Identification of quantitative trait loci influencing foliar concentrations of terpenes and formylated phloroglucinol compounds in Eucalyptus nitens

Leaves of eucalypt species contain a variety of plant secondary metabolites, including terpenoids and formylated phloroglucinol compounds (FPCs). Both terpene and FPC concentrations are quantitative traits that can show large variation within a population and have been shown to be heritable. The molecular genetic basis of this variation is currently unknown. Progeny from a field trial of a three-generation mapping pedigree of Eucalyptus nitens were assayed for terpenes and FPCs. Quantitative trait loci (QTL) analyses were conducted using a map constructed from 296 markers to locate regions of the genome influencing foliar concentrations of these plant secondary compounds. A large number of significant QTL for 14 traits were located across nine linkage groups, with significant clustering of QTL on linkage groups 7, 8 and 9. As expected, QTL for biosynthetically related compounds commonly colocated, but QTL for unrelated monterpenes and FPCs also mapped closely together. Colocation of these QTL with mapped candidate genes from the various biosynthetic pathways, and subsequent use of these genes in association mapping, will assist in determining the causes of variation in plant secondary metabolites in eucalypts.     

Gut Symbionts from Distinct Hosts Exhibit Genotoxic Activity via Divergent Colibactin Biosynthesis Pathways

Secondary metabolites produced by nonribosomal peptide synthetase (NRPS) or polyketide synthase (PKS) pathways are chemical mediators of microbial interactions in diverse environments. However, little is known about their distribution, evolution, and functional roles in bacterial symbionts associated with animals. A prominent example is colibactin, a largely unknown family of secondary metabolites produced by Escherichia coli via a hybrid NRPS-PKS biosynthetic pathway that inflicts DNA damage upon eukaryotic cells and contributes to colorectal cancer and tumor formation in the mammalian gut. Thus far, homologs of this pathway have only been found in closely related Enterobacteriaceae, while a divergent variant of this gene cluster was recently discovered in a marine alphaproteobacterial Pseudovibrio strain. Herein, we sequenced the genome of Frischella perrara PEB0191, a bacterial gut symbiont of honey bees and identified a homologous colibactin biosynthetic pathway related to those found in Enterobacteriaceae. We show that the colibactin genomic island (GI) has conserved gene synteny and biosynthetic module architecture across F. perrara, Enterobacteriaceae, and the Pseudovibrio strain. Comparative metabolomics analyses of F. perrara and E. coli further reveal that these two bacteria produce related colibactin pathway-dependent metabolites. Finally, we demonstrate that F. perrara, like E. coli, causes DNA damage in eukaryotic cells in vitro in a colibactin pathway-dependent manner. Together, these results support that divergent variants of the colibactin biosynthetic pathway are widely distributed among bacterial symbionts, producing related secondary metabolites and likely endowing its producer with functional capabilities important for diverse symbiotic associations.     

植物萜类次生代谢及其调控

植物次生代谢在植物生长发育、环境适应、抵御病虫害等方面发挥着重要作用,这些天然产物组成地球上最丰富的有机化合物的宝库．萜类是植物代谢产物中种类最多的一类,具有重要的生理和生态功能,一些成分还有应用价值．近十几年来,人们在萜类化合物的分离、鉴定、应用、生物合成、相关基因与基因族、酶蛋白结构和功能、代谢调控以及代谢工程等各方面取得了重大进展．本文概述了植物萜类化合物代谢及其调控领域的研究进展与发展趋势．     

Natural products and enzymes from plant cell cultures

Plants represent an unlimited source of natural products. Many of the recently detected phytochemicals exhibit remarkable bioactivities, ranging from anticancer activity, phosphodiesterase inhibition to cytotoxicity against HIV-infected cells. Cultivated plant cells produce at their unorganized, dedifferentiated stage secondary metabolites, but in very different amounts in so far as new compounds are concerned. In fact, more than 140 novel natural products are presently known from plant cell cultures, which also include new metabolites formed by biotransformation. The biotransformation capacity of suspended cells is described and recent high yielding transformations, like the formation of arbutin by hydroquinone-transformation withRauwolfia cells are discussed. As an example of alkaloid production by cell suspensions, the pattern of monoterpenoid indole alkaloids of the Indian medicinal plantRauwolfia serpentina Benth. is described and the so far 30 identified compounds are divided into eight groups which are biosynthetically closely related. Some of the key biosynthetic reactions leading to theRauwolfia alkaloids are discussed and an overview of the enzymes involved in the formation of the alkaloid ajmaline and proteins catalyzing side reactions of the ajmaline pathway are given.     

