Endogenous Secondary Metabolites Research Articles

Comparative study on the morpho-physiological responses and alkaloids content in Eschscholzia californica cham to foliar applied α-Fe2O3 NPs and ferric ions

The effect of iron oxide nanoparticles on medicinal plants biomass biosynthesis and antioxidant defense compounds should be assessed critically to ensure their safe and sustainable use within the agricultural fields and food chains. Current focus has been given to the effects of hematite nanoparticles (α-Fe2O3 NPs, ∼ 27 nm) and ferric ions (Fe3+) at different concentrations [(0, control), 10, 100 and 1000 mg L−1)] on morpho-physiological and biochemical parameters and benzophenanthridine alkaloids content in Eschscholzia californica plants. Upon exposure to α-Fe2O3 NPs, a significant (P < 0.05) inhibition (by 26.6%) of root biomass was observed at the high concentration (1000 mg L−1) over the control. As for the total dry weight of plants, significant (P < 0.01) increase (by 22.7%) was observed at all exposures, except that of 1000 mg L−1 α-Fe2O3 NPs/and Fe+3 treatment which was reduced by 12.8%. The treatment of 100 mg L−1 α-Fe2O3 NPs and Fe3+ caused significant increase in chlorophyll a (29.2% and 40%,), chlorophyll b (59.8% and 49.3%,) and carotenoids (14.3% and 25.4%%,) content when compared to control, respectively. However, accumulation of the lipid peroxidation product, malondialdehyde (MDA), in root and leaf tissues was similar (73.5% vs control) that was induced by 1000 mg L−1 α-Fe2O3 NPs. No significant change in MDA content was observed in leaves and roots of Fe+3 treated plants at all exposure doses. Furthermore, total phenolics and flavonoids content of the leaves and roots exposed to α-Fe2O3 NPs at 100 mg L−1 were significantly (P < 0.01) increased by 41–45%, 27–29%, and 44–48%, respectively, over the control. All obtained extracts revealed strong and significant scavenging effects on DPPH˙ as compared to control, but only exposure to 100 mg L−1 Fe+3 and α-Fe2O3 NPs treatment revealed the highest free radical scavenging/antioxidant activity by 44.2% and 37.65%, respectively. The alkaloid accumulation in leaves exhibited significant (P < 0.05) differences between the control plants and those treated with α-Fe2O3 NPs and Fe+3 of all concentrations. The eshchschollzine and sanguinarine contents showed no significant change in plants treated with Fe+3 concentrations; however, the exposure of high Fe+3 concentration increased (33.4%) the content of chelerythrine compared to control. Foliar applied α-Fe2O3 NPs enhanced the content of californidine (35.4%), eshchschollzine (22.3%), and eshchschollzine (19.1%) compared to control. The observed toxicity was dose-dependent for both α-Fe2O3 NPs and Fe+3. The synthesized endogenous secondary metabolites (e.g., phenolics, flavonoids and benzophenanthridine alkaloids) induce defense mechanism in exposed plants. These results suggest that specific concentrations (10–100 mg L−1) of α-Fe2O3 NPs have extra benefits both as a nanoregulator and nanoelicitor for plant growth promotion and biosynthesis of alkaloids and cellular antioxidants.

Functional characterization of MATE gene family under abiotic stresses and melatonin-mediated tolerance in dragon fruit (Selenicereus undatus L.)

