Abstract:Plant‐derived diterpenoids serve as important pharmaceuticals, food additives, and fragrances, yet their low natural abundance and high structural complexity limits their broader industrial utilization. By mimicking the modularity of diterpene biosynthesis in plants, we constructed 51 functional combinations of class I and II diterpene synthases, 41 of which are “new‐to‐nature”. Stereoselective biosynthesis of over 50 diterpene skeletons was demonstrated, including natural variants and novel enantiomeric or di… Show more
“…This secondary product is likely resulting from thermal degradation of copalol during GC-MS analysis as previously observed in transient expression analyses of other class II diTPSs [21, 24, 37, 46]. In contrast to IrTPS5, sequential activity of IrTPS3 was only detected with MvELS yielding miltiradiene (product e), whereas no product formation was detected after co-expression with GrEKS (Fig 3B).…”
Plants produce an immense diversity of natural products (i.e. secondary or specialized metabolites) that offer a rich source of known and potentially new pharmaceuticals and other desirable bioproducts. The Traditional Chinese Medicinal plant Isodon rubescens (Lamiaceae) contains an array of bioactive labdane-related diterpenoid natural products. Of these, the ent-kauranoid oridonin is the most prominent specialized metabolite that has been extensively studied for its potent antimicrobial and anticancer efficacy. Mining of a previously established transcriptome of I. rubescens leaf tissue identified seven diterpene synthase (diTPSs) candidates. Here we report the functional characterization of four I. rubescens diTPSs. IrTPS5 and IrTPS3 were identified as an ent-copalyl diphosphate (CPP) synthase and a (+)-CPP synthase, respectively. Distinct transcript abundance of IrTPS5 and the predicted ent-CPP synthase IrTPS1 suggested a role of IrTPS5 in specialized ent-kaurene metabolism possibly en route to oridonin. Nicotiana benthamiana co-expression assays demonstrated that IrTPS4 functions sequentially with IrTPS3 to form miltiradiene. In addition, IrTPS2 converted the IrTPS3 product (+)-CPP into the hydroxylated tricyclic diterpene nezukol not previously identified in I. rubescens. Metabolite profiling verified the presence of nezukol in I. rubescens leaf tissue. The proposed IrTPS2-catalyzed reaction mechanism proceeds via the common ionization of the diphosphate group of (+)-CPP, followed by formation of an intermediary pimar-15-en-8-yl+ carbocation and neutralization of the carbocation by water capture at C-8 to yield nezukol, as confirmed by nuclear magnetic resonance (NMR) analysis. Oxygenation activity is rare for the family of class I diTPSs and offers new catalysts for developing metabolic engineering platforms to produce a broader spectrum of bioactive diterpenoid natural products.
“…This secondary product is likely resulting from thermal degradation of copalol during GC-MS analysis as previously observed in transient expression analyses of other class II diTPSs [21, 24, 37, 46]. In contrast to IrTPS5, sequential activity of IrTPS3 was only detected with MvELS yielding miltiradiene (product e), whereas no product formation was detected after co-expression with GrEKS (Fig 3B).…”
Plants produce an immense diversity of natural products (i.e. secondary or specialized metabolites) that offer a rich source of known and potentially new pharmaceuticals and other desirable bioproducts. The Traditional Chinese Medicinal plant Isodon rubescens (Lamiaceae) contains an array of bioactive labdane-related diterpenoid natural products. Of these, the ent-kauranoid oridonin is the most prominent specialized metabolite that has been extensively studied for its potent antimicrobial and anticancer efficacy. Mining of a previously established transcriptome of I. rubescens leaf tissue identified seven diterpene synthase (diTPSs) candidates. Here we report the functional characterization of four I. rubescens diTPSs. IrTPS5 and IrTPS3 were identified as an ent-copalyl diphosphate (CPP) synthase and a (+)-CPP synthase, respectively. Distinct transcript abundance of IrTPS5 and the predicted ent-CPP synthase IrTPS1 suggested a role of IrTPS5 in specialized ent-kaurene metabolism possibly en route to oridonin. Nicotiana benthamiana co-expression assays demonstrated that IrTPS4 functions sequentially with IrTPS3 to form miltiradiene. In addition, IrTPS2 converted the IrTPS3 product (+)-CPP into the hydroxylated tricyclic diterpene nezukol not previously identified in I. rubescens. Metabolite profiling verified the presence of nezukol in I. rubescens leaf tissue. The proposed IrTPS2-catalyzed reaction mechanism proceeds via the common ionization of the diphosphate group of (+)-CPP, followed by formation of an intermediary pimar-15-en-8-yl+ carbocation and neutralization of the carbocation by water capture at C-8 to yield nezukol, as confirmed by nuclear magnetic resonance (NMR) analysis. Oxygenation activity is rare for the family of class I diTPSs and offers new catalysts for developing metabolic engineering platforms to produce a broader spectrum of bioactive diterpenoid natural products.
