Kegg Pathway: Biosynthesis of Secondary Metabolites

Molecules. 2009; 14(10): 3922-3941
James JT, Dubery IA

Centella asiatica accumulates large quantities of pentacyclic triterpenoid saponins, collectively known as centelloids. These terpenoids include asiaticoside, centelloside, madecassoside, brahmoside, brahminoside, thankuniside, sceffoleoside, centellose, asiatic-, brahmic-, centellic- and madecassic acids. The triterpene saponins are common Secondary plant Metabolites and are synthesized via the isoprenoid pathway to produce a hydrophobic triterpenoid structure (aglycone) containing a hydrophilic sugar chain (glycone). The biological activity of saponins has been attributed to these characteristics. In planta, the Centella triterpenoids can be regarded as phytoanticipins due to their antimicrobial activities and protective role against attempted pathogen infections. Preparations of C. asiatica are used in traditional and alternative medicine due to the wide spectrum of pharmacological activities associated with these Secondary Metabolites. Here, the Biosynthesis of the centelloid triterpenoids is reviewed; the range of Metabolites found in C. asiatica, together with their known biological activities and the chemotype variation in the production of these Metabolites due to growth conditions are summarized. These plant-derived pharmacologically active compounds have complex structures, making chemical synthesis an economically uncompetitive option. Production of Secondary Metabolites by cultured cells provides a particularly important benefit to manipulate and improve the production of desired compounds; thus biotechnological approaches to increase the concentrations of the Metabolites are discussed.

J Biol Chem. 2009 Nov 18;
Lee TV, Johnson LJ, Johnson RD, Koulman A, Lane GA, Lott JS, Arcus VL

Non-ribosomal peptide synthetases (NRPSs) are large, multi-domain proteins that are involved in the Biosynthesis of an array of Secondary Metabolites. We report the structure of the third adenylation domain from the siderophore-synthesizing NRPS, SidN, from the endophytic fungus, Neotyphodium lolii. This is the first structure of a eukaryotic NRPS domain and it reveals a large binding pocket required to accommodate the unusual amino acid substrate, N(delta)-cis-anhydromevalonyl-N(delta)-hydroxy-L-ornithine (cis-AMHO). The specific activation of cis-AMHO was confirmed biochemically and an AMHO moiety was unambiguously identified as a component of the fungal siderophore using mass spectroscopy. The protein structure shows that the substrate-binding pocket is defined by 17 amino acid residues, in contrast to both prokaryotic adenylation domains and to previous predictions based on modeling. Existing substrate prediction methods for NRPS adenylation domains fail for domains from eukaryotes due to the divergence of their signature sequences from those of prokaryotes. Thus, this new structure will provide a basis for improving prediction methods for eukaryotic NRPS enzymes which play important and diverse roles in the biology of fungi.

J Exp Bot. 2009 Nov 18;
Scheerer U, Haensch R, Mendel RR, Kopriva S, Rennenberg H, Herschbach C

Sulphate assimilation provides reduced sulphur for the synthesis of cysteine, methionine, and numerous other essential Metabolites and Secondary compounds. The key step in the pathway is the reduction of activated sulphate, adenosine 5'-phosphosulphate (APS), to sulphite catalysed by APS reductase (APR). In the present study, [(35)S]sulphur flux from external sulphate into glutathione (GSH) and proteins was analysed to check whether APR controls the flux through the sulphate assimilation pathway in poplar roots under some stress conditions and in transgenic poplars. (i) O-Acetylserine (OAS) induced APR activity and the sulphur flux into GSH. (ii) The herbicide Acetochlor induced APR activity and results in a decline of GSH. Thereby the sulphur flux into GSH or protein remained unaffected. (iii) Cd treatment increased APR activity without any changes in sulphur flux but lowered sulphate uptake. Several transgenic poplar plants that were manipulated in sulphur metabolism were also analysed. (i) Transgenic poplar plants that overexpressed the gamma-glutamylcysteine synthetase (gamma-ECS) gene, the enzyme catalysing the key step in GSH formation, showed an increase in sulphur flux into GSH and sulphate uptake when gamma-ECS was targeted to the cytosol, while no changes in sulphur flux were observed when gamma-ECS was targeted to plastids. (ii) No effect on sulphur flux was observed when the sulphite oxidase (SO) gene from Arabidopsis thaliana, which catalyses the back reaction of APR, that is the reaction from sulphite to sulphate, was overexpressed. (iii) When Lemna minor APR was overexpressed in poplar, APR activity increased as expected, but no changes in sulphur flux were observed. For all of these experiments the flux control coefficient for APR was calculated. APR as a controlling step in sulphate assimilation seems obvious under OAS treatment, in gamma-ECS and SO overexpressing poplars. A possible loss of control under certain conditions, that is Cd treatment, Acetochlor treatment, and in APR overexpressing poplar, is discussed.

