Details
| Original language | English |
|---|---|
| Title of host publication | Engineering Natural Product Biosynthesis |
| Subtitle of host publication | Methods and Protocols |
| Publisher | Humana Press |
| Pages | 395-420 |
| Number of pages | 26 |
| Edition | 1 |
| ISBN (electronic) | 978-1-0716-2273-5 |
| ISBN (print) | 978-1-0716-2272-8 |
| Publication status | Published - 7 May 2022 |
Publication series
| Name | Methods in molecular biology (Clifton, N.J.) |
|---|---|
| Publisher | Humana Press |
| ISSN (Print) | 1064-3745 |
Abstract
Metabolic engineering of heterologous hosts requires coexpression of multiple genes, often more than ten for a single pathway. Traditional approaches to create genetic constructs for this purpose are highly inflexible and suffer from very low throughput. In this book chapter, we describe a powerful method to overcome this bottleneck, namely, combinatorial co-expression in the Australian tobacco plant Nicotiana benthamiana. This system is based on Agrobacterium tumefaciens-mediated transient gene expression, often called agroinfiltration. Herein, instead of creating complex multigenic constructs, coexpression is achieved by simply mixing different plasmid-bearing Agrobacterium strains without the need for different selection markers. We present a practical guide for coexpressing multiple biosynthetic genes followed by GC-MS analysis, using production of the plant triterpene β-amyrin as an example. Our chapter provides a guideline to harness the potential of this versatile expression system in the natural product community for studying and engineering metabolic pathways.
Keywords
- Agrobacterium tumefaciens, Agroinfiltration, Combinatorial coexpression, Eukaryotic host, Heterologous expression, Metabolic engineering, Nicotiana benthamiana, Transient expression
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
- Molecular Biology
- Biochemistry, Genetics and Molecular Biology(all)
- Genetics
Cite this
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Engineering Natural Product Biosynthesis: Methods and Protocols. 1. ed. Humana Press, 2022. p. 395-420 (Methods in molecular biology (Clifton, N.J.)).
Research output: Chapter in book/report/conference proceeding › Contribution to book/anthology › Research › peer review
}
TY - CHAP
T1 - Rapid Combinatorial Coexpression of Biosynthetic Genes by Transient Expression in the Plant Host Nicotiana benthamiana.
AU - Chuang, L
AU - Franke, J
N1 - Funding Information: We gratefully acknowledge financial support by the Emmy Noether programme of the Deutsche Forschungsgemeinschaft (FR 3720/ 3-1) and the SMART BIOTECS alliance between the Technische Universit€at Braunschweig and the Leibniz Universit€at Hannover, supported by the Ministry of Science and Culture (MWK) of Lower Saxony. We thank Yvonne Leye and Miriam Fent for excellent horticultural support. We thank Lorenzo Caputi (Max Planck Institute for Chemical Ecology, Jena) and Annika Stein for helpful cloning advice and Dave Biedermann for GC-MS support.
PY - 2022/5/7
Y1 - 2022/5/7
N2 - Metabolic engineering of heterologous hosts requires coexpression of multiple genes, often more than ten for a single pathway. Traditional approaches to create genetic constructs for this purpose are highly inflexible and suffer from very low throughput. In this book chapter, we describe a powerful method to overcome this bottleneck, namely, combinatorial co-expression in the Australian tobacco plant Nicotiana benthamiana. This system is based on Agrobacterium tumefaciens-mediated transient gene expression, often called agroinfiltration. Herein, instead of creating complex multigenic constructs, coexpression is achieved by simply mixing different plasmid-bearing Agrobacterium strains without the need for different selection markers. We present a practical guide for coexpressing multiple biosynthetic genes followed by GC-MS analysis, using production of the plant triterpene β-amyrin as an example. Our chapter provides a guideline to harness the potential of this versatile expression system in the natural product community for studying and engineering metabolic pathways.
AB - Metabolic engineering of heterologous hosts requires coexpression of multiple genes, often more than ten for a single pathway. Traditional approaches to create genetic constructs for this purpose are highly inflexible and suffer from very low throughput. In this book chapter, we describe a powerful method to overcome this bottleneck, namely, combinatorial co-expression in the Australian tobacco plant Nicotiana benthamiana. This system is based on Agrobacterium tumefaciens-mediated transient gene expression, often called agroinfiltration. Herein, instead of creating complex multigenic constructs, coexpression is achieved by simply mixing different plasmid-bearing Agrobacterium strains without the need for different selection markers. We present a practical guide for coexpressing multiple biosynthetic genes followed by GC-MS analysis, using production of the plant triterpene β-amyrin as an example. Our chapter provides a guideline to harness the potential of this versatile expression system in the natural product community for studying and engineering metabolic pathways.
KW - Agrobacterium tumefaciens
KW - Agroinfiltration
KW - Combinatorial coexpression
KW - Eukaryotic host
KW - Heterologous expression
KW - Metabolic engineering
KW - Nicotiana benthamiana
KW - Transient expression
UR - http://www.scopus.com/inward/record.url?scp=85129999577&partnerID=8YFLogxK
U2 - 10.1007/978-1-0716-2273-5_20
DO - 10.1007/978-1-0716-2273-5_20
M3 - Contribution to book/anthology
C2 - 35524061
SN - 978-1-0716-2272-8
T3 - Methods in molecular biology (Clifton, N.J.)
SP - 395
EP - 420
BT - Engineering Natural Product Biosynthesis
PB - Humana Press
ER -