Multidrug and toxic compound extrusion (MATE) are transporter proteins exist widely in all living organisms which are involved in toxins detoxification. In plants, MATE proteins function in detoxification of endogenous secondary metabolites, exogenous agents, and other plant developmental processes. In this study, the identification and expression analysis of MATE gene family was conducted to analyse their response against heavy metal and salt stresses in pitaya seedlings. We have identified and analysed 35 MATE gene family members from pitaya genome which were mapped on all 11 chromosomes. All members of this family were named from HuMATE-1 to HuMATE-35, divided into 14 groups based on phylogenetic analysis, tree topology and motif's structure. The subcellular localization of 35 proteins was predicted and data showed that 62 % of the total gene members were localized on plasma membrane. The syntenic analysis showed 14 collinearity gene pairs in which two gene pairs showed tandem duplication and twelve pairs showed segmental duplication on the chromosomes. Genes motif composition and exon-intron structures were found more similar within the same group. Cis-acting element in promoter regions predicted their regulatory function in plant toxin detoxification and defence processes. RNA-Seq analysis of HuMATE candidate genes exhibit higher expression under copper and salt stresses individually as well as both in combination. Melatonin applications regulate HuMATE gene expression effectively for both copper and salt stresses, thus enhancing pitaya seedling growth and development. Moreover, RT-qPCR analysis of highly expressed 10 HuMATE genes at different developmental stages of pitaya validates the expression and RNA-Seq results. Our finding predicts thatHuMATE-7/11/12/28 genes are putative candidates and might play a key role in plant toxin detoxification produced by heavy metals accumulation and high soil salinity. Furthermore, our results provide the foundation for development of stress-tolerant genotypes under various climate scenarios through forward and reverse genetic breeding programs.

Structural and Functional Characterization at the Molecular Level of the MATE Gene Family in Wheat in Silico.

A series of multidrug extransporters known as the multidrug and potentially toxic extrusion (MATE) genes are found in all living things and are crucial for the removal of heavy metal ions, metalloids, exogenous xenobiotics, endogenous secondary metabolites, and other toxic substances from the cells. However, there has only been a small amount of them in silico analysis of the MATE family of genes in plant species. In the current study, the MATE gene family was characterized in silico where two families and seven subfamilies based on their evolutionary relationships were proposed. Plant breeders may use TraesCS1D02G030400, TraesCS4B02G244400, and TraesCS1A02G029900 genes for marker-assisted or transgenic breeding to develop novel cultivars since these genes have been hypothesized from protein-protein interaction study to play a critical role in the transport of toxic chemicals across cells. The exon number varies from 01 to 14. One exon has TraesCS1A02G188100, TraesCS5B02G562500, TraesCS6A02G256400, and TraesCS6D02G384300 genes, while 14 exons have only two genes that are TraesCS6A02G418800 and TraesCS6D02G407900. Biological stress (infestations of disease) affects the expression of most of the MATE genes, with the gene TraesCS5D02G355500 having the highest expression level in the wheat expression browser tool. Using the Grain interpretation search engine tool, it is found that the vast bulk of MATE genes are voiced throughout biotic environmental stresses caused by disease pests, with the genotype TraesCS5B02G326600.1 from family 1 exhibiting the greatest level of expression throughout Fusarium head blight infection by Fusarium graminearum after 4 days of infection. The researchers constructed 39 ternary plots, each with a distinct degree of expression under biotic and abiotic stress settings, and observed that 44% of the triplets have imbalanced outputs (extreme values) due to their higher tissue specificity and increased intensity.

Heterologous production of ascofuranone and ilicicolin A in Aspergillus sojae.

Ascofuranone and its precursor, ilicicolin A, are secondary metabolites with various pharmacological activities that are produced by Acremonium egyptiacum. In particular, ascofuranone strongly inhibits trypanosome alternative oxidase and represents a potential drug candidate against African trypanosomiasis. However, difficulties associated with industrial production of ascofuranone by A. egyptiacum, specifically the co-production of ascochlorin, which inhibits mammalian respiratory chain complex III at low concentrations, has precluded its widespread application. Therefore, in this study, ascofuranone biosynthetic genes (ascA-E and H-J) were heterologously expressed in Aspergillus sojae, which produced very low-levels of endogenous secondary metabolites under conventional culture conditions. As a result, although we obtained transformants producing both ilicicolin A and ascofuranone, they were produced only when an adequate concentration of chloride ions was added to the medium. In addition, we succeeded in increasing the production of ilicicolin A, by enhancing the expression of the rate-determining enzyme AscD, using a multi-copy integration system. The heterologous expression approach described here afforded the production of both ascofuranone and ilicicolin A, allowing for their development as therapeutics.

