The biosynthesis of the anti‐microbial diterpenoid leubethanol in Leucophyllum frutescens proceeds via an all‐cis prenyl intermediate

Abstract

SUMMARY Serrulatane diterpenoids are natural products found in plants from a subset of genera within the figwort family (Scrophulariaceae). Many of these compounds have been characterized as having anti-microbial properties and share a common diterpene backbone. One example, leubethanol from Texas sage ( Leucophyllum frutescens ) has demonstrated activity against multi-drug-resistant tuberculosis. Leubethanol is the only serrulatane diterpenoid identified from this genus; however, a range of such compounds have been found throughout the closely related Eremophila genus. Despite their potential therapeutic relevance, the biosynthesis of serrulatane diterpenoids has not been previously reported. Here we leverage the simple product profile and high accumulation of leubethanol in the roots of L . frutescens and compare tissue-specific transcriptomes with existing data from Eremophila serrulata to decipher the biosynthesis of leubethanol. A short-chain cis -prenyl transferase ( Lf CPT1) first produces the rare diterpene precursor nerylneryl diphosphate, which is cyclized by an unusual plastidial terpene synthase ( Lf TPS1) into the characteristic serrulatane diterpene backbone. Final conversion to leubethanol is catalyzed by a cytochrome P450 (CYP71D616) of the CYP71 clan. This pathway documents the presence of a short-chain cis -prenyl diphosphate synthase, previously only found in Solanaceae, which is likely involved in the biosynthesis of other known diterpene backbones in Eremophila . Lf TPS1 represents neofunctionalization of a compartment-switching terpene synthase accepting a novel substrate in the plastid. Biosynthetic access to leubethanol will enable pathway discovery to more complex serrulatane diterpenoids which share this common starting structure and provide a platform for the production and diversification of this class of promising anti-microbial therapeutics in heterologous systems.

Biological Potential of Silver Nanoparticles Mediated by Leucophyllum frutescens and Russelia equisetiformis Extracts

“…Although not investigated herein, the chemical components of both plants were identified previously. The natural serrulatane diterpenoids are well distributed in plants from the Scrophulariaceae family [ 49 ]. About 27 and 20 chemical compounds were identified in essential oils from L. frutescens and R. equisetiformis , respectively, by the GC-MS technique [ 33 ].…”

Genome-Wide Identification and Functional Characterization of the Trans-Isopentenyl Diphosphate Synthases Gene Family in Cinnamomum camphora

“…IPP and DMAPP can be condensed (head to tail) by prenyltransferase (PTS) or isoprenyl diphosphate synthase (IDS), resulting in a series of prenyl diphosphates with various chain lengths. These linear precursors are then catalyzed by terpene synthase (TPS) and other modifying enzymes to form a variety of different terpenoids ( Tholl and Lee, 2011 ; Athanasakoglou and Grypioti, 2019 ; Johnson et al, 2019 ; Johnson and Bhat, 2019 ; Adal and Mahmoud, 2020 ; Hivert et al, 2020 ; Miller and Bhat, 2020 ).…”

“…Isoprenyl diphosphate synthase is located at the branch point of the terpenoid biosynthetic pathway, and plays a vital role in the formation of diverse terpenoid structures ( Jia and Chen, 2016 ). Differences in terpenoid synthase gene expression and the supply of precursors determine the terpenoid composition produced by plants ( Dudareva et al, 2000 ; Johnson et al, 2019 ; Johnson and Bhat, 2019 ; Adal and Mahmoud, 2020 ; Miller and Bhat, 2020 ). Both trans- and cis- isomers of the products of IDS exist ( Liang et al, 2002 ).…”

Autoxidation of a C2-Olefinated Dihydroartemisinic Acid Analogue to Form an Aromatic Ring: Application to Serrulatene Biosynthesis

“…An isomer of α-terpinene, α-phellandrine, is also known to undergo endoperoxide formation as well as multiple ene reactions to form a mixture of allylic hydroperoxides and an aromatization to form p -cymene . Dihydroserrulatene has been suggested to undergo aromatization to serrulatene (Figure B, 10 to 11 ) . Miltiradiene has been postulated to autoxidize to form the aromatized compound 13 (Figure C, 12 to 13 ) .…”

“…This study suggests that the monoalkene functional group of DHAA is a requirement for the spontaneous endoperoxide-forming reaction to occur to yield artemisinin (Figure , 1 to 2 ). Furthermore, these results have implications in enhancing the understanding of other natural products that contain aromatic rings, which are formed by diene precursors, erogorgiaene and leubethanol, two natural products possessing antibacterial properties. A group of the compounds synthesized in this study were tested and had antimicrobial activity against S. aureus and C. neoformans with MIC values at 12.5 μg/mL (Figure , compounds 27 , 28 , 32 , and 33 ); these compounds all possessed a C4-monoalkene in the bicyclic ring system, which have a different three-dimensional structure compared to the 2,4-diene (Figure ) and the aromatic ring systems.…”