Molecular identification and expression of sesquiterpene pathway genes responsible for patchoulol biosynthesis and regulation in Pogostemon cablin

Abstract

Background Many commercially important drug and flavor compounds are secondary metabolites of terpenoid origin. Pogostemon cablin , a commercially important industrial and medicinal crop, accumulates abundant patchouli oil comprised of more than 24 unique sesquiterpene compounds, with the most abundant being patchouli alcohol. Results In this study, we analyzed the P. cablin transcriptome library, obtaining 74 terpenoid biosynthesis-related genes, and identified their expression patterns in leaves, stems, and flowers. These genes are members of 15 different families, and we detected all the enzymes involved in the sesquiterpenes pathway that are responsible for patchoulol biosynthesis. Sequence structure, homology, conserved domain properties, and phylogeny of certain identified genes were systematically investigated. Color complementation assay was used to verify the functional activity of the MEP pathway proteins. Exogenous hormone treatment revealed that patchoulol synthesis is induced by methyl jasmonate (MeJA). Quantitative reverse-transcription PCR analysis indicated that the MVA pathway genes (acetoacetyl-CoA thiolase, 3-hydroxy-3-methylglutaryl-coenzyme A reductase, mevalonate diphosphate decarboxylase, and farnesyl diphosphate synthase) participate in patchoulol biosynthesis and are mediated by MeJA. Conclusions Taken together, this is the first report of integrated analysis of P. cablin MVA and MEP pathway related genes, providing a better understanding of terpenoid and/or patchoulol biosynthesis in P. cablin, and the basis for improving patchoulol production through genetic engineering. Electronic supplementary material The online version of this article (10.1186/s40529-019-0259-9) contains supplementary material, which is available to authorized users.

Optimization of factors influencing enzyme activity and product selectivity and the role of proton transfer in the catalytic mechanism of patchoulol synthase

“…The reaction mechanism of (−)‐patchoulol formation by the PTS has not yet been clarified in detail . More particularly, there is no proof in the literature on further reprotonation steps as postulated by Faraldos et al Because the formation of PTS products is obviously influenced by the pH‐value, it is possible that protons from the reaction solution are directly involved in the mechanism of terpene formation.…”

“…Optimization of pH during bioconversion was tested by measuring product ratio with optimal activity buffer (50 mM MES, 10 mM MgCl 2 , 10% (v/v) glycerol) at pH 5.6, 5.8, 6.0, 6.2, 6.4, and 6.6 and 38 C. After evaluation of optimal pH, the biotransformation was analyzed for optimal temperature with optimal activity buffer (50 mM MES, 10 mM MgCl 2 , 10% (v/v) glycerol, pH 6.4) at 30,32,34,36,38,40,42, and 44 C. For each measuring point a glass vial was filled with activity buffer, substrate (FPP = 30 μM) and 0.5 μM enzyme to a total volume of 500 μl, overlaid with 150 μl of isooctane and incubated for 10 min in a water bath. After stopping reaction by shaking for 30 s, reaction volume was centrifuged, 80 μl of the organic phase was transferred to a GC vial and measured in GC-FID (see…”

“…The reaction mechanism of (−)-patchoulol formation by the PTS has not yet been clarified in detail. 32 FPP for their experiments to establish the reaction mechanism. 14 The use of deuterated water for the reaction buffer instead allows to study the influence of protons from the reaction medium in the reaction mechanism.…”

Transcriptome analysis of two Pogostemon cablin chemotypes reveals genes related to patchouli alcohol biosynthesis

“… Chen et al (2020) found that overexpression of PatMYB46 in P. cablin could increase the content of PA. Li et al (2021) found that overexpression of PatGT-1 in P. cablin could reduce the content of PA and revealed that PatGT-1 negatively regulates PA biosynthesis. Recently, the molecular dynamics of P. cablin have been gradually revealed, especially the genes controlling the synthesis of PA ( Tang et al, 2019 ). However, there is limited information on the regulation of its TFs to date, especially PA biosynthesis in P. cablin .…”

PatJAZ6 Acts as a Repressor Regulating JA-Induced Biosynthesis of Patchouli Alcohol in Pogostemon Cablin

“…Patchouli alcohol, a natural tricyclic sesquiterpene compound that is a bioactive ingredient in P. cablin [21], is used worldwide in decorative cosmetics, toilet soaps, perfume industries [22] and medical treatments [23]. With the increasing market demand in the world, a metabolic engineering approach has been considered an effective approach to increase useful metabolites in medical plants, and several strategies have been reported to promote the industrialization process of patchouli alcohol production using Saccharomyces cerevisiae [24], and gene isolation and cloning in MVA and MEP pathways participating in patchouli alcohol biosynthesis have been reported [25]. In addition, full-length transcriptome data reported provide a valuable genetic resource in P. cablin [18].…”