
  • Front Cover
  • Synthetic Biology and Metabolic Engineering in Plants and Microbes Part B: Metabolism in Plants
  • Copyright
  • Contents
  • Contributors
  • Preface
  • Chapter One: Comparative Biochemistry and In Vitro Pathway Reconstruction as Powerful Partners in Studies of Metabolic Di ...
  • 1. Introduction
  • 2. Characterization of Specialized Metabolite Diversity Using LC/MS and NMR
  • 2.1. Metabolite Structure Elucidation by LC/MS with Multiplexed CID
  • 2.1.1. Example of Acylsugar Structure Profiling by LC/MS
  • 2.1.2. Protocol for Plant Trichome Acylsugar Extraction and LC/MS
  • 2.2. Sample Purification for NMR Spectroscopy
  • 2.2.1. Protocol for Purifying Individual Acylsugars from Trichomes for NMR Analysis
  • 3. Phylogeny-Driven Analysis of the Biochemical Basis of Specialized Metabolite Diversity
  • 3.1. Phylogeny-Based Screening of Metabolic Phenotypes
  • 3.2. The Biochemical Basis for Differences in Furanose Ring Acylation
  • 3.2.1. Protocol to Clone and Characterize S. habrochaites ASAT3 Isoforms
  • 3.3. Identifying a Key Polymorphism Driving Enzyme Function Divergence
  • 3.3.1. Discovery of an ASAT3 Polymorphism Associated with In Vitro Functional Variation
  • 3.3.2. Protocol Used to Identify Key Polymorphisms Contributing to Variation in ASAT3 Acyl-CoA Substrate Specificity
  • 4. Conclusions
  • Acknowledgments
  • References
  • Chapter Two: De Novo Deep Transcriptome Analysis of Medicinal Plants for Gene Discovery in Biosynthesis of Plant Natural ...
  • 1. Methods for Transcriptomic Study Prior to High-Throughput Sequencing
  • 2. Deep Transcriptome Outline
  • 3. Preparation of the Plant Materials and RNA Extraction
  • 4. cDNA Library Construction
  • 5. High-Throughput Sequencing
  • 6. Data Interpretation
  • 6.1. Trimming and Quality Validation of Raw Reads
  • 6.2. De Novo Assembly
  • 6.3. Expression Analysis of Transcripts
  • 6.4. Sequence Similarity Search
  • 6.5. Gene Annotation
  • 6.6. An Example of RNA-Seq
  • 7. Application of RNA-Seq on Medicinal Plants and Perspectives
  • Acknowledgments
  • References
  • Chapter Three: Genomics-Based Discovery of Plant Genes for Synthetic Biology of Terpenoid Fragrances: A Case Study in San ...
  • 1. Introduction
  • 2. Prior Knowledge of Sandalwood TPSs and P450s and Development of a Hypothesis
  • 3. Replication, Sampling, and Statistical Design
  • 4. Defining Temporal and Spatial Variables for Tissue Sampling
  • 5. Tissue Sampling
  • 6. Metabolite Profiling
  • 7. Isolation of High-Quality RNA from Recalcitrant Tissues
  • 8. Transcriptome Sequencing and De Novo Assembly
  • 9. Transcriptome Mining and Annotation
  • 10. Expression Analysis and Candidate Gene Selection
  • 11. Functional Characterization of Candidate Genes
  • 11.1. Yeast In Vivo Assays
  • 11.2. Microsomes In Vitro Assays
  • 12. Product Identification
  • 12.1. MS-MS
  • 12.2. Nuclear Magnetic Resonance
  • Acknowledgments
  • References
  • Chapter Four: A Workflow for Studying Specialized Metabolism in Nonmodel Eukaryotic Organisms
  • 1. Introduction
  • 2. ``Omics´´-Based Novel Specialized Metabolic Pathway Discovery
  • 2.1. De Novo Transcriptome Sequencing and Assembly
  • 2.2. Making Use of the De Novo Transcriptome Data
  • 2.3. De Novo Genome Sequencing
  • 2.4. Metabolomics Approaches for Defining Intermediates of an Unknown Metabolic Pathway
  • 3. Structure-Function Analysis of Specialized Metabolic Enzymes
  • 3.1. Evolutionary Approaches for Candidate Enzyme Identification
  • 3.2. Recombinant Expression and Purification of Candidate Enzymes
  • 3.3. Comparative Biochemical Analysis of Enzyme Functions
  • 3.4. Structural Basis for Specialized Metabolic Enzyme Evolution
  • 4. Reconstitution of Specialized Metabolic Pathways in Heterologous Systems
  • 4.1. Escherichia coli
  • 4.2. Saccharomyces cerevisiae
  • 4.3. Nicotiana benthamiana
  • 5. Summary
  • Acknowledgments
  • References
  • Chapter Five: Gene Discovery for Synthetic Biology: Exploring the Novel Natural Product Biosynthetic Capacity of Eukaryot ...