Secondary transport as an efficient membrane transport mechanism for plant secondary metabolites

Plants produce a large number of secondary metabolites, such as alkaloids, terpenoids, and phenolic compounds. Secondary metabolites have various functions including protection against pathogens and UV light in plants, and have been used as natural medicines for humans utilizing their diverse biological activities. Many of these natural compounds are accumulated in a particular compartment such as vacuoles, and some are even translocated from source cells to sink organs via long distance transport. Both primary and secondary transporters are involved in such compartmentation and translocation, and many transporter genes, especially genes belonging to the multidrug and toxin extrusion type transporter family, which consists of 56 members in Arabidopsis, have been identified as responsible for the membrane transport of secondary metabolites. Better understandings of these transporters as well as the biosynthetic genes of secondary metabolites will be important for metabolic engineering aiming to increase the production of commercially valuable secondary metabolites in plant cells.     

Genome mining of Streptomyces ambofaciens

Since the discovery of the streptomycin produced by Streptomyces griseus in the middle of the last century, members of this bacterial genus have been largely exploited for the production of secondary metabolites with wide uses in medicine and in agriculture. They have even been recognized as one of the most prolific producers of natural products among microorganisms. With the onset of the genomic era, it became evident that these microorganisms still represent a major source for the discovery of novel secondary metabolites. This was highlighted with the complete genome sequencing of Streptomyces coelicolor A3(2) which revealed an unexpected potential of this organism to synthesize natural products undetected until then by classical screening methods. Since then, analysis of sequenced genomes from numerous Streptomyces species has shown that a single species can carry more than 30 secondary metabolite gene clusters, reinforcing the idea that the biosynthetic potential of this bacterial genus is far from being fully exploited. This review highlights our knowledge on the potential of Streptomyces ambofaciens ATCC 23877 to synthesize natural products. This industrial strain was known for decades to only produce the drug spiramycin and another antibacterial compound, congocidine. Mining of its genome allowed the identification of 23 clusters potentially involved in the production of other secondary metabolites. Studies of some of these clusters resulted in the characterization of novel compounds and of previously known compounds but never characterized in this Streptomyces species. In addition, genome mining revealed that secondary metabolite gene clusters of phylogenetically closely related Streptomyces are mainly species-specific.     

Evolution of secondary metabolites in legumes (Fabaceae)

Legumes produce a high diversity of secondary metabolites which serve as defence compounds against herbivores and microbes, but also as signal compounds to attract pollinating and fruit-dispersing animals. As nitrogen-fixing organisms, legumes produce more nitrogen containing secondary metabolites than other plant families. Compounds with nitrogen include alkaloids and amines (quinolizidine, pyrrolizidine, indolizidine, piperidine, pyridine, pyrrolidine, simple indole, Erythrina, simple isoquinoline, and imidazole alkaloids; polyamines, phenylethylamine, tyramine, and tryptamine derivatives), non-protein amino acids (NPAA), cyanogenic glucosides, and peptides (lectins, trypsin inhibitors, antimicrobial peptides, cyclotides). Secondary metabolites without nitrogen are phenolics (phenylpropanoids, flavonoids, isoflavones, catechins, anthocyanins, tannins, lignans, coumarins and furanocoumarins), polyketides (anthraquinones), and terpenoids (especially triterpenoid, steroidal saponins, tetraterpenes). While some secondary metabolites have a wide distribution (flavonoids, triterpenes, pinitol), however, others occur in a limited number of taxa. The distributions of secondary metabolites with an irregular occurrence are mapped on a molecular phylogeny of the Fabaceae, reconstructed from a combined data set of nucleotide sequences from rbcL, matK and ITS genes. In most cases, the distribution patterns of secondary metabolites do not agree with the phylogeny of the plants producing them. In contrary, the distribution of many secondary metabolites is patchy and irregular. Thus, the use of phytochemical data to reconstruct a phylogeny of plants is often not informative and can be misleading. The patchy distribution may be due to convergent evolution, a contribution of endophytic fungi or more likely, to an early acquisition of the key genes of secondary metabolism in the evolution of land plants among others by horizontal gene transfer from bacteria. Thus it would be a matter of gene regulation whether these genes are active in some but not all taxa.     

