Optimization of Biochemical Screening Methods for Volatile and Unstable Sesquiterpenoids Using HS-SPME-GC-MS

Abstract

HS-SPME-GC-MS has been suggested as a fast and robust analytical platform for the product characterization of sesquiterpene synthases. The choice of fiber and injection temperature can have a significant effect on the observed product profile, due to the chemical rearrangements that can occur on the fiber material. Here we present a systematic study on the effects of fiber choice and injection port temperature on the observed sesquiterpenoid profile of four sesquiterpene synthases expressed in Nicotiana benthamiana. We found that the absorbent material PDMS was much less likely to support acid-induced rearrangement of sesquiterpenoids when compared to the adsorbent materials PDMS/DVB, PDMS/CAR, and PDMS/CAR/DVB. Furthermore, utilizing an injection port temperature at 160 °C almost eliminated the inherent thermal instability of germacrene sesquiterpenoids. Thus, for fast screening of sesquiterpene synthases, the results suggest that PDMS fibers and an injection temperature of 160 °C provide a fast and reproducible HS-SPME GC-MS method when using H2 as carrier gas.

Additional diterpenes from Physcomitrella patens synthesized by copalyl diphosphate/kaurene synthase ( Pp CPS/KS)

“…These detection patterns may reflect a volatile nature of 1, 2 and a less volatile nature 5 at 60°C (temperature used during fiber exposure). Our results highlight the importance of using complementary methods to reveal metabolites with different volatility and further supports the use of HS-SPME as a valuable tool for discovery and characterisation of volatile metabolites [16,17]. A lack of head-space sampling in former studies of PpCPS/KS may explain why 1, 2 have not previously been found [5,6].…”

“…Further supporting that non-enzymatic conversion of terpenoids can occur, a recent study has demonstrated instability of another hydroxylated terpene synthase product [17]. More studies are needed to determine whether 3 is a true enzymatic product of PpCPS/KS and what the rate of conversion of 5 to 3 is.…”

“…Cultivation conditions were 16 hour light/8 hour dark cycle at 25°C. After 2-3 weeks cultivation, the headspace volatiles of the WT and KO line were sampled by incubating a SPME fibre (DVB/CAR/PDMS; Supelco 57298-U) in the vials for 30 min at room temperature directly a rack on the CTC AOC-5000 auto sampler and analysed by GC-MS [17].…”

“…for 20 min at 60˚C [17]. In addition, liquid extractions of two leaf discs (ø=3cm) with 1 ml hexane were done in capped glass vials by gentle shaking for 30 min at room temperature.…”

“…GCMS analysis utilizing SPME fibers was previously published [16,17], but briefly the injection port temperature was set to 230°C, with a sampling time of 1 min. The flow control mode was pressure control with a total flow of 2.3 mL/min with H 2 as carrier gas, and a purge flow of 1.0 mL/min.…”