Syzygium samarangense Volatile Oil Inhibited Bacteria Growth and Extracellular Protease of Salmonella typhimurium.

Medicinal plants are fast becoming essential pharmaceuticals for disease and infection management. The vast antimicrobial properties of these plants reside in the inhibitory properties of their endogenous secondary metabolites. Therefore, this study aimed to assess if the volatile oil of Syzygium samarangense inhibits enteric bacteria growth and its effect against the caseinolytic activity of the extracellular protease of Salmonella typhimurium. The volatile oil was extracted by hydrodistillation, while the antimicrobial assay was assessed with the microdilution method. The extracellular protease was partially purified by salting out, followed by size-exclusion chromatography. The mode of inhibition of this enzyme was deduced from the enzyme-substrate kinetics using a line-weaver burke plot. The antimicrobial properties of the oil were reported against ten enteric bacteria. Proteus vulgaris has the highest IC50 value of 0.75±0.004% v/v, while S. typhimurium, the most sensitive bacterium, showed the lowest IC50 value of 0.17±0.005% v/v. The extracellular protease of S. typhimurium was partially purified to achieve 3.73 purification fold and 314.2 μmol min-1 mg-1 protein. The optimal caseinolytic activity of this enzyme was found at pH 7.5 and 40 °C. The protease showed significantly higher activity in the presence of Zn2+ (9.3±0.33 U min-1) as compared to the control (7.0±0.58 U min-1) (p<0.05), however, K+, Ca2+, Co2+, Ba2+, Pb2+ and Hg2+ considerably reduced the enzyme activity. The activity of this enzyme was competitively inhibited by the volatile oil as an inhibitor. The volatile oil of S. samarangense inhibited a wide range of enteric bacteria and, therefore proposed as a potential antimicrobial agent. Inhibiting the extracellular protease of S. typhimurium may be one of its modes of action against these pathogens.

Indole-3-acetic-acid and ACC deaminase producing Leclercia adecarboxylata MO1 improves Solanum lycopersicum L. growth and salinity stress tolerance by endogenous secondary metabolites regulation

BackgroundThe utilization of plant growth-promoting microbes is an environment friendly strategy to counteract stressful condition and encourage plants tolerance. In this regards, the current study was designed to isolate ACC deaminase and indole-3-acetic acid (IAA) producing halotolerant bacteria to promote tomato (Solanum lycopersicum L.) growth and tolerance against salinity stress.ResultsThe selected bacterial isolate MO1 was identified as Leclercia adecarboxylata and IAA quantification results revealed that MO1 produced significant amount of IAA (9.815 ± 0.6293 μg mL− 1). The MO1 showed the presence of ACC (1-Aminocyclopropane-1-Carboxylate) deaminase responsible acdS gene and tolerance against salinity stress. A plant microbe interaction experiment using tomato (Solanum lycopersicum L.) with glycine betaine (GB) as a positive control was carried out to investigate the positive role MO1 in improving plant growth and stress tolerance. The results indicated that MO1 inoculation and GB application significantly increased growth attributes under normal as well as saline condition (120 mM NaCl). The MO1 inoculation and GB treatment approach conferred good protection against salinity stress by significantly improving glucose by 17.57 and 18.76%, sucrose by 34.2 and 12.49%, fructose by 19.9 and 10.9%, citric acid by 47.48 and 34.57%, malic acid by 52.19 and 28.38%, serine by 43.78 and 69.42%, glycine by 14.48 and 22.76%, methionine by 100 and 124.99%, threonine by 70 and 63.08%, and proline by 36.92 and 48.38%, respectively, while under normal conditions MO1 inoculation and GB treatment also enhanced glucose by 19.83 and 13.19%, sucrose by 23.43 and 15.75%, fructose by 15.79 and 8.18%, citric acid by 43.26 and 33.14%, malic acid by 36.18 and 14.48%, serine by 46.5 and 48.55%, glycine by 19.85 and 29.77%, methionine by 22.22 and 38.89%, threonine by 21.95 and 17.07%, and proline by 29.61 and 34.68% compared to levels in non-treated plants, respectively. In addition, the endogenous abscisic acid (ABA) level was noticeably lower in MO1-inoculated (30.28 and 30.04%) and GB-treated plants (45 and 35.35%) compared to their corresponding control plants under normal condition as well as salinity stress, respectively.ConclusionThe current findings suggest that the IAA- and ACC-deaminase-producing abilities MO1 can improve plants tolerance to salinity stress.