The use of high-performance liquid chromatography and diode array detection in fungal chemotaxonomy based on profiles of secondary metabolites

FRISVAD, J. C, 1989. The use of high-performance liquid chromatography and diode array detection in fungal chemotaxonomy based on profiles of secondary metabolites. Fungal chemotaxonomy (that part dealing with secondary metabolites) has often been based on thin layer chromatography (TLC) and visual or UV inspection of separated spots, before and after different chemical treatments. The identity of a small proportion of the spots can be suggested based on known internal and external standards. In most chemotaxonomical studies it is impossible to isolate, purify and identify all secondary metabolites produced, due to restraints of time and resources. High performance liquid chromatography (HPLC) of fungal extracts may have some advantages over TLC, but the problems mentioned above remain. These problems have been approached by using an alkylphenone retention time index in a reversed phase HPLC system combined with the use of a diode array UV-VIS detector. High performance thin layer chromatography is used for further confirmation of identity of the secondary metabolites. A particular advantage of this method is that the number of biosynthetic families or groups ('chemosyndromes') can be detected, as biosynthetically related metabolites usually have the same chromophores and UV-VIS spectra. Results obtained from Penicillium, Aspergillus and Fusanum species have shown that each species produces 5 to 15 different biosynthetic families of secondaiy metabolites, indicating that good chromatography data may be sufficient to identify species in the three genera. The use of the technique is exemplified by data on Aspergillus and Talaromyces species.     

Secondary metabolism in cannabis

Cannabis sativa L. is an annual dioecious plant from Central Asia. Cannabinoids, flavonoids, stilbenoids, terpenoids, alkaloids and lignans are some of the secondary metabolites present in C. sativa. Earlier reviews were focused on isolation and identification of more than 480 chemical compounds; this review deals with the biosynthesis of the secondary metabolites present in this plant. Cannabinoid biosynthesis and some closely related pathways that involve the same precursors are disscused.     

The use of Aspergillus niger cultures for biotransformation of terpenoids

Aspergillus niger is a well-known fungus that has been used for many different biotransformations of organic compounds. The terpenoids include a large variety of natural hydrocarbons and their derivatives, mostly obtained from plant essential oils, but some obtained from animals or fungi. They may be acyclic or have one or more rings of various sizes, and they show a variety of biological activities that include antibacterial, antifungal, antiparasitic, antiviral, and anticancer activities. Terpenoids are classified as monoterpenoids (C₁₀), sesquiterpenoids (C₁₅), diterpenoids (C₂₀), triterpenoids (C₃₀), and others. This review summarizes experimental processes that use cultures of various A. niger strains to carry out stereoselective biochemical reactions in terpenoids, including related epoxides, lactones, N-phenylcarbamates, and saponins, to produce metabolites that may be useful as flavors and fragrances or as new experimental drug candidates. Cultures of A. niger that add hydroxyl, carbonyl, and other groups at specific positions or reduce double bonds have resulted in the production of valuable new compounds.     

Modules of co-regulated metabolites in turmeric (Curcuma longa) rhizome suggest the existence of biosynthetic modules in plant specialized metabolism

Turmeric is an excellent example of a plant that produces largenumbers of metabolites from diverse metabolic pathways or networks.It is hypothesized that these metabolic pathways or networkscontain biosynthetic modules, which lead to the formation ofmetabolite modules—groups of metabolites whose productionis co-regulated and biosynthetically linked. To test whethersuch co-regulated metabolite modules do exist in this plant,metabolic profiling analysis was performed on turmeric rhizomesamples that were collected from 16 different growth and developmenttreatments, which had significant impacts on the levels of 249volatile and non-volatile metabolites that were detected. Importantly,one of the many co-regulated metabolite modules that were indeedreadily detected in this analysis contained the three majorcurcuminoids, whereas many other structurally related diarylheptanoidsbelonged to separate metabolite modules, as did groups of terpenoids.The existence of these co-regulated metabolite modules supportedthe hypothesis that the 3-methoxyl groups on the aromatic ringsof the curcuminoids are formed before the formation of the heptanoidbackbone during the biosynthesis of curcumin and also suggestedthe involvement of multiple polyketide synthases with differentsubstrate selectivities in the formation of the array of diarylheptanoidsdetected in turmeric. Similar conclusions about terpenoid biosynthesiscould also be made. Thus, discovery and analysis of metabolitemodules can be a powerful predictive tool in efforts to understandmetabolism in plants. Key words: Biosynthesis, Curcuma longa, curcumin, metabolite module, metabolomics, rhizome, specialized metabolism Received 16 June 2008; Revised 29 September 2008 Accepted 2 October 2008     

Red Soils Harbor Diverse Culturable Actinomycetes That Are Promising Sources of Novel Secondary Metabolites

