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Carotenoids : carotenoid and apocarotenoid biosynthesis, metabolic engineering and synthetic biology /

Carotenoids: Carotenoid and Apocarotenoid Biosynthesis, Metabolic Engineering and Synthetic Biology, Volume 671, the latest release in the Methods of Enzymology series highlights new advances in the field with chapters on Metabolomics-based analysis of carotenoids and related metabolites in various...

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Bibliographic Details
Corporate Author: ScienceDirect (Online service)
Other Authors: Wurtzel, Eleanore T.
Format: eBook
Language:English
Published: Cambridge, MA : Academic Press, 2022.
Series:Methods in enzymology ; v. 671.
Subjects:
Carotenoids > Biotechnology.
Carotenoids > Analysis.
Carotenoids > metabolism
Carotenoids > analysis
Biosynthetic Pathways > physiology
Caroténoïdes > Biotechnologie.
Caroténoïdes > Analyse.
Carotenoids > Analysis
Carotenoides.
Biosíntesi.
Electronic books.
Llibres electrònics.
Online Access:Connect to the full text of this electronic book
Table of Contents:
  • 4.6. FEC inoculation with A. tumefaciens strain LBA4404 * TIMING: 4 days
  • 4.7. Washing and recovery * TIMING: 6 days
  • 4.8. Selection and regeneration * TIMING: 5 months
  • 5. Expected outcomes
  • 6. Optimization and troubleshooting
  • 7. Safety considerations and standards
  • 8. Alternative methods/procedures
  • References
  • Chapter Three: Elevating fruit carotenoid content in apple (Malus x domestica Borkh)
  • 1. Introduction
  • 2. Understanding the apple carotenoid pathway
  • 2.1. Identification of apple carotenoid genes
  • 2.1.1. Genome resources in the public domain
  • 2.2. Promoter screening assay to identify transcription factors
  • 2.2.1. Materials
  • 2.2.2. Agrobacterium infiltration of Nicotiana sp. leaves
  • 2.2.3. Fluorescence assay with dual luciferase reporter system
  • 2.3. Gene expression analysis of apple carotenoid genes
  • 2.3.1. RNA extraction protocol
  • 2.3.1.1. Laboratory equipment
  • 2.3.1.2. Chemicals and reagents
  • 2.3.1.3. Preparation of CTAB extraction buffer
  • 3. Functional testing of genes encoding carotenoid enzymes
  • 3.1. Rapid functional gene testing
  • 3.1.1. Apple callus transformation
  • 3.1.1.1. Induction of apple callus
  • 3.1.1.2. Transformation of apple callus
  • 3.2. Stable transformation of apple
  • 3.2.1. Resources required
  • 4. Analysis of carotenoid pigments in apple
  • 4.1. Carotenoid extraction
  • 4.1.1. Saponified extraction
  • 4.1.2. Unsaponified extraction
  • 4.2. HPLC analysis
  • 4.3. Fluorescence confocal microscopy
  • 4.3.1. Key resources
  • 5. Conclusion
  • Acknowledgments
  • References
  • Chapter Four: The breeder´s tool-box for enhancing the content of esterified carotenoids in wheat: From extraction and pr ...
  • 1. Introduction
  • 1.1. The importance of carotenoids and biofortification programs
  • 1.2. The role of the H genome for enhancing carotenoid content of tritordeum.
  • 1.3. Genetic bases of carotenoid esterification in wheat and H. chilense
  • 2. Crossing scheme and selection steps
  • 2.1. Notes (Fig. 1)
  • 3. Marker assisted selection (MAS) protocol
  • 3.1. DNA isolation protocol
  • 3.1.1. Equipment and labware
  • 3.1.2. Chemicals
  • 3.1.3. Protocols
  • 3.1.3.1. DNA extraction (microscale)
  • 3.1.3.2. DNA extraction (macroscale, two 96-well plates simultaneously)
  • 3.1.4. Notes
  • 3.2. Identification of XAT-7Hch in the wheat background
  • 3.2.1. Equipment and labware
  • 3.2.2. Reagents
  • 3.2.3. Protocol
  • 4. Analysis of carotenoids and carotenoid esters in grains
  • 4.1. Extraction of carotenoids
  • 4.1.1. Equipment and labware
  • 4.1.2. Chemicals
  • 4.1.3. Protocols
  • 4.1.3.1. Extraction of carotenoids by a standard procedure
  • 4.1.3.2. Extraction of carotenoids by one-step grinding-extraction procedure
  • 4.2. Analysis by high performance liquid chromatography (HPLC)
  • 4.2.1. Equipment and labware
  • 4.2.2. Chemicals
  • 4.2.3. Protocol
  • 4.2.4. Preparation of calibration curves
  • 4.3. Notes
  • 5. Conclusions
  • Acknowledgments
  • References
  • Chapter Five: Understanding carotenoid biosynthetic pathway control points using metabolomic analysis and natural genetic ...