Appl Microbiol Biotechnol. 2009 Nov 14;
Tisch D, Schmoll M

Light represents a major carrier of information in nature. The molecular machineries translating its electromagnetic energy (photons) into the chemical language of cells transmit vital signals for adjustment of virtually every living organism to its habitat. Fungi react to illumination in various ways, and we found that they initiate considerable adaptations in their metabolic pathways upon growth in light or after perception of a light pulse. Alterations in response to light have predominantly been observed in carotenoid metabolism, polysaccharide and carbohydrate metabolism, fatty acid metabolism, nucleotide and nucleoside metabolism, and in regulation of production of Secondary Metabolites. Transcription of genes is initiated within minutes, abundance and activity of metabolic enzymes are adjusted, and subsequently, levels of Metabolites are altered to cope with the harmful effects of light or to prepare for reproduction, which is dependent on light in many cases. This review aims to give an overview on metabolic pathways impacted by light and to illustrate the physiological significance of light for fungi. We provide a basis for assessment whether a given metabolic pathway might be subject to regulation by light and how these properties can be exploited for improvement of biotechnological processes.

Curr Top Med Chem. 2009 Nov 11;
Acuña UM, Jancovski N, Kennelly EJ

Many new polyisoprenylated benzophenones with a bicyclo[3.3.1]-nonane-2,4,9-trione core structure have been isolated from plants in the Clusiaceae family, and their potent biological properties have been the subject of several studies. This review summarizes the biological activities reported for these Secondary Metabolites including cytotoxic, antimicrobial, antioxidant, and anti-inflammatory activities. Our efforts during the past years have foremost been directed towards isolating new polyisoprenylated benzophenones, as well as understanding the possible target and mechanism of action through which these compounds arrest cancer cells and inhibit the progression of the cell-cycle. The transcription of genes is affected in cancer cells treated with polyisoprenylated benzophenones; the oncogene c-Myb is down-regulated and endoplasmatic stress genes XBP1, ATF4, and DDIT3/CHOP are turned on. Consequently, the expression of iNOS and cell cycle regulators such as cyclin D and E are reduced. Evidence presented by independent investigators suggests that polyisoprenylated benzophenones affect the mediators in the Akt/mTOR stress pathway, although the specific target remains to be discovered. In addition, benzophenones isolated from plants display high antioxidant capacity and protect cells from oxidative stress and the formation of ROS involved during the inflammatory process. Since antiviral activity was initially reported for guttiferone A, potent synthetic analogues have been developed as effective new non-nucleoside reverse transcriptase inhibitors (NNRTI) to treat drug resistant HIV-1. In addition, benzophenones exert antimicrobial effects particularly against MRSA. The structure-activity relationships of polyisoprenylated benzophenones from natural sources and those of synthetic analogues are included in this review. Absorption, metabolism, and elimination of benzophenones are also discussed.

Biotechnol Prog. 2009 Nov 6;
Shaikh AS, Tang YJ, Mukhopadhyay A, Martín HG, Gin J, Benke PI, Keasling JD

Microbial production of many commercially important Secondary Metabolites occurs during stationary phase, and methods to measure metabolic flux during this growth phase would be valuable. Metabolic flux analysis is often based on isotopomer information from proteinogenic amino acids. As such, flux analysis primarily reflects the metabolism pertinent to the growth phase during which most proteins are synthesized. To investigate central metabolism and amino acids synthesis activity during stationary phase, addition of fully (13)C-labeled glucose followed by induction of green fluorescent protein (GFP) expression during stationary phase was used. Our results indicate that Escherichia coli was able to produce new proteins (i.e., GFP) in the stationary phase, and the amino acids in GFP were mostly from degraded proteins synthesized during the exponential growth phase. Among amino acid biosynthetic pathways, only those for serine, alanine, glutamate/glutamine, and aspartate/asparagine had significant activity during the stationary phase. (c) 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010.

