Carotenoids : carotenoid and apocarotenoid biosynthesis, metabolic engineering and synthetic biology / Eleanore Wurtzel, editor.

Format:

Book

Contributor:

Wurtzel, Eleanore T., editor.

Series:

Methods in enzymology ; Volume six hundred and seventy one.

Methods in enzymology ; Volume six hundred and seventy one

Language:

English

Subjects (All):

Carotenoids.

Physical Description:

1 online resource (580 pages)

Edition:

First edition.

Place of Publication:

London : Academic Press, [2022]

Summary:

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 species via quantitative trait loci and genome.

Contents:

Intro

Carotenoids: Carotenoid and Apocarotenoid Biosynthesis, Metabolic Engineering and Synthetic Biology

Copyright

Contents

Contributors

Preface

Chapter One: Golden Rice-Lessons learned for inspiring future metabolic engineering strategies and synthetic biology solu ...

1. The need for Golden Rice

2. Initial phase for Golden Rice generation

2.1. Fragmented knowledge of the carotenoid pathway

2.2. Knowledge of carotenoid precursors in endosperm

3. Decision on genes required for Golden Rice development

4. Assessment with today´s technologies

4.1. Similarities and dissimilarities between maize and rice endosperm

4.2. Development of Golden Rice 1 (GR1)

4.2.1. Considerations for phytoene desaturation and isomerization

4.2.2. Gene combinations

4.3. Assessment with today´s technologies

4.4. Development of Golden Rice 2 (GR2)

4.4.1. Reconsidering gene selection for GR2

4.4.2. Intrinsic properties of PSY variants

5. Possible strategies for improvement of Golden Rice

6. Non-mevalonate pathway

7. Phytoene synthase

7.1. Metabolons

7.2. Desaturation

8. Improved storage stability through β-carotene crystals

9. Application of the OR protein

Acknowledgments

References

Chapter Two: Metabolic engineering of cassava to improve carotenoids

1. Introduction

2. Before you begin

2.1. Reagents

2.2. Reagent setup. (All stock volumes are final volumes)

3. Materials and equipment

3.1. Materials

3.2. Equipment

4. Protocol

4.1. In vitro plant propagation * TIMING: 30 days

4.2. Primary somatic embryogenesis (SE) * TIMING: 25 days

4.3. Secondary somatic embryogenesis * TIMING: 22 days

4.4. Friable embryogenic callus (FEC) induction and purification * TIMING: 3 months 10 days

4.5. Growing bacteria * TIMING: 4 days.

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

Chapter Three: Elevating fruit carotenoid content in apple (Malus x domestica Borkh)

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

Chapter Four: The breeder´s tool-box for enhancing the content of esterified carotenoids in wheat: From extraction and pr ...

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

Chapter Five: Understanding carotenoid biosynthetic pathway control points using metabolomic analysis and natural genetic ...

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

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

Chapter Seven: Genomics-based strategies toward the identification of a Z-ISO carotenoid biosynthetic enzyme suitable for ...

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

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

Chapter Nine: Production and structural characterization of the cytochrome P450 enzymes in carotene ring hydroxylation

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

Chapter Ten: Preparation of carotenoid cleavage dioxygenases for X-ray crystallography

2. Methods overview

3. Expression of native CCDs in Escherichia coli and their purification

3.1. Overview

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.

5. Production of metal-substituted CCDs.

Notes:

Includes bibliographical references.

Description based on print version record.

Description based on publisher supplied metadata and other sources.

Other Format:

Print version: Wurtzel, Eleanore Carotenoids: Carotenoid and Apocarotenoid Biosynthesis, Metabolic Engineering and Synthetic Biology

ISBN:

0-323-91354-7

OCLC:

1337947069