CYP99A3: functional identification of a diterpene oxidase from the momilactone biosynthetic gene cluster in rice

Rice (Oryza sativa) produces momilactone diterpenoids as both phytoalexins and allelochemicals. Strikingly, the rice genome contains a biosynthetic gene cluster for momilactone production, located on rice chromosome 4, which contains two cytochromes P450 mono-oxygenases, CYP99A2 and CYP99A3, with undefined roles; although it has been previously shown that RNAi double knock-down of this pair of closely related CYP reduced momilactone accumulation. Here we attempted biochemical characterization of CYP99A2 and CYP99A3, which ultimately was achieved by complete gene recoding, enabling functional recombinant expression in bacteria. With these synthetic gene constructs it was possible to demonstrate that, while CYP99A2 does not exhibit significant activity with diterpene substrates, CYP99A3 catalyzes consecutive oxidations of the C19 methyl group of the momilactone precursor syn-pimara-7,15-diene to form, sequentially, syn-pimaradien-19-ol, syn-pimaradien-19-al and syn-pimaradien-19-oic acid. These are presumably intermediates in momilactone biosynthesis, as a C19 carboxylic acid moiety is required for formation of the core 19,6-γ-lactone ring structure. We further were able to detect syn-pimaradien-19-oic acid in rice plants, which indicates physiological relevance for the observed activity of CYP99A3. In addition, we found that CYP99A3 also oxidized synstemod- 13(17)-ene at C19 to produce, sequentially, syn-stemoden-19-ol, syn-stemoden-19-al and syn-stemoden-19-oic acid, albeit with lower catalytic efficiency than with syn-pimaradiene. Although the CYP99A3 syn-stemodene derived products were not detected in planta, these results nevertheless provide a hint at the currently unknown metabolic fate of this diterpene in rice. Regardless of any wider role, our results strongly indicate that CYP99A3 acts as a multifunctional diterpene oxidase in momilactone biosynthesis

Characterizing the Metabolic Fingerprint and Anti-inflammatory Activity of Hypericum gentianoides

In this paper we characterize the metabolic fingerprint and first reported anti-inflammatory activity of Hypericum gentianoides. H. gentianoides has a history of medical use by Native Americans, but it has been studied very little for biological activity. High-performance liquid chromatography (HPLC) and liquid chromatography−electrospray ionization−mass spectrometry (LC-ESI-MS) analyses of a methanol extract show that H. gentianoides contains a family of over nine related compounds that have retention times, mass spectra, and a distinctive UV absorption spectra characteristic of certain acyl-phloroglucinols. These metabolites are abundant relative to other secondary products present in H. gentianoides, accounting for approximately 0.2 g per gram of dry plant tissue. H. gentianoides methanol extracts and a specific semipreparative HPLC fraction from these extracts containing the putative acyl-phloroglucinols reduce prostaglandin E2 synthesis in mammalian macrophages

Combining Metabolomics and Transcriptomics to Characterize Tanshinone Biosynthesis in Salvia Miltiorrhiza

Plant natural products have been co-opted for millennia by humans for various uses such as flavor, fragrances, and medicines. These compounds often are only produced in relatively low amounts and are difficult to chemically synthesize, limiting access. While elucidation of the underlying biosynthetic processes might help alleviate these issues (e.g., via metabolic engineering), investigation of this is hindered by the low levels of relevant gene expression and expansion of the corresponding enzymatic gene families. However, the often-inducible nature of such metabolic processes enables selection of those genes whose expression pattern indicates a role in production of the targeted natural product. Here, we combine metabolomics and transcriptomics to investigate the inducible biosynthesis of the bioactive diterpenoid tanshinones from the Chinese medicinal herb, Salvia miltiorrhiza(Danshen). Untargeted metabolomics investigation of elicited hairy root cultures indicated that tanshinone production was a dominant component of the metabolic response, increasing at later time points. A transcriptomic approach was applied to not only define a comprehensive transcriptome (comprised of 20,972 non-redundant genes), but also its response to induction, revealing 6,358 genes that exhibited differential expression, with significant enrichment for up-regulation of genes involved in stress, stimulus and immune response processes. Consistent with our metabolomics analysis, there appears to be a slower but more sustained increased in transcript levels of known genes from diterpenoid and, more specifically, tanshinone biosynthesis. Among the co-regulated genes were 70 transcription factors and 8 cytochromes P450, providing targets for future investigation. Our results indicate a biphasic response of Danshen terpenoid metabolism to elicitation, with early induction of sesqui- and tri- terpenoid biosynthesis, followed by later and more sustained production of the diterpenoid tanshinones. Our data provides a firm foundation for further elucidation of tanshinone and other inducible natural product metabolism in Danshen