J Appl Microbiol. 2009 Nov 6;
Kurumbang NP, Liou K, Sohng JK

Abstract Aims: Paromamine is a vital and common intermediate in the Biosynthesis of 4,5 and 4,6-disubstituted 2-deoxystreptamine (DOS)-containing aminoglycosides. Our aim is to develop an engineered Escherichia coli system for heterologous production of paromamine. Methods and Results: We have constructed a mutant of E. coli BL21 (DE3) by disrupting glucose-6-phosphate isomerase (pgi) of primary metabolic pathway to increase glucose-6-phosphate pool inside the host. Disruption was carried out by lambda Red/ET recombination following the protocol mentioned in the kit. Recombinants bearing 2-deoxy-scyllo-inosose (DOI), DOS and paromamine producing genes were constructed from butirosin gene cluster and heterologously expressed in engineered host designed as E. coli BL21 (DE3) Deltapgi. Secondary Metabolites produced by the recombinants fermentated in 2YTG medium were extracted, and analysis of the extracts showed there is formation of DOI, DOS and paromamine. Conclusions: Escherichia coli system is engineered for heterologous expression of paromamine derivatives of aminoglycoside Biosynthesis. Significance and Impact of the Study: This is the first report of heterologous expression of paromamine gene set in E. coli. Hence a new platform is established in E. coli system for the production of paromamine which is useful for the exploration of novel aminoglycosides by combinatorial Biosynthesis of 4,5- and 4,6-disubtituted route of DOS-containing aminoglycosides.

Plant Cell Physiol. 2009 Nov 4;
Kim HJ, Ono E, Morimoto K, Yamagaki T, Okazawa A, Kobayashi A, Satake H

Lignans are a large class of Secondary Metabolites in plants, with numerous biological effects in mammals, including anti-tumor and anti-oxidant activities. Sesamin, the most abundant furofuran-class lignan in sesame seeds (Sesamum plants), is produced by the cytochrome P450 enzyme CYP81Q1 from the precursor lignan, pinoresinol. In contrast, Forsythia plants produce dibenzylbutyrolactone-class lignans, such as matairesinol, from pinoresinol via the catalysis of pinoresinol/lariciresinol reductase (PLR) and secoisolariciresinol dehydrogenase. Here we present the engineering of lignan Biosynthesis in Forsythia cell suspension cultures for the development of an efficient production method of beneficial lignans. A suspension cell culture prepared from leaves of Forsythia koreana produced lignans, mainly pinoresinol and matairesinol glucosides, at levels comparable to that obtained from the leaves. In an attempt to increase the pinoresinol content in Forsythia, we generated a transgenic cell line overexpressing an RNA interference (RNAi) construct of PLR (PLR-RNAi). Down-regulation of PLR expression led to a complete loss of matairesinol and an accumulation of approximately 20-fold pinoresinol in its glucoside form in comparison with the non-transformant. Moreover, the Forsythia transgenic cells co-expressing CYP81Q1 and PLR-RNAi exhibited production of sesamin as well as accumulation of pinoresinol glucoside. These data suggest Forsythia cell suspension to be a promising tool for the engineering of lignan production. To the best of our knowledge, this is the first report on transgenic production of an exogenous lignan in a plant species.

J Microbiol Biotechnol. 2009 Oct; 19(10): 1142-1149
Keum YS, Lee YJ, Lee YH, Kim JH

Several bioactive Metabolites, including pyrrolnitrin, Nacylhomoserine lactones, and polyhydroxyalkanoates were isolated from Burkholderia sp. O33. Effects of various nutrients, including sugars, gluconolactone, glycerol, tryptophan, chloride, and zinc were investigated in relation to the production of these Metabolites. Logarithmic increase of pyrrolnitrin was observed between 2-5 days and reached a maximum at 7-10 days. Tryptophan concentration reached the maximum at 3 days, whereas 7-chlorotryptophan was gradually increased throughout the studies. Among various carbon sources, gluconolactone, trehalose, and glycerol enhanced pyrrolnitrin production, whereas strong inhibitory effects were found with glucose. Relative concentrations of pyrrolnitrin and its precursors were in the order of pyrrolnitrin >> dechloroaminopyrrolnitrin or aminopyrrolnitrin throughout the experiments. Among three N-acylhomoserine lactones, the N-octanoyl analog was the most abundant quorum sensing signal, of which the concentrations reached the maximum in 2-3 days, followed by a rapid dissipation to trace level. No significant changes in pyrrolnitrin Biosynthesis were observed by external addition of N-acylhomoserine lactones. Polyhydroxyalkanoates accumulated up to 3-4 days and decreased slowly thereafter. According to the kinetic analyses, no strong correlations were found between the levels of pyrrolnitrin, N-acylhomoserine lactones,and Polyhydroxyalkanoates.

Phytochemistry. 2009 Oct 31;
Yang CQ, Lu S, Mao YB, Wang LJ, Chen XY

Cytochrome P450 monooxygenases (P450s) are commonly involved in Biosynthesis of endogenous compounds and catabolism of xenobiotics, and their activities rely on a partner enzyme, cytochrome P450 reductase (CPR, E.C.1.6.2.4). Two CPR cDNAs, GhCPR1 and GhCPR2, were isolated from cotton (Gossypium hirsutum). They are 71% identical to each other at the amino acid sequence level and belong to the Class I and II of dicotyledonous CPRs, respectively. The recombinant enzymes reduced cytochrome c, ferricyanide and dichlorophenolindophenol (DCPIP) in an NADPH-dependent manner, and supported the activity of CYP73A25, a cinnamate 4-hydroxylase of cotton. Both GhCPR genes were widely expressed in cotton tissues, with a reduced expression level of GhCPR2 in the glandless cotton cultivar. Expression of GhCPR2, but not GhCPR1, was inducible by mechanical wounding and elicitation, indicating that the GhCPR2 is more related to defense reactions, including Biosynthesis of Secondary Metabolites.

