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Plant J. 2015 Nov;84(3):478-90. doi: 10.1111/tpj.13012.

Identification and genome organization of saponin pathway genes from a wild crucifer, and their use for transient production of saponins in Nicotiana benthamiana.

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Department of Plant and Environmental Sciences, Copenhagen Plant Science Center, University of Copenhagen, Thorvaldsensvej 40, Frederiksberg C, DK-1871, Denmark.
Department of Food Science, University of Copenhagen, Rolighedsvej 26, Frederiksberg, DK-1958, Denmark.
Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Osaka, 565-0871, Japan.
EMBL, Heidelberg, Germany.
Keygene, Agro Business Park 90, Wageningen, 6708 PW, The Netherlands.
Danziger Innovations, Mishmar HaShiva, 5029700, Israel.


The ability to evolve novel metabolites has been instrumental for the defence of plants against antagonists. A few species in the Barbarea genus are the only crucifers known to produce saponins, some of which make plants resistant to specialist herbivores, like Plutella xylostella, the diamondback moth. Genetic mapping in Barbarea vulgaris revealed that genes for saponin biosynthesis are not clustered but are located in different linkage groups. Using co-location with quantitative trait loci (QTLs) for resistance, transcriptome and genome sequences, we identified two 2,3-oxidosqualene cyclases that form the major triterpenoid backbones. LUP2 mainly produces lupeol, and is preferentially expressed in insect-susceptible B. vulgaris plants, whereas LUP5 produces β-amyrin and α-amyrin, and is preferentially expressed in resistant plants; β-amyrin is the backbone for the resistance-conferring saponins in Barbarea. Two loci for cytochromes P450, predicted to add functional groups to the saponin backbone, were identified: CYP72As co-localized with insect resistance, whereas CYP716As did not. When B. vulgaris sapogenin biosynthesis genes were transiently expressed by CPMV-HT technology in Nicotiana benthamiana, high levels of hydroxylated and carboxylated triterpenoid structures accumulated, including oleanolic acid, which is a precursor of the major resistance-conferring saponins. When the B. vulgaris gene for sapogenin 3-O-glucosylation was co-expressed, the insect deterrent 3-O-oleanolic acid monoglucoside accumulated, as well as triterpene structures with up to six hexoses, demonstrating that N. benthamiana further decorates the monoglucosides. We argue that saponin biosynthesis in the Barbarea genus evolved by a neofunctionalized glucosyl transferase, whereas the difference between resistant and susceptible B. vulgaris chemotypes evolved by different expression of oxidosqualene cyclases (OSCs).


Barbarea vulgaris; Nicotiana benthamiana; P450; UDP-glycosyltransferases; evolution; metabolic engineering; oxidosqualene cyclases; synthetic biology

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