“…In root cork extracts, ferruginol was detected together with dehydroabietadiene. Presence of ferruginol was confirmed by comparison to an authentic standard (Ignea et al, 2016a), while presence of miltiradiene ( B ) and dehydroabietadiene ( C ) were confirmed by comparison of m/z spectra with previously characterized compounds (Andersen-Ranberg et al, 2016). For every combination, extracts from leaves of three different N. benthamiana plants have been analyzed and representative chromatograms are shown. DOI:
http://dx.doi.org/10.7554/eLife.23001.01710.7554/eLife.23001.018Figure 7.GC-MS analysis of 13 R -manoyl oxide-derived diterpenoids obtained by transient expression of CYP76AHs in N. benthamiana leaves.( A ) Total ion chromatograms (TIC) of extracts transiently expressing CfCXS, CfGGPPS, CfTPS2 and CfTPS3 (13 R -manoyl oxide biosynthesis genes) in combination with water (-), CfCYP76AH15, RoCYP76AH4, RoFS1 and SpFS are shown.…”
Section: Resultsmentioning
confidence: 87%
“…Utilizing this system, all candidate P450 genes were heterologously expressed in combination with genes necessary for the production of high amounts of 13 R -manoyl oxide (Andersen-Ranberg et al, 2016; Pateraki et al, 2014). Of the CYPs tested, six efficiently converted 13 R -manoyl oxide into oxygenated derivatives (Figures 2A and 3 and Figure 3—figure supplements 1 and 2).…”
Section: Resultsmentioning
confidence: 99%
“…A comparison of the total intermediate profiles of C. forskohlii root cork versus the fermenter grown EVST21543 yeast strain is shown in (Figure 10—figure supplement 1). 10.7554/eLife.23001.021Figure 10.Forskolin production in S. cerevisiae following stable genomic integration of codon-optimized C.forskohlii genes.( A ) Forskolin ( 16c ) accumulation in a fermenter batch using the EVST21543 strain (expressing Cf CYP76AH15, Cf CYP76HA11, Cf CYP76AH16 and Cf ACT1-8 encoding genes in the EFSC4498 S. cerevisiae strain, optimized for the production of 13 R -manoyl oxide [Andersen-Ranberg et al, 2016]). ( B ) 13 R -manoyl oxide ( 1 ) accumulation in EVST21543 strain.…”
Section: Resultsmentioning
confidence: 99%
“…( A ) Scheme showing the structures of the diterpene precursor 13 R -manoyl oxide, deacetylforskolin and forskolin on a background of root cork cells with forskolin containing oil bodies. ( B ) Transcript profiles of biosynthetic candidate genes in selected tissues of C. forskohlii as shown on the illustrations. DOI:
http://dx.doi.org/10.7554/eLife.23001.00310.7554/eLife.23001.004cDNAs identified in the C. forskohlii root cork transcriptome and cloned during this work, with the GeneBank accession numbers. DOI:
http://dx.doi.org/10.7554/eLife.23001.00410.7554/eLife.23001.005Table of FPKM (Fragments Per Kilobase of transcript per Million mapped reads) values of the first 20 most abundant cDNAs identified in the root cork transcriptome library.cDNAs involved in terpenoid metabolism are marked in bold. DOI:
http://dx.doi.org/10.7554/eLife.23001.00510.7554/eLife.23001.006Table of primers used in this study.Construction of plasmids for expression of Cf TPS2, Cf TPS3, Cf TPS1 is described in Andersen-Ranberg et al (2016). U (uracil, marked in bold), represents the cleavage site, used in the USER cloning (Nour-Eldin et al, 2006).…”
Forskolin is a unique structurally complex labdane-type diterpenoid used in the treatment of glaucoma and heart failure based on its activity as a cyclic AMP booster. Commercial production of forskolin relies exclusively on extraction from its only known natural source, the plant Coleus forskohlii, in which forskolin accumulates in the root cork. Here, we report the discovery of five cytochrome P450s and two acetyltransferases which catalyze a cascade of reactions converting the forskolin precursor 13R-manoyl oxide into forskolin and a diverse array of additional labdane-type diterpenoids. A minimal set of three P450s in combination with a single acetyl transferase was identified that catalyzes the conversion of 13R-manoyl oxide into forskolin as demonstrated by transient expression in Nicotiana benthamiana. The entire pathway for forskolin production from glucose encompassing expression of nine genes was stably integrated into Saccharomyces cerevisiae and afforded forskolin titers of 40 mg/L.DOI:
http://dx.doi.org/10.7554/eLife.23001.001
Die Substratflexibilität von acht rekombinant erzeugten Sesquiterpencyclasen wurde unter Anwendung von sechs neuen Heteroatom‐modifizierten Farnesylpyrophosphaten untersucht. Als Ergebnis werden sechs neue makro‐ oder tricyclische, Heteroatom‐modifizierte Sesquiterpene beschrieben. Die GC‐O‐Analyse zeigte, dass das tricyclische Tetrahydrofuran etherische, pfeffrige und campherartige Geruchsnoten aufweist.
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