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Plant Cell Environ. 2018 Jan;41(1):39-49. doi: 10.1111/pce.12901. Epub 2017 Mar 9.

Mechanistic studies of sesquiterpene cyclases based on their carbon isotope ratios at natural abundance.

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Max Planck Institute for Chemical Ecology, Beutenberg Campus, Hans-Knöll-Str. 8, 07745, Jena, Germany.


During the process of terpene biosynthesis, C-C bond breaking and forming steps are subjected to kinetic carbon isotope effects, leading to distinct carbon isotopic signatures of the products. Accordingly, carbon isotopic signatures could be used to reveal the 'biosynthetic history' of the produced terpenoids. Five known sesquiterpene cyclases, regulating three different pathways, representing simple to complex biosynthetic sequences, were heterologously expressed and used for in vitro assays with farnesyl diphosphate as substrate. Compound specific isotope ratio mass spectrometry measurements of the enzyme substrate farnesyl diphosphate (FDP) and the products of all the five cyclases were performed. The calculated δ13 C value for FDP, based on δ13 C values and relative amounts of the products, was identical with its measured δ13 C value, confirming the reliability of the approach and the precision of measurements. The different carbon isotope ratios of the products reflect the complexity of their structure and are correlated with the frequency of carbon-carbon bond forming and breaking steps on their individual biosynthetic pathways. Thus, the analysis of carbon isotopic signatures of terpenes at natural abundance can be used as a powerful tool in elucidation of associated biosynthetic mechanisms of terpene synthases and in future in vivo studies even without 'touching' the plant.


biosynthetic pathway; carbon isotope effect; carbon isotopic signatures; gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS); gas chromatography-mass spectrometry (GC-MS); sesquiterpene; terpene cyclase; δ13C value

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