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 -