Genome-wide analysis of MATE transporters and response to metal stress in Cajanus cajan

ABSTRACT Multidrug and Toxic Compound Extrusion (MATEs) is one of the characteristic transporter families, which plays a key role in the detoxication of endogenous secondary metabolites and exogenous agents in both animal and plant cells. In this study, we identified a total of 67 MATE genes (CcMATEs) from the pigeonpea genome, on which we performed bioinformatics analysis and we group them by phylogenetic analysis. Finally, eight represented CcMATE genes were selected for further qRT-PCR analysis of tissue specificity and response to metal stress in pigeonpea. The results showed that both CcMATE34 and 45 genes were significantly up-regulated and the CcMATE4 gene was only up-regulated in the roots under the stress of Al, Mn and Zn. We speculated that the function of CcMATE34 and 45 might be related to the transport of alkaloids and harmful substances and the function of CcMATE4 might be related to the delivery of flavonoids.

Crucifer glucosinolate production in legume-crucifer cover crop mixtures

Cover crops grown in rotation with cash crops provide ecosystem services by reducing water pollution and anthropogenic inputs. Bispecific crucifer–legume cover crop mixtures are seen as a solution to increase biodiversity and to combine ecosystem services of both species. Legumes fix nitrogen while crucifers have the capacity to suppress pathogens due to the biocidal hydrolysis products of the endogenous secondary metabolites called glucosinolates (GSL). However there is a lack of information on the impact of plant–plant interactions in the crucifer-legume mixtures on crucifer GSL production compared to sole crop. The aim of our study was to assess GSL production of a wide range of bispecific crucifer-legume mixtures in comparison to sole crops. Experiments were conducted at two sites (near Toulouse and Orleans, France) over two years. Various cultivars from eight crucifer (Brassicaceae) species (rape, white mustard, Indian mustard, Ethiopian mustard, turnip, turnip rape, radish and rocket) and nine legume (Fabaceae) species (Egyptian clover, crimson clover, common vetch, purple vetch, hairy vetch, pea, soya bean, faba bean, and white lupin) were tested in sole crop and bispecific mixtures (substitutive design of 50%–50% sole crops). We show that for a wide range of species, crucifers in cover crop mixtures had the same GSL types, concentrations and proportions (aliphatic, aromatic and indole) as in sole crops. Crucifers in mixtures tended to have more biomass per plant, both for shoots and roots, than in sole crops so that the total GSL production per plant generally increased in the mixture compared to the sole crop. Thus despite halving the crucifer density in the mixtures the GSL production on an area basis declined by only 20%. These results have been validated for a wide range of crucifer species and provide support for crucifer-legume mixtures to produce nutrient-related services while potentially maintaining high GSL production. Specific experiments are needed to evaluate whether this double effect of unchanged GSL concentration and small reduction in GSL production has an impact on the biofumigation potential of cover crop mixtures compared to sole crucifers.