Characterization of an Orphan Diterpenoid Biosynthetic Operon from Salinispora arenicola

While more commonly associated with plants than microbes, diterpenoid natural products have been reported to have profound effects in marine microbe–microbe interactions. Intriguingly, the genome of the marine bacterium Salinispora arenicola CNS-205 contains a putative diterpenoid biosynthetic operon, terp1. Here recombinant expression studies are reported, indicating that this three-gene operon leads to the production of isopimara-8,15-dien-19-ol (4). Although 4 is not observed in pure cultures of S. arenicola, it is plausible that the terp1 operon is only expressed under certain physiologically relevant conditions such as in the presence of other marine organisms

Minimum Information about a Biosynthetic Gene cluster

A wide variety of enzymatic pathways that produce specialized metabolites in bacteria, fungi and plants are known to be encoded in biosynthetic gene clusters. Information about these clusters, pathways and metabolites is currently dispersed throughout the literature, making it difficult to exploit. To facilitate consistent and systematic deposition and retrieval of data on biosynthetic gene clusters, we propose the Minimum Information about a Biosynthetic Gene cluster (MIBiG) data standard.Chemistry and Chemical Biolog

Biosynthesis of Ambiguine Indole Alkaloids in Cyanobacterium <i>Fischerella ambigua</i>

Ambiguines belong to a family of hapalindole-type indole alkaloid natural products, with many of the members possessing up to eight consecutive carbon stereocenters in a fused pentacyclic 6-6-6-5-7 ring scaffold. Here, we report the identification of a 42 kbp ambiguine (<i>amb</i>) biosynthetic gene cluster that harbors 32 protein-coding genes in its native producer Fischerella ambigua UTEX1903. Association of the <i>amb</i> cluster with ambiguine biosynthesis was confirmed by both bioinformatic analysis and <i>in vitro</i> characterizations of enzymes responsible for 3-((<i>Z</i>)-2′-isocyanoethenyl) indole and geranyl pyrophosphate biosynthesis and a C-2 indole dimethylallyltransferase that regiospecifically tailors hapalindole G to ambiguine A. The presence of five nonheme iron-dependent oxygenase coding genes (including four Rieske-type oxygenases) within the <i>amb</i> cluster suggests late-stage C–H activations are likely responsible for the structural diversities of ambiguines by regio- and stereospecific chlorination, hydroxylation, epoxidation, and sp²–sp³ C–C bond formation

CYP701A8: A Rice ent-Kaurene Oxidase Paralog Diverted to More Specialized Diterpenoid Metabolism1[W][OA]

All higher plants contain an ent-kaurene oxidase (KO), as such a cytochrome P450 (CYP) 701 family member is required for gibberellin (GA) phytohormone biosynthesis. While gene expansion and functional diversification of GA-biosynthesis-derived diterpene synthases into more specialized metabolism has been demonstrated, no functionally divergent KO/CYP701 homologs have been previously identified. Rice (Oryza sativa) contains five CYP701A subfamily members in its genome, despite the fact that only one (OsKO2/CYP701A6) is required for GA biosynthesis. Here we demonstrate that one of the other rice CYP701A subfamily members, OsKOL4/CYP701A8, does not catalyze the prototypical conversion of the ent-kaurene C4α-methyl to a carboxylic acid, but instead carries out hydroxylation at the nearby C3α position in a number of related diterpenes. In particular, under conditions where OsKO2 catalyzes the expected conversion of ent-kaurene to ent-kaurenoic acid required for GA biosynthesis, OsKOL4 instead efficiently reacts with ent-sandaracopimaradiene and ent-cassadiene to produce the corresponding C3α-hydroxylated diterpenoids. These compounds are expected intermediates in biosynthesis of the oryzalexin and phytocassane families of rice antifungal phytoalexins, respectively, and can be detected in rice plants under the appropriate conditions. Thus, it appears that OsKOL4 plays a role in the more specialized diterpenoid metabolism of rice, and our results provide evidence for divergence of a KO/CYP701 family member from GA biosynthesis. This further expands the range of enzymes recruited from the ancestral GA primary pathway to the more complex and specialized labdane-related diterpenoid metabolic network found in rice