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

1st Edition, Volume 671 - July 23, 2022
Editor: Eleanore T Wurtzel
Language: English
Hardback ISBN:
9 7 8 - 0 - 3 2 3 - 9 1 3 5 3 - 9
eBook ISBN:
9 7 8 - 0 - 3 2 3 - 9 1 3 5 4 - 6

Carotenoids: Carotenoid and Apocarotenoid Biosynthesis, Metabolic Engineering and Synthetic Biology, Volume 671, the latest release in the Methods of Enzymology series highlight… Read more

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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 wide association mapping approaches, Using bacteria for functional analysis of genes encoding carotenoid biosynthetic enzymes, Rice Callus as a High Throughput Platform for Synthetic Biology and Metabolic Engineering of Carotenoids, Transient expression in Nicotiana benthamiana: A simple platform to investigate genes encoding carotenoid biosynthesis enzymes from diverse algal lineages, and much more.

Additional chapters in this new release cover Protein-protein interaction techniques to investigate post-translational regulation of carotenogenesis, The isolation of sub-chromoplast structures from tomato and capsicum fruit, Carrot protoplasts as a suitable method for protein subcellular localization, High throughput production and characterization of carotenoid enzymes for structural and functional studies, Production and structural characterization of the cytochrome P450 enzymes in carotene ring hydroxylation, and more.

Cover image
Title page
Table of Contents
Copyright
Contributors
Preface
Chapter One: Golden Rice—Lessons learned for inspiring future metabolic engineering strategies and synthetic biology solutions
Abstract
1: The need for Golden Rice
2: Initial phase for Golden Rice generation
3: Decision on genes required for Golden Rice development
4: Assessment with today's technologies
5: Possible strategies for improvement of Golden Rice
6: Non-mevalonate pathway
7: Phytoene synthase
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
Abstract
1: Introduction
2: Before you begin
3: Materials and equipment
4: Protocol
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)
Abstract
1: Introduction
2: Understanding the apple carotenoid pathway
3: Functional testing of genes encoding carotenoid enzymes
4: Analysis of carotenoid pigments in apple
5: Conclusion
Acknowledgments
References
Chapter Four: The breeder's tool-box for enhancing the content of esterified carotenoids in wheat: From extraction and profiling of carotenoids to marker-assisted selection of candidate genes
Abstract
1: Introduction
2: Crossing scheme and selection steps
3: Marker assisted selection (MAS) protocol
4: Analysis of carotenoids and carotenoid esters in grains
5: Conclusions
Acknowledgments
References
Chapter Five: Understanding carotenoid biosynthetic pathway control points using metabolomic analysis and natural genetic variation
Abstract
1: Introduction
2: Genetic analysis leveraging natural variation
3: Metabolomic methods used in carotenoid analysis
4: Case studies of genetic analysis of natural variation in carotenoids
5: Summary
References
Chapter Six: Enzymatic isomerization of ζ-carotene mediated by the heme-containing isomerase Z-ISO
Abstract
1: Introduction
2: Materials
3: Methods
4: Notes
Acknowledgments
References
Chapter Seven: Genomics-based strategies toward the identification of a Z-ISO carotenoid biosynthetic enzyme suitable for structural studies
Abstract
1: Introduction
2: Selection of targets
3: Equipment, chemicals, and reagents
4: Preparation of gene fragments for cloning
5: Protein ortholog expression screening
6: Summary
Acknowledgments
References
Chapter Eight: Metalloenzymes involved in carotenoid biosynthesis in plants
Abstract
1: Introduction: Metalloenzymes discovered in carotenoid biosynthesis in plants
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
Abstract
1: Introduction
2: Materials
3: Production of Arabidopsis CYP97s
4: CYP97 activity assays
5: Crystallization and structural characterization
6: Concluding remark
Acknowledgments
References
Chapter Ten: Preparation of carotenoid cleavage dioxygenases for X-ray crystallography
Abstract
1: Introduction
2: Methods overview
3: Expression of native CCDs in Escherichia coli and their purification
4: Spectrophotometric assay of CCD activity
5: Production of metal-substituted CCDs
6: Quantification of CCD metal content
7: Crystallization of CCDs (with and without ligands)
8: Summary
Acknowledgments
References
Chapter Eleven: Carrot protoplasts as a suitable method for protein subcellular localization
Abstract
1: Introduction
2: Materials and equipment
3: Methods
4: Summary
References
Chapter Twelve: Isolation and characterization of sub-plastidial fractions from carotenoid rich fruits
Abstract
1: Introduction
2: Experimental approach and design
3: Subchromoplast fractionation of tomato and capsicum (pepper) fruit
4: Extraction of carotenoids, isoprenoids, and proteins from sub-chromoplast fractions
5: Spatial proteomic analysis and sub-chromoplast fractions
6: Expected outcomes
7: Summary and perspectives
Acknowledgments
References
Chapter Thirteen: Protein–protein interaction techniques to investigate post-translational regulation of carotenogenesis
Abstract
1: Introduction
2: Experimental design
3: Membrane-based split ubiquitin yeast two-hybrid system
4: Bimolecular fluorescence complementation
5: Pull-down assay
6: Summary
Acknowledgments
References
Chapter Fourteen: Tracking subplastidic localization of carotenoid metabolic enzymes with proteomics
Abstract
1: Introduction
2: Isolation and proteomic analysis of chloroplast sub-compartments
3: Protocol
Acknowledgments
References
Chapter Fifteen: Use of directed enzyme evolution to create novel biosynthetic pathways for production of rare or non-natural carotenoids
Abstract
1: Introduction
2: General scheme of directed evolution experiments with carotenoid enzymes
3: Designing a colony-based screening assay for carotenoid enzyme activity in E. coli
4: Directed evolution of a bacterial phytoene desaturase CrtI for C50-phytoene desaturation
5: Materials and equipment
6: Step-by-step method details
7: Next steps
References
Chapter Sixteen: A synthetic biology approach to study carotenoid production in Corynebacterium glutamicum: Read-out by a genetically encoded biosensor combined with perturbing native gene expression by CRISPRi
Abstract
1: Introduction
2: Protocols
Acknowledgment
Funding
References
Chapter Seventeen: Imaging retinaldehyde-protein binding in plants using a merocyanine reporter
Abstract
1: Introduction
2: Materials and equipment
3: Step-by-step method details
Acknowledgments
References
Chapter Eighteen: Synthesis and characterization of abscisic acid receptor modulators
Abstract
1: Introduction
2: Synthesis of opabactin
3: Synthesis of OP-azide (OPZ) and alkynes 19–20
4: Synthesis of antabactin and TAMRA-ANT
5: Expression and purification of Arabidopsis receptors and HAB1
6: Characterization of HAB1 enzymatic activity
7: Characterization of ABA receptor activity using TAMRA-ANT
8: In vitro characterization of agonists using receptor-mediated phosphatase inhibition assays
9: In vitro characterization of antagonists using receptor-mediated phosphatase inhibition assays
10: Summary
11: Notes
References
Chapter Nineteen: Heterologous complementation in bacteria for functional analysis of genes encoding carotenoid biosynthetic enzymes
Abstract
1: Introduction
2: Materials and equipment
3: Methods
4: Summary
Acknowledgments
References
Chapter Twenty: An in vivo plant platform to assess genes encoding native and synthetic enzymes for carotenoid biosynthesis
Abstract
1: Introduction
2: Before you begin
3: Key resources table
4: Materials and equipment
5: Step-by-step method details
6: Expected outcomes
7: Quantification and statistical analysis
8: Advantages
9: Limitations
10: Optimization and troubleshooting
11: Safety considerations and standards
References
Chapter Twenty-One: Rice callus as a high-throughput platform for synthetic biology and metabolic engineering of carotenoids
Abstract
1: Introduction
2: Functional characterization of the Arabidopsis orange gene using a heterologous rice callus platform
3: Metabolic engineering strategies using native and synthetic genes
4: Cereal endosperm specific promoters
5: Targeting efficiency and mutations through gene editing
6: Rice transformation
7: Carotenoid quantification
8: Summary
Acknowledgments
References
Chapter Twenty-Two: Characterizing cytochrome P450 enzymes involved in plant apocarotenoid metabolism by using an engineered yeast system
Abstract
1: Introduction
2: Materials
3: Methods
Acknowledgments
References