CoIN: co-inducible nitrate expression system for secondary metabolites in Aspergillus nidulans

BackgroundSequencing of fungal species has demonstrated the existence of thousands of putative secondary metabolite gene clusters, the majority of them harboring a unique set of genes thought to participate in production of distinct small molecules. Despite the ready identification of key enzymes and potential cluster genes by bioinformatics techniques in sequenced genomes, the expression and identification of fungal secondary metabolites in the native host is often hampered as the genes might not be expressed under laboratory conditions and the species might not be amenable to genetic manipulation. To overcome these restrictions, we developed an inducible expression system in the genetic model Aspergillus nidulans.ResultsWe genetically engineered a strain of A. nidulans devoid of producing eight of the most abundant endogenous secondary metabolites to express the sterigmatocystin Zn(II)2Cys6 transcription factor-encoding gene aflR and its cofactor aflS under control of the nitrate inducible niiA/niaD promoter. Furthermore, we identified a subset of promoters from the sterigmatocystin gene cluster that are under nitrate-inducible AflR/S control in our production strain in order to yield coordinated expression without the risks from reusing a single inducible promoter. As proof of concept, we used this system to produce β-carotene from the carotenoid gene cluster of Fusarium fujikuroi.ConclusionUtilizing one-step yeast recombinational cloning, we developed an inducible expression system in the genetic model A. nidulans and show that it can be successfully used to produce commercially valuable metabolites.

Genomewide analysis of MATE-type gene family in maize reveals microsynteny and their expression patterns under aluminum treatment.

Multidrug and toxic compound extrusion (MATE) proteins are a group of secondary active transporters, which widely exist in all living organisms and play important role in the detoxication of endogenous secondary metabolites and exogenous agents. However, to date, no systematic and comprehensive study of this family is reported in maize. Here, a total of 49 MATE genes (ZmMATE) were identified and divided into seven groups by phylogenetic analysis. Conserved intro-exon structures and motif compositions were investigated in these genes. Results by gene locations indicated that these genes were unevenly distributed among all 10 chromosomes. Tandem and segmental duplications appeared to contribute to the expansion and evolution of this gene family. The Ka/Ks ratios suggested that the ZmMATE has undergone large-scale purifying selection on the maize genome. Interspecies microsynteny analysis revealed that there were independent gene duplication events of 10 ZmMATE. In addition, most maize MATE genes exhibited different expression profiles in diverse tissues and developmental stages. Sixteen MATE genes were chosen for further quantitative real-time polymerase chain reaction analysis showed differential expression patterns in response to aluminum treatment. These results provide a useful clue for future studies on the identification of MATE genes and functional analysis of MATE proteins in maize.

LcGST4 is an anthocyanin-related glutathione S-transferase gene in Litchi chinensis Sonn.

A novel LcGST4 was identified and characterized from Litchi chinensis . Expression and functional analysis demonstrated that it might function in anthocyanin accumulation in litchi. Glutathione S-transferases (GSTs) have been defined as detoxification enzymes for their ability to recognize reactive electrophilic xenobiotic molecules as well as endogenous secondary metabolites. Anthocyanins are among the few endogenous substrates of GSTs for vacuolar accumulation. The gene encoding a GST protein that is involved in anthocyanin sequestration from Litchi chinensis Sonn. has not been reported. Here, LcGST4, an anthocyanin-related GST, was identified and characterized. Phylogenetic analysis showed that LcGST4 was clustered with other known anthocyanin-related GSTs in the same clade. Expression analysis revealed that the expression pattern of LcGST4 was strongly correlated with anthocyanin accumulation in litchi. ABA- and light-responsive elements were found in the LcGST4 promoter, which is in agreement with the result that the expression of LcGST4 was induced by both ABA and debagging treatment. A GST activity assay in vitro verified that the LcGST4 protein shared universal activity with the GST family. Functional complementation of an Arabidopsis mutant tt19 demonstrated that LcGST4 might function in anthocyanin accumulation in litchi. Dual luciferase assay revealed that the expression of LcGST4 was activated by LcMYB1, a key R2R3-MYB transcription factor that regulates anthocyanin biosynthesis in litchi.

Translating biosynthetic gene clusters into fungal armor and weaponry.