  • 1. Introduction
  • 2. Natural Product Synthases
  • 2.1. Polyketide Synthases
  • 2.2. Nonribosomal Peptide Synthetases
  • 3. Genome Mining for the Identification of Natural Products
  • 3.1. Identification of PKSs
  • 3.2. Identification of NRPSs
  • 3.3. Hybrid NRPS/PKSs
  • 3.4. Identification of Other Components of Microalgal Natural Product Biosynthetic Pathways
  • 4. Natural Product Discovery
  • 5. Conclusions
  • Acknowledgments
  • References
  • Chapter Six: cis-Prenyltransferase and Polymer Analysis from a Natural Rubber Perspective
  • 1. Introduction
  • 2. Rationale: Observation of Revertants from rer2 Mutant
  • 3. Generation of rer2 and srt1 Double Knockout Yeast Strain
  • 3.1. Background on rer2Delta Strain
  • 3.2. Deletion of SRT1 on rer2Delta Strain
  • 3.2.1. Preparation of Homologous DNA
  • 3.2.2. Yeast Transformation with Linear DNA
  • 4. Complementation of rer2Delta srt1Delta with CPT and CBP
  • 4.1. Construct Generation
  • 4.2. Yeast Transformation with Plasmids
  • 4.3. Selection in 5-FOA
  • 4.4. Results
  • 5. CPT Biochemical Assay Using Yeast Microsomes
  • 5.1. In Vitro CPT Assays
  • 5.2. Yeast Microsome Preparation
  • 5.2.1. Yeast Culture
  • 5.2.2. Bead Beating
  • 5.2.3. Microsome Preparation
  • 5.3. cis-Prenyltransferase Enzyme Assays Using Yeast Microsomes and 14C-IPP
  • 5.4. Dephosphorylation
  • 5.4.1. Enzymatic Dephosphorylation
  • 5.4.2. Chemical Dephosphorylation
  • 5.5. Thin Layer Chromatography
  • 5.6. Results
  • 6. General Discussion
  • Acknowledgments
  • References
  • Chapter Seven: Generation and Functional Evaluation of Designer Monoterpene Synthases
  • 1. Introduction
  • 2. Equipment
  • 3. Materials
  • 3.1. Molecular Biology
  • 3.1.1. TE Stock Solution
  • 3.1.2. DNA Gel Loading Dye
  • 3.1.3. IPTG Solution
  • 3.2. Recombinant Enzyme Purification
  • 3.2.1. MOPSO Buffer
  • 3.2.2. Phosphate Buffer
  • 3.3. Protein Gel Electrophoresis
  • 3.3.1. Protein Gel Loading Dye
  • 3.3.2. Protein Gel Running Buffer
  • 3.3.3. Protein Staining Dye
  • 3.3.4. Protein Gel Destaining Solution
  • 3.4. Enzyme Assays
  • 3.4.1. Geranyl Diphosphate Stock Solution
  • 3.4.2. Enzyme Assay Buffer
  • 4. Step 1-Generation of Expression Constructs
  • 4.1. Quick Change PCR
  • 4.1.1. Amplification of Mutated cDNA Sequence
  • 4.1.2. Transformation of Vector Containing Mutated cDNA into Host Cells
  • 4.2. Overlap Extension Mutagenesis
  • 4.2.1. Introduction of Point Mutations
  • 4.2.2. Self-Annealing of Amplicons to Generate a Full-Length Mutated cDNA
  • 5. Step 2-Production of Purified, Recombinant Target Enzyme
  • 5.1. E. coli Cultivation and Induction of Target Enzyme Expression
  • 5.2. Isolation of Target Enzyme and Assessment of Purity
  • 5.2.1. Cell Disruption
  • 5.2.2. Batch Purification
  • 5.2.3. Total Protein Quantitation
  • 5.2.4. Assessment of Recombinant Enzyme Purity
  • 6. Step 3-Functional Evaluation of Recombinant Monoterpene Synthases
  • 6.1. Enzymatic Reaction
  • 6.2. Product Quantitation
  • 6.3. Analysis of Kinetic Data
  • 7. Conclusions
  • Acknowledgments
  • References
  • Chapter Eight: Prequels to Synthetic Biology: From Candidate Gene Identification and Validation to Enzyme Subcellular Loc ...