Red soils, which are widely distributed in tropical and subtropical regions of southern China, are characterized by low organic carbon, high content of iron oxides, and acidity and, hence, are likely to be ideal habitats for acidophilic actinomycetes. However, the diversity and biosynthetic potential of actinomycetes in such habitats are underexplored. Here, a total of 600 actinomycete strains were isolated from red soils collected in Jiangxi Province in southeast China. 16S rRNA gene sequence analysis revealed a high diversity of the isolates, which were distributed into 26 genera, 10 families, and 7 orders within the class Actinobacteria; these taxa contained at least 49 phylotypes that are likely to represent new species within 15 genera. The isolates showed good physiological potentials for biosynthesis and biocontrol. Chemical screening of 107 semirandomly selected isolates spanning 20 genera revealed the presence of at least 193 secondary metabolites from 52 isolates, of which 125 compounds from 39 isolates of 12 genera were putatively novel. Macrolides, polyethers, diketopiperazines, and siderophores accounted for most of the known compounds. The structures of six novel compounds were elucidated, two of which had a unique skeleton and represented characteristic secondary metabolites of a putative novel Streptomyces phylotype. These results demonstrate that red soils are rich reservoirs for diverse culturable actinomycetes, notably members of the families Streptomycetaceae, Pseudonocardiaceae, and Streptosporangiaceae, with the capacity to synthesize novel bioactive compounds.     

Metabolic Regulation of Floral Scent in Petunia axillaris Lines: Biosynthetic Relationship between Dihydroconiferyl Acetate and iso-Eugenol

Aromatic scent-related compounds in flowers were comprehensively analyzed by high-performance liquid chromatography (HPLC) based on their absorption spectra to understand regulation of metabolism leading to floral scent diversity in Petunia axillaris lines. An unknown compound occurring at similar levels to scent compounds in some plant lines was identified to be dihydroconiferyl acetate. Based on the structure, dihydroconiferyl acetate is likely to be a biosynthetically closely related compound to aromatic scent compounds, especially iso-eugenol. Similar time-course changes of the concentrations suggest that the metabolism of dihydroconiferyl acetate is underlaid by the similar regulation to aromatic scent compounds. Dihydroconiferyl acetate and iso-eugenol occurred almost exclusively, implying that metabolism of the common precursors to each compound is selectively regulated in these plants. The branching of the biosynthetic pathway into dihydroconiferyl acetate and iso-eugenol is probably one of regulatory steps leading to scent diversity in P. axillaris lines.     

From genetic diversity to metabolic unity: studies on the biosynthesis of aurafurones and aurafuron-like structures in myxobacteria and streptomycetes

The myxobacterial polyketide secondary metabolites aurafuron A and B were identified by genome mining in the myxobacterial strain Stigmatella aurantiaca DW4/3-1. The compounds contain an unusual furanone moiety and resemble metabolites isolated from soil-dwelling and marine actinobacteria, a fungus and mollusks. We describe here the cloning and functional analysis of the aurafuron biosynthetic gene cluster, including site-directed mutagenesis and feeding studies using labeled precursors. The polyketide core of the aurafurones is assembled by a modular polyketide synthase (PKS). As with many such systems described from myxobacteria, the aurafuron PKS exhibits a number of unusual features, including the apparent iterative use of a module, redundant modules and domains, a trans acting dehydratase and the absence of a terminal thioesterase domain. Four oxidoreductases are encoded within the gene locus, some of which likely participate in formation of the furanone moiety via a Baeyer-Villiger type oxidation. Indeed, inactivation of a gene encoding a cytochrome P₄₅₀ monooxygenase completely abolished production of both compounds. We also compare the complete gene locus to biosynthetic gene clusters from two Streptomyces sp., which produce close structural analogues of the aurafurones. A portion of the post-PKS biosynthetic machinery is strikingly similar in all three cases, in contrast to the PKS genes, which are highly divergent. Phylogenetic analysis of the ketosynthase domains further indicates that the PKSs have developed independently (polyphyletically) during evolution. These findings point to a currently unknown but important biological function of aurafuron-like compounds for the producing organisms.     

Labdane-type diterpenoids from hairy root cultures of Coleus forskohlii,possible intermediates in the biosynthesis of forskolin

Significant attention has been devoted to studying hairy root cultures as a promising strategy for production of various valuable secondary metabolites. These offer many advantages, such as high growth rate, genetic stability and being hormone-free. In this study, a detailed phytochemical investigation of the secondary metabolites of Coleus forskohlii hairy root cultures was undertaken and which resulted in the isolation of 22 compounds, including four forskolin derivatives and a monoterpene. Their structures were elucidated by extensive spectroscopic analyses. These compounds could be classified into four groups viz.: labdane-type diterpenes, monoterpenes, triterpenes and phenylpropanoid dimers. Apart from one compound, all labdane type diterpenes are oxygenated at C-11 as in forskolin and a scheme showing their biosynthetic relationships is proposed.