Biosynthesis of Diterpenoids in Tripterygium Adventitious Root Cultures

Abstract

ORCID IDs: 0000-0003-4828-958X (M.H.); 0000-0001-7934-7987 (N.S.); 0000-0002-4080-8608 (A.N.P.); 0000-0003-4691-8477 (R.J.P.); 0000-0001-6565-9584 (B.M.L.).Adventitious root cultures were developed from Tripterygium regelii, and growth conditions were optimized for the abundant production of diterpenoids, which can be collected directly from the medium. An analysis of publicly available transcriptome data sets collected with T. regelii roots and root cultures indicated the presence of a large gene family (with 20 members) for terpene synthases (TPSs). Nine candidate diterpene synthase genes were selected for follow-up functional evaluation, of which two belonged to the TPS-c, three to the TPS-e/f, and four to the TPS-b subfamilies. These genes were characterized by heterologous expression in a modular metabolic engineering system in Escherichia coli. Members of the TPS-c subfamily were characterized as copalyl diphosphate (diterpene) synthases, and those belonging to the TPS-e/f subfamily catalyzed the formation of precursors of kaurane diterpenoids. The TPS-b subfamily encompassed genes coding for enzymes involved in abietane diterpenoid biosynthesis and others with activities as monoterpene synthases. The structural characterization of diterpenoids accumulating in the medium of T. regelii adventitious root cultures, facilitated by searching the Spektraris online spectral database, enabled us to formulate a biosynthetic pathway for the biosynthesis of triptolide, a diterpenoid with pharmaceutical potential. Considering the significant enrichment of diterpenoids in the culture medium, fast-growing adventitious root cultures may hold promise as a sustainable resource for the large-scale production of triptolide.

Identification and functional characterization of diterpene synthases for triptolide biosynthesis from Tripterygium wilfordii

“…These were found to consist of not only the expected class II diterpene cyclases that produce CPP, termed here TwTPS7v2 and TwTPS9v2, but also the unusual finding of a subsequently acting class I diterpene synthase, TwTPS27v2, which is not from the TPS-e subfamily that typically provides such enzymes in labdane-related diterpenoid biosynthesis (Zi et al, 2014), rather being derived from the phylogenetically distinct TPS-b subfamily instead. While similar biochemical characterization has been very recently reported for both presumably allelic copies of these T. wilfordii diterpene synthases (Hansen et al, 2017a), as well as the orthologs from T. regelii (Inabuy et al, 2017), the data reported here go beyond this to unambiguously demonstrate the role of the characterized enzymes in triptolide biosynthesis. Most critically, in addition to showing subcellular localization of these enzymes to the plastid, where such diterpene biosynthesis occurs in planta, this includes genetic evidence, with RNAi knock-down of these genes leading to reduced triptolide levels.…”

“…The ability of T. wilfordii suspension cell cultures to produce valuable terpenoids, particularly the abeo ‐abietane tri‐expoxide triptolide, has long been appreciated (Kutney et al ., ). The suspension cell cultures used here constitutively produce 53 ± 3 μg g −1 of triptolide in the suspension cells and 4.0 ± 0.2 mg l −1 in the medium, which is slightly lower than that of recently reported Tripterygium adventitious root cultures (90 ± 80 μg g −1 in the adventitious roots and 4.7 ± 0.9 mg l −1 in the medium; Inabuy et al ., ). Here the utility of these cell cultures for investigation of triptolide biosynthesis was first demonstrated by not only finding that miltiradiene can be detected therein but also that feeding this olefin leads to increased accumulation, as well as the presence of several other plausible biosynthetic intermediates.…”

“…To further investigate the potential role of miltiradiene in triptolide biosynthesis, this abietane Figure 1. Diterpenoid biosynthetic network in Tripterygium wilfordii as elucidated by the work described here and others (Forman et al, 2017;Inabuy et al, 2017). [Colour figure can be viewed at wileyonlinelibrary.com].…”

“…Diterpenoid biosynthetic network in Tripterygium wilfordii as elucidated by the work described here and others (Forman et al ., ; Inabuy et al ., ). [Colour figure can be viewed at wileyonlinelibrary.com].…”

Friedelane‐type triterpene cyclase in celastrol biosynthesis from Tripterygium wilfordii and its application for triterpenes biosynthesis in yeast

“…However, the biosynthetic pathway of celastrol remains unknown. The pharmaceutical potential of triptolide has led many researchers to investigate the biosynthetic pathway of this compound (Forman et al ., ; Hansen et al ., ; Inabuy et al ., ; Su et al ., ), but there have been few reports on the biosynthetic pathway of celastrol.…”