  • 1. Introduction
  • 2. Identification of Candidate Genes Through Transcriptomic Data Mining and Analysis
  • 2.1. Transcriptome Assembly, Annotation, and Transcript Abundance Estimation
  • 2.1.1. Transcriptome Assembly
  • 2.1.2. Transcriptome Annotation
  • 2.1.3. Transcript Abundance Estimation
  • 2.1.3.1. Prepare Reference for Abundance Estimation on CDF97
  • 2.1.3.2. Align Paired-End Reads to Reference Transcriptome with bowtie2
  • 2.1.3.3. Combine RSEM Results Files to Generate Raw Count or FPKM Matrix
  • 2.2. Transcriptome Postassembly Analysis
  • 2.2.1. Differential Expression
  • 2.2.2. Correlation Analysis
  • 2.2.3. Clustering Procedures
  • 2.2.3.1. Partitioning
  • 2.2.3.2. Hierarchical Clustering
  • 2.2.3.3. HOPACH
  • 3. Validation of Candidate Gene Function by Biolistic-Mediated VIGS
  • 3.1. Plant Material and Growth Condition Pretransformation
  • 3.2. Silencing Constructs for VIGS
  • 3.3. Biolistic-Mediated Transformation of C. roseus
  • 3.3.1. Particle Preparation
  • 3.3.2. Coating of Plasmids onto Particles
  • 3.3.3. Particle Bombardment Procedure
  • 3.4. Posttransformation Treatments and Analysis
  • 4. Studying the Subcellular Localization of Biosynthetic Pathway Enzymes in Plant and Yeast Cells to Alleviate Bottleneck ...
  • 4.1. Protein Subcellular Localization in C. roseus Cells
  • 4.1.1. Constructs Expressing Fusions with Fluorescent Proteins in Plant Cells
  • 4.1.2. Cell Culture and Plating
  • 4.1.3. Transient Cell Transformation by Biolistic
  • 4.1.4. Fluorescent Protein Imaging and Epifluorescence Microscopy
  • 4.1.5. The Importance of Being Correctly Fused
  • 4.2. Protein Subcellular Localization in Yeast Cells
  • 4.2.1. Constructs Expressing Fusions with Fluorescent Proteins in Yeast Cells
  • 4.2.2. Preparation of Yeast Competent Cells
  • 4.2.3. Protocol of Yeast Cell Transformation
  • 4.2.4. Correct and Incorrect Plant Protein Targeting in Yeast
  • 5. Concluding Remarks
  • Acknowledgments
  • References
  • Chapter Nine: Functional Expression and Characterization of Plant ABC Transporters in Xenopus laevis Oocytes for Transpor ...
  • 1. Introduction
  • 2. Preparation of cDNA of Plant ABC Transporter Genes by In Planta ``Exon Engineering´´
  • 3. ABC Transporter Expression in Xenopus Oocytes
  • 4. Optimization of Transport Assay for Diffusible ABA in Xenopus Oocytes
  • 5. Case Study: Characterization of the ABA Exporter at ABCG25 in Xenopus Oocytes
  • 6. Conclusions
  • Acknowledgments
  • References
  • Chapter Ten: Quantifying the Metabolites of the Methylerythritol 4-Phosphate (MEP) Pathway in Plants and Bacteria by Liqu ...