Eleanore T Wurtzel

As a Ph.D student, Eleanore Wurtzel innovated gene tagging and isolated the first genes for two-component signaling in bacteria, laying the foundation for study of signaling mechanisms found throughout nature, including plants. With an NSF postdoctoral fellowship, Dr. Wurtzel boldly changed fields from bacterial membrane biochemistry to plant biology, when maize was the only model system. She established some of the first experiments on plant chromatin structure as an NSF Plant Biology postdoctoral fellow at Brookhaven National Laboratory. She then joined Cold Spring Harbor Laboratory and began research on maize carotenoid biosynthesis, then a poorly studied area. Dr. Wurtzel next joined the Biological Sciences Department at Lehman College, City University of New York, where she is currently a Full Professor and on the faculty of the CUNY Biology and Biochemistry PhD programs. Eleanore Wurtzel has made fundamental and longstanding contributions to the field of plant carotenoid biosynthesis, plant biochemistry, and plant metabolic engineering which are enabling improvement of crops for sustainable solutions to global vitamin A deficiency affecting the health and mortality of 250 million children worldwide. Dr. Wurtzel is grateful to the many students, postdocs, and visiting scientists who have contributed to her laboratory’s research for which she has been recognized as a Fellow of AAAS, Fellow of ASPB, and most recently as a Fellow of the International Carotenoid Society. Dr. Wurtzel serves as a Monitoring Editor of Plant Physiology. Dr. Wurtzel has also been a long-standing elected member of the Gordon Research Conferences (GRC) Board of Trustees. She has been instrumental at GRC in developing and contributing to programs for women in science. She also founded and chaired the first GRC on Plant Metabolic Engineering and founded the GRC seminar for early career scientists for both the GRC Plant Metabolic Engineering community and the GRC Carotenoids community.

Affiliations and expertise

Professor, Department of Biological Sciences, Lehman College and the Graduate Center, The City University of New York, USA