Plant Cell. 2009 Oct 30;
Horbach R, Graf A, Weihmann F, Antelo L, Mathea S, Liermann JC, Opatz T, Thines E, Aguirre J, Deising HB

In filamentous fungi, Sfp-type 4'-phosphopantetheinyl transferases (PPTases) activate enzymes involved in primary (alpha-aminoadipate reductase [AAR]) and Secondary (polyketide synthases and nonribosomal peptide synthetases) metabolism. We cloned the PPTase gene PPT1 of the maize anthracnose fungus Colletotrichum graminicola and generated PPTase-deficient mutants (Deltappt1). Deltappt1 strains were auxotrophic for Lys, unable to synthesize siderophores, hypersensitive to reactive oxygen species, and unable to synthesize polyketides (PKs). A differential analysis of Secondary Metabolites produced by wild-type and Deltappt1 strains led to the identification of six novel PKs. Infection-related morphogenesis was affected in Deltappt1 strains. Rarely formed appressoria of Deltappt1 strains were nonmelanized and ruptured on intact plant. The hyphae of Deltappt1 strains colonized wounded maize (Zea mays) leaves but failed to generate necrotic anthracnose disease symptoms and were defective in asexual sporulation. To analyze the pleiotropic pathogenicity phenotype, we generated AAR-deficient mutants (Deltaaar1) and employed a melanin-deficient mutant (M1.502). Results indicated that PPT1 activates enzymes required at defined stages of infection. Melanization is required for cell wall rigidity and appressorium function, and Lys supplied by the AAR1 pathway is essential for necrotrophic development. As PPTase-deficient mutants of Magnaporthe oryzea were also nonpathogenic, we conclude that PPTases represent a novel fungal pathogenicity factor.

Zhongguo Zhong Yao Za Zhi. 2009 Jul; 34(13): 1636-9
Zhang Z, Yan R, Qiu X, Zeng Q, You H, Zhu D

OBJECTIVE: To analyze and compare the cell growth and accumulation of flavonoids and chlorogenic acid in the callus and suspension cell of Eucommia ulmoides. METHOD: The callus induced from the leaf of E. ulmoides seedlings were suspended in liquid medium. The time courses of cell growth and yields of flavonoids and chlorogenic acid were studied. RESULT: The highest contents of flavonoids and chlorogenic acid in the callus were 13.46, 1.712 mg x g(-1), respectively, while the contents of these two Secondary Metabolites were 16.63, 3.93 mg x g(-1) in suspension cell culture correspondingly. CONCLUSION: Comparing with callus, the suspension cell showed a short growth period and high growth rate with a remarkable high content of flavonoids and chlorogenic acid.

Plant Physiol. 2009 Oct 23;
Badri DV, Quintana N, El Kassis EG, Kim HK, Choi YH, Sugiyama A, Verpoorte R, Martinoia E, Manter DK, Vivanco JM

Root exudates influence the surrounding soil microbial community and recent evidence demonstrates the involvement of ABC transporters in root secretion of phytochemicals. In this study, we examined effects of seven Arabidopsis ABC transporter mutants on the microbial community in native soils. After two generations, only the Arabidopsis abcg30 (Atpdr2) mutant had significantly altered both the fungal and bacterial communities compared with the wild type using automated ribosomal intergenic spacer analysis (ARISA). Similarly, root exudate profiles differed between the mutants; however, the largest variance from the wild type (Col-0) was observed in abcg30, which showed increased phenolics and decreased sugars. In support of this biochemical observation, whole genome expression analyses of abcg30 roots revealed that some genes involved in Biosynthesis and transport of Secondary Metabolites were up-regulated while some sugar transporters were down-regulated compared with genome expression in wild type roots. Microbial taxa associated with Col-0 and abcg30 cultured soils determined by pyrosequencing, revealed that exudates from abcg30 cultivated a microbial community with a relatively greater abundance of potentially beneficial bacteria (i.e. PGPRs, nitrogen fixers) and were specifically enriched in bacteria involved in heavy metal remediation. In summary, this is the first report of a single gene mutation from a functional plant mutant influencing the surrounding community of soil organisms, showing that genes are not only important for intrinsic plant physiology but for the interactions with the surrounding community of organisms.

Phytochemistry. 2009 Oct 21;
Peluffo L