  • 1. Introduction
  • 2. Preparation of Stable Isotope-Labeled Internal Standards
  • 2.1. Growing E. coli with Labeled Glucose as a Carbon Source
  • 2.2. Isolation of MEcDP by Export from Transgenic E. coli
  • 3. Extraction of Methylerythritol Phosphate Pathway Intermediates from Biological Sources
  • 3.1. Extraction of Methylerythritol Phosphate Pathway Intermediates from Plant Materials
  • 3.2. Extraction of Methylerythritol Phosphate Pathway Intermediates from Bacterial Cultures
  • 4. Analysis of Methylerythritol Phosphate Pathway Metabolites by LC-MS/MS
  • 4.1. Separation by HILIC
  • 4.2. Detection by Tandem Mass Spectrometry
  • 4.3. Quantification
  • 5. Discussion and Summary
  • References
  • Chapter Eleven: Establishing the Architecture of Plant Gene Regulatory Networks
  • 1. Introduction
  • 2. The cis-Regulatory Apparatus
  • 2.1. Establishing TSSs
  • 2.1.1. Cap Analysis of Gene Expression
  • 2.1.2. Other Methods
  • 2.2. Promoters and Enhancers
  • 2.3. cis-Regulatory Variation
  • 3. The Trans-Acting Factors
  • 3.1. Transcription Factors
  • 3.1.1. Functional Assays to Determine Transcriptional Regulatory Activity
  • 3.1.2. TFs Function as Activators and/or Repressors of Transcription
  • 3.1.3. Functional Methods to Identify TFs Controlling Agronomic Traits
  • 3.2. Cofactors
  • 4. Transcription Factor Centered Approaches
  • 4.1. ChIP Approaches
  • 4.2. In Vitro TF-DNA Interaction Approaches
  • 4.2.1. SELEX and SELEX-Seq
  • 4.2.2. Protein Binding Microarrays (PBMs)
  • 4.2.3. Chemically Regulated Gene Expression Systems
  • 4.3. In Silico TF-Location Prediction Approaches
  • 5. Gene-Centered Approaches
  • 5.1. Y1H Approaches
  • 5.1.1. Principle and Work Flow of Y1H
  • 5.1.2. Comparing cDNA and TFome Libraries for Y1H
  • 5.1.3. Comparison of Transformation and Mating Y1H Strategies
  • 5.1.4. Limitations of Y1H-Based Screens
  • 5.1.4.1. False Negatives
  • 5.1.4.2. False Positives
  • 5.2. Electrophoretic Mobility Shift Assay (EMSA) Based Methods
  • 6. Resources for Studying Plant GRNs
  • 6.1. Plant Transcription Factor ORFeome Collections (TFomes)
  • 6.2. Databases
  • 7. Conclusions
  • Acknowledgment
  • References
  • Chapter Twelve: Engineering of Tomato Glandular Trichomes for the Production of Specialized Metabolites
  • 1. Introduction
  • 1.1. Trichomes on Plants
  • 1.2. Specialized Metabolites and Application
  • 1.3. Production of Specialized Metabolites in Tomato Trichomes
  • 1.4. Modifying Terpene Production
  • 1.5. Focus
  • 2. Materials and Technology
  • 2.1. Transient Transformation of Tomato Trichomes by Microparticle Bombardment
  • 2.1.1. Preparation of the pSlTPS5: GUS-sYFP1 Construct
  • 2.1.2. Particle Coating with DNA
  • 2.1.3. Plant Material and Growing Conditions
  • 2.1.4. Bombardment Conditions
  • 2.1.5. GUS Expression Assay
  • 2.1.6. Analysis of Promoter Trichome Specificity
  • 2.2. Stable Transformation of Tomato Trichomes
  • 2.2.1. Preparation of Construct pSlTPS9: GUS-sYFP1
  • 2.2.2. Stable Tomato Transformation
  • 2.2.3. Transgene Expression
  • 3. Proof of Concept: Targeted Expression of a Terpene Precursor Gene in Tomato Glandular Trichomes
  • 3.1. Stable Trichome-Specific Expression of FPS
  • 3.1.1. Preparation of pMKS1:ssu-SlFPS and pMKS1:ssu-GgFPS Transgenic Tomatoes
  • 3.1.2. Expression Analysis of the Different FPS Transgenes
  • 3.2. Functional Validation of Trichome Engineering
  • 3.2.1. Redirecting FPS to the Plastid Alters Terpenoid Production in Glandular Trichomes
  • 3.2.2. Biological Relevance of Changing Specialized Metabolites in Trichomes
  • 4. Summary
  • Acknowledgments
  • References
  • Chapter Thirteen: Tomato Fruits-A Platform for Metabolic Engineering of Terpenes
  • 1. Introduction
  • 2. Terpenoid Formation in Tomato Fruits
  • 3. Transgene Expression in Ripening Tomato Fruits
  • 3.1. Stable Transformation Under Fruit Ripening-Specific Promoters
  • 3.2. Transient Transformation via Agrobacterium Injection into Fruits
  • 4. Overexpression of Terpene Biosynthetic Genes in Tomato Fruits
  • 4.1. MEP and MVA Pathway Genes
  • 4.2. Prenyltransferases
  • 4.3. Terpene Synthases
  • 4.4. Pyramiding Multiple Terpene Biosynthetic Genes
  • 5. Analysis of Terpenes in Tomato Fruits
  • 5.1. Analysis of Emitted Terpenes
  • 5.2. Analysis of Internal Terpene Pools
  • 6. Conclusions
  • Acknowledgments
  • References
  • Chapter Fourteen: Libraries of Synthetic TALE-Activated Promoters: Methods and Applications
  • 1. Introduction
  • 2. Construction of Libraries of Synthetic Promoters Using Golden Gate Cloning
  • 3. Analyzing Promoter Activity in Transient Assays
  • 4. Conclusion
  • References
  • Author Index
  • Subject Index
  • Back Cover

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