The terpene synthase gene family in Tripterygium wilfordii harbors a labdane‐type diterpene synthase among the monoterpene synthase TPS‐b subfamily

Abstract

SUMMARYTripterygium wilfordii (Celastraceae) is a medicinal plant with anti-inflammatory and immunosuppressive properties. Identification of a vast array of unusual sesquiterpenoids, diterpenoids and triterpenoids in T. wilfordii has spurred investigations of their pharmacological properties. The tri-epoxide lactone triptolide was the first of many diterpenoids identified, attracting interest due to the spectrum of bioactivities. To probe the genetic underpinning of diterpenoid diversity, an expansion of the class II diterpene synthase (diTPS) family was recently identified in a leaf transcriptome. Following detection of triptolide and simple diterpene scaffolds in the root, we sequenced and mined the root transcriptome. This allowed identification of the root-specific complement of TPSs and an expansion in the class I diTPS family. Functional characterization of the class II diTPSs established their activities in the formation of four C-20 diphosphate intermediates, precursors of both generalized and specialized metabolism and a novel scaffold for Celastraceae. Functional pairs of the class I and II enzymes resulted in formation of three scaffolds, accounting for some of the terpenoid diversity found in T. wilfordii. The absence of activity-forming abietane-type diterpenes encouraged further testing of TPSs outside the canonical class I diTPS family. TwTPS27, close relative of mono-TPSs, was found to couple with TwTPS9, converting normal-copalyl diphosphate to miltiradiene. The phylogenetic distance to established diTPSs indicates neo-functionalization of TwTPS27 into a diTPS, a function not previously observed in the TPS-b subfamily. This example of evolutionary convergence expands the functionality of TPSs in the TPS-b family and may contribute miltiradiene to the diterpenoids of T. wilfordii.

Biosynthesis of Diterpenoids in Tripterygium Adventitious Root Cultures

“…The previously reported metabolites can be grouped into two major classes: four-ring ent-kauranoic acids/alcohols and five-ring oxygen bridge-containing ent-kauranes (Duan et al, 1999(Duan et al, , 2001Tanaka et al, 2004). In the closely related species T. wilfordii, recent studies also identified an ent-CPS (TwTPS3) and several isoforms of entkaurene/ent-isokaurene synthase (TwTPS2, TwTPS16, TwTPS17, and TwTPS18; Zerbe et al, 2013;Hansen et al, 2017). Various biological activities of Tripterygium ent-kauranes have been documented in in vitro assays (for review, see Brinker et al, 2007), but the in vivo Figure 3.…”

“…Genes from T. wilfordii were characterized previously as coding for ent-copal-8-ol diphosphate synthase (termed TwTPS21) or kolavenyl diphosphate synthase (TwTPS10, TwTPS14, and TwTPS28; AndersenRanberg et al, 2016;Hansen et al, 2017). However, the stereochemistry at C8 and C9 (8S, 9S) is the opposite of that of all labdane-type diterpenoids characterized from Tripterygium thus far (8R, 9R; Duan et al, 1999Duan et al, , 2001).…”

“…Based on phylogenetic analyses, Hansen et al (2017) concluded that TwTPS27 diversified from members of the TPS-b subfamily, more specifically from terpene synthases that catalyze the formation of acyclic monoterpenes. The location of TwTPS27 on the phylogenetic tree of angiosperm terpene synthases was a surprise because all angiosperm diterpene synthases involved in abietane/labdane biosynthesis characterized until then belonged to the TPS-e/f subfamily (Chen et al, 2011).…”

“…This evolutionary history also applies to TrTPS8, which is very closely related to TwTPS27. Hansen et al (2017) expressed TwTPS23, TwTPS24, and TwTPS26 transiently in Nicotiana benthamiana and then subjected volatiles to a head-space analysis. The authors did not have authentic standards for monoterpenes and, therefore, could only use spectral comparisons for tentative identification.…”

“…Several diterpene synthases, some of which are relevant to triptolide formation, have now been characterized in T. wilfordii (Zerbe et al, 2013;Andersen-Ranberg et al, 2016;Hansen et al, 2017). However, the remaining genes involved in the biosynthesis of the highly functionalized triptolide structure have remained enigmatic.…”