  • 1. Introduction
  • 2. Genetic analysis leveraging natural variation
  • 2.1. Linkage analysis
  • 2.2. Genome-wide association studies
  • 3. Metabolomic methods used in carotenoid analysis
  • 3.1. Carotenoid extraction, isolation and identification
  • 3.1.1. Liquid chromatography
  • 3.1.2. Mass spectrometry (MS)
  • 3.1.3. Quantitative and qualitative analysis by LC-MS
  • 3.2. Workflow of metabolomics-based analysis of carotenoids
  • 3.2.1. Sample collection
  • 3.2.2. Sample preservation
  • 3.2.3. Qualitative and quantitative analyses
  • 4. Case studies of genetic analysis of natural variation in carotenoids
  • 5. Summary
  • References.
  • Chapter Six: Enzymatic isomerization of zeta-carotene mediated by the heme-containing isomerase Z-ISO
  • 1. Introduction
  • 2. Materials
  • 2.1. Functional analysis of Z-ISO in E. coli
  • 2.2. Carotenoid extraction for analysis of zeta-carotene
  • 2.3. Separation of zeta-carotene by HPLC
  • 2.4. Expression and purification of the maltose binding protein::Z-ISO fusion protein
  • 2.5. Preparation of the Z-ISO substrate
  • 2.6. Preparation of substrate-containing liposomes
  • 2.7. In vitro reactions
  • 2.8. Extraction of zeta-carotene isomers for separation by HPLC
  • 3. Methods
  • 3.1. Functional analysis of Z-ISO in E. coli
  • 3.2. Carotenoid extraction for analysis of zeta-carotene
  • 3.3. Separation of zeta-carotene by HPLC
  • 3.4. Expression and purification of the maltose binding protein::Z-ISO fusion protein
  • 3.5. Preparation of the Z-ISO substrate
  • 3.6. Preparation of substrate-containing liposomes
  • 3.7. In vitro reactions and separation of zeta-carotene isomers
  • 4. Notes
  • Acknowledgments
  • References
  • Chapter Seven: Genomics-based strategies toward the identification of a Z-ISO carotenoid biosynthetic enzyme suitable for ...
  • 1. Introduction
  • 2. Selection of targets
  • 2.1. Identification of orthologs of Zm Z-ISO
  • 2.2. Identification of disordered regions and N-terminal deletions
  • 2.3. Refinement of the Z-ISO target set
  • 2.4. Selection of expression system and design of constructs for expression screening
  • 2.4.1. Screening targets in E. coli
  • 2.4.2. Screening targets in eukaryotic cells
  • 3. Equipment, chemicals, and reagents
  • 4. Preparation of gene fragments for cloning
  • 4.1. Primer design and PCR amplification of targets for LIC
  • 4.2. Cloning
  • 4.3. Screening and sequence verification
  • 5. Protein ortholog expression screening
  • 5.1. Small-scale expression testing in E. coli.
  • 5.2. Expression screening of orthologs in eukaryotic cells
  • 5.3. Scale-up expression and purification in E. coli
  • 5.4. Scale-up expression and purification in eukaryotic systems
  • 6. Summary
  • Acknowledgments
  • References
  • Chapter Eight: Metalloenzymes involved in carotenoid biosynthesis in plants
  • 1. Introduction: Metalloenzymes discovered in carotenoid biosynthesis in plants
  • 1.1. Synthesis of IPP from glucose
  • 1.2. Lycopene synthesis and modification
  • 1.3. Heme-dependent enzymes involved in carotenoid biosynthesis
  • 2. Z-ISO: A case study
  • 3. Concluding remarks
  • Acknowledgment
  • References
  • Chapter Nine: Production and structural characterization of the cytochrome P450 enzymes in carotene ring hydroxylation
  • 1. Introduction
  • 2. Materials
  • 3. Production of Arabidopsis CYP97s
  • 3.1. Expression of CYP97A3, CYP97B3, and CYP97C1
  • 3.2. Protein purification
  • 4. CYP97 activity assays
  • 4.1. Retinaldehyde hydroxylation assay
  • 4.2. Ligand-binding assay
  • 5. Crystallization and structural characterization
  • 5.1. Crystallization
  • 5.2. Structural determination and analysis
  • 6. Concluding remark
  • Acknowledgments
  • References
  • Chapter Ten: Preparation of carotenoid cleavage dioxygenases for X-ray crystallography
  • 1. Introduction
  • 2. Methods overview
  • 3. Expression of native CCDs in Escherichia coli and their purification
  • 3.1. Overview
  • 3.2. Equipment
  • 3.3. Reagents, cells, and other consumables
  • 3.4. Protocol
  • 3.4.1. CCD expression
  • 3.4.2. Native CCD purification methods
  • 3.4.2.1. Ammonium sulfate fractionation
  • 3.4.2.2. Anion exchange chromatography
  • 3.4.2.3. Size-exclusion chromatography
  • 3.5. Expected results
  • 4. Spectrophotometric assay of CCD activity
  • 4.1. Overview
  • 4.2. Equipment
  • 4.3. Reagents
  • 4.4. Other materials
  • 4.5. Protocol
  • 4.6. Expected results.