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Research article - Peer-reviewed, 2020

Transient expression and purification of beta-caryophyllene synthase in Nicotiana benthamiana to produce beta-caryophyllene in vitro

Muthusamy, Saraladevi; Vetukuri, Ramesh R.; Lundgren, Anneli; Ganji, Suresh; Zhu, Li-Hua; Brodelius, Peter E.; Kanagarajan, Selvaraju


The sesquiterpene beta-caryophyllene is an ubiquitous component in many plants that has commercially been used as an aroma in cosmetics and perfumes. Recent studies have shown its potential use as a therapeutic agent and biofuel. Currently, beta-caryophyllene is isolated from large amounts of plant material. Molecular farming based on the Nicotiana benthamiana transient expression system may be used for a more sustainable production of beta-caryophyllene. In this study, a full-length cDNA of a new duplicated beta-caryophyllene synthase from Artemisia annua (AaCPS1) was isolated and functionally characterized. In order to produce beta-caryophyllene in vitro, the AaCPS1 was cloned into a plant viral-based vector pEAQ-HT. Subsequently, the plasmid was transferred into the Agrobacterium and agroinfiltrated into N. benthamiana leaves. The AaCPS1 expression was analyzed by quantitative PCR at different time points after agroinfiltration. The highest level of transcripts was observed at 9 days post infiltration (dpi). The AaCPS1 protein was extracted from the leaves at 9 dpi and purified by cobalt-nitrilotriacetate (Co-NTA) affinity chromatography using histidine tag with a yield of 89 mg kg(-1). fresh weight of leaves. The protein expression of AaCPS1 was also confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analyses. AaCPS1 protein uses farnesyl diphosphate (FPP) as a substrate to produce p-caryophyllene. Product identification and determination of the activity of purified AaCPS1 were done by gas chromatography-mass spectrometry (GC-MS). GC-MS results revealed that the AaCPS1 produced maximum 26.5 +/- 1 mg of P-caryophyllene per kilogram fresh weight of leaves after assaying with FPP for 6 h. Using AaCPS1 as a proof of concept, we demonstrate that N. benthamiana can be considered as an expression system for production of plant proteins that catalyze the formation of valuable chemicals for industrial applications.


Artemisia annua; Sesquiterpene synthase; AaCPS1; Phylogenetics; Terpenoids; Nicotiana benthamiana; Transient expression; beta-caryophyllene synthase

Published in

2020, volume: 8, article number: e8904
Publisher: PEERJ INC

Authors' information

Muthusamy, Saraladevi
Linnaeus Univ
Vetukuri, Ramesh (Ramesh, Vetukuri)
Swedish University of Agricultural Sciences, Department of Plant Breeding
Lundgren, Anneli
Linnaeus Univ
Ganji, Suresh
Linnaeus Univ
Swedish University of Agricultural Sciences, Department of Plant Breeding
Brodelius, Peter E.
Linnaeus Univ
Linnaeus University
Swedish University of Agricultural Sciences, Department of Plant Breeding

Sustainable Development Goals

SDG12 Ensure sustainable consumption and production patterns

UKÄ Subject classification

Biochemistry and Molecular Biology

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