Filamentous fungi are renowned for the production of a diverse array of secondary metabolites (SMs) where the genetic material required for synthesis of a SM is typically arrayed in a biosynthetic gene cluster (BGC). These natural products are valued for their bioactive properties stemming from their functions in fungal biology, key among those protection from abiotic and biotic stress and establishment of a secure niche. The producing fungus must not only avoid self-harm from endogenous SMs but also deliver specific SMs at the right time to the right tissue requiring biochemical aid. This review highlights functions of BGCs beyond the enzymatic assembly of SMs, considering the timing and location of SM production and other proteins in the clusters that control SM activity. Specifically, self-protection is provided by both BGC-encoded mechanisms and non-BGC subcellular containment of toxic SM precursors; delivery and timing is orchestrated through cellular trafficking patterns and stress- and developmental-responsive transcriptional programs.

8-Methoxypsoralen is a competitive inhibitor of glutathione S-transferase P1-1.

The blood-brain barrier (BBB) is known to protect healthy brain cells from potentially dangerous chemical agents, but there are many evidences supporting the idea that this protective action is extended to tumor cells. Since the process of angiogenesis in brain tumors leads to BBB breakdown, biochemical characteristics of the BBB seem to be more relevant than physical barriers to protect tumor cells from chemotherapy. In fact, a number of resistance related factors were already demonstrated to be component of both BBB and tumor cells. The enzyme glutathione S-transferases (GST) detoxify electrophilic xenobiotics and endogenous secondary metabolites formed during oxidative stress. A role has been attributed to GST in the resistance of cancer cells to chemotherapeutic agents. This study characterized 8-methoxypsoralen (8-MOP) as a human GST P1-1 (hGST P1-1) inhibitor. To identify and characterize the potential inhibitory activity of 8-MOP, we studied the enzyme kinetics of the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) with GSH catalyzed by hGST P1-1. We report here that 8-MOP competitively inhibited hGST P1-1 relative to CDNB, but there was an uncompetitive inhibition relative to GSH. Chromatographic analyses suggest that 8-MOP is not a substrate. Molecular docking simulations suggest that 8-MOP binds to the active site, but its position prevents the GSH conjugation. Thus, we conclude that 8-MOP is a promising prototype for new GST inhibitors pharmacologically useful in the treatment of neurodegenerative disorders and the resistance of cancer to chemotherapy.

RNA-Seq analysis reveals a six-gene SoxR regulon in Streptomyces coelicolor.

The redox-regulated transcription factor SoxR is conserved in diverse bacteria, but emerging studies suggest that this protein plays distinct physiological roles in different bacteria. SoxR regulates a global oxidative stress response (involving >100 genes) against exogenous redox-cycling drugs in Escherichia coli and related enterics. In the antibiotic producers Streptomyces coelicolor and Pseudomonas aeruginosa, however, SoxR regulates a smaller number of genes that encode membrane transporters and proteins with homology to antibiotic-tailoring enzymes. In both S. coelicolor and P. aeruginosa, SoxR-regulated genes are expressed in stationary phase during the production of endogenously-produced redox-active antibiotics. These observations suggest that SoxR evolved to sense endogenous secondary metabolites and activate machinery to process and transport them in antibiotic-producing bacteria. Previous bioinformatics analysis that searched the genome for SoxR-binding sites in putative promoters defined a five-gene SoxR regulon in S. coelicolor including an ABC transporter, two oxidoreductases, a monooxygenase and an epimerase/dehydratase. Since this in silico screen may have missed potential SoxR-targets, we conducted a whole genome transcriptome comparison of wild type S. coelicolor and a soxR-deficient mutant in stationary phase using RNA-Seq. Our analysis revealed a sixth SoxR-regulated gene in S. coelicolor that encodes a putative quinone oxidoreductase. Knowledge of the full complement of genes regulated by SoxR will facilitate studies to elucidate the function of this regulatory molecule in antibiotic producers.

The potential of Physcomitrella patens as a platform for the production of plant-based vaccines

The moss Physcomitrella patens has a number of advantages for the production of biopharmaceuticals, including: i) availability of standardized conditions for cultivation in bioreactors; ii) not being part of the food chain; iii) high biosafety; iv) availability of highly efficient transformation methods; v) a haploid, fully sequenced genome providing genetic stability and uniform expression; vi) efficient gene targeting at the nuclear level allows for the generation of mutants with specific post-translational modifications (e.g., glycosylation patterns); and vii) oral formulations are a viable approach as no toxic effects are attributed to ingestion of this moss. In the light of this panorama, this opinion paper analyzes the possibilities of using P. patens for the production of oral vaccines and presents some specific cases where its use may represent significant progress in the field of plant-based vaccine development. The advantages represented by putative adjuvant effects of endogenous secondary metabolites and producing specific glycosylation patterns are highlighted.

Genetic enhancement of Brassica napus seed quality

The ultimate value of the Brassica napus (canola) seed is derived from the oil fraction, which has long been recognized for its premium dietary attributes, including its low level of saturated fatty acids, high content of monounsaturated fatty acids, and favorable omega-3 fatty acid profile. However, the protein (meal) portion of the seed has also received favorable attention for its essential amino acids, including abundance of sulfur-containing amino acids, such that B. napus protein is being contemplated for large scale use in livestock and fish feed formulations. Efforts to optimize the composition of B. napus oil and protein fractions are well documented; therefore, this article will review research concerned with optimizing secondary metabolites that affect the quality of seed oil and meal, from undesirable anti-nutritional factors to highl value beneficial products. The biological, agronomic, and economic values attributed to secondary metabolites have brought much needed attention to those in Brassica oilseeds and other crops. This review focuses on increasing levels of beneficial endogenous secondary metabolites (such as carotenoids, choline and tochopherols) and decreasing undesirable antinutritional factors (glucosinolates, sinapine and phytate). Molecular genetic approaches are given emphasis relative to classical breeding.

Cloning and functional analysis of CDS_CCI2: a Tanacetum cinerariaefolium chrysanthemyl diphosphate synthase gene

Although irregular monoterpenes are important and common in the Asteraceae family, little is known about their biosynthesis at the genetic level via the MEP pathway. Chrysanthemyl diphosphate (CPP) is an intermediate in the biosynthesis of pyrethrins which are irregular monoterpenes with excellent insecticidal activity in Tanacetum cinerariaefolium (T. cinerariaefolium). In this study, a chrysanthemyl diphosphate synthase (CDS) gene named CDS_CCI2 (GenBank accession no. HQ235057) was isolated from T. cinerariaefolium. It was homologous to T. cinerariaefolium CPP gene family, and proved to be located in the plastid by the in situ subcellular localization. CDS_CCI2 was found to be expressed in roots, stems, leaves, buds and flowers. Moreover, the expression of CDS_CCI2 can be up-regulated by abscisic acid (ABA), methyl jasmonate and ethrel treatment. Phenotypic and molecular analysis showed that overexpression of CDS_CCI2 in micro-tom tomato resulted in dwarf phenotypes characterized with infertile flowers and seedless fruits. Furthermore, overexpression of CDS_CCI2 altered the production of endogenous secondary metabolites. Our data indicate that CPP affects the synthesis of gibberellic acid (GA) and ABA.

Genome mining in Streptomyces avermitilis: cloning and characterization of SAV_76, the synthase for a new sesquiterpene, avermitilol.

The terpene synthase encoded by the sav76 gene of Streptomyces avermtilis was expressed in Escherichia coli as an N-terminal-His(6)-tag protein, using a codon-optimized synthetic gene. Incubation of the recombinant protein, SAV_76, with farnesyl diphosphate (1, FPP) in the presence of Mg(2+) gave a new sesquiterpene alcohol avermitilol (2), whose structure and stereochemistry were determined by a combination of (1)H, (13)C, COSY, HMQC, HMBC, and NOESY NMR, along with minor amounts of germacrene A (3), germacrene B (4), and viridiflorol (5). The absolute configuration of 2 was assigned by (1)H NMR analysis of the corresponding (R)- and (S)-Mosher esters. The steady state kinetic parameters were k(cat) 0.040 +/- 0.001 s(-1) and K(m) 1.06 +/- 0.11 microM. Individual incubations of recombinant avermitilol synthase with [1,1-(2)H(2)]FPP (1a), (1S)-[1-(2)H]-FPP (1b), and (1R)-[1-(2)H]-FPP (1c) and NMR analysis of the resulting avermitilols supported a cyclization mechanism involving the loss of H-1(re) to generate the intermediate bicyclogermacrene (7), which then undergoes proton-initiated anti-Markovnikov cyclization and capture of water to generate 2. A copy of the sav76 gene was reintroduced into S. avermitilis SUKA17, a large deletion mutant from which the genes for the major endogenous secondary metabolites had been removed, and expressed under control of the native S. avermitilis promoter rpsJp (sav4925). The resultant transformants generated avermitilol (2) as well as the derived ketone, avermitilone (8), along with small amounts of 3, 4, and 5. The biochemical function of all four terpene synthases found in the S. avermtilis genome have now been determined.

Secondary metabolite profiling of the model legume Lotus japonicus during its symbiotic interaction with Mesorhizobium loti

Plant secondary metabolites, particularly flavonoids, are key components in the early stages of nitrogen-fixing symbiosis. Despite their importance, the endogenous secondary metabolites involved in symbiosis have not yet been identified in the model legume Lotus japonicus. We therefore determined changes in the secondary metabolic profile of Lotus japonicus roots in response to its symbiont. Analysis of the root secondary metabolite profiles 1 week after inoculation with Mesorhizobium loti revealed quantitative changes in the level of 14 phenolic peaks when compared with non-inoculated control plants. These changes affected compounds from most phenolic classes, possibly resulting from interconversion between classes since the total phenolic level remained constant. In addition, the use of 2 M. loti strains differing only in their capacity to synthesise Nod factor revealed that, although Nod factor signalling induced accumulation of a specific subset of 4 phenolic peaks, most changes were induced in response to both rhizobial strains.

Genome-minimized Streptomyces host for the heterologous expression of secondary metabolism

To construct a versatile model host for heterologous expression of genes encoding secondary metabolite biosynthesis, the genome of the industrial microorganism Streptomyces avermitilis was systematically deleted to remove nonessential genes. A region of more than 1.4 Mb was deleted stepwise from the 9.02-Mb S. avermitilis linear chromosome to generate a series of defined deletion mutants, corresponding to 83.12-81.46% of the wild-type chromosome, that did not produce any of the major endogenous secondary metabolites found in the parent strain. The suitability of the mutants as hosts for efficient production of foreign metabolites was shown by heterologous expression of three different exogenous biosynthetic gene clusters encoding the biosynthesis of streptomycin (from S. griseus Institute for Fermentation, Osaka [IFO] 13350), cephamycin C (from S. clavuligerus American type culture collection (ATCC) 27064), and pladienolide (from S. platensis Mer-11107). Both streptomycin and cephamycin C were efficiently produced by individual transformants at levels higher than those of the native-producing species. Although pladienolide was not produced by a deletion mutant transformed with the corresponding intact biosynthetic gene cluster, production of the macrolide was enabled by introduction of an extra copy of the regulatory gene pldR expressed under control of an alternative promoter. Another mutant optimized for terpenoid production efficiently produced the plant terpenoid intermediate, amorpha-4,11-diene, by introduction of a synthetic gene optimized for Streptomyces codon usage. These findings highlight the strength and flexibility of engineered S. avermitilis as a model host for heterologous gene expression, resulting in the production of exogenous natural and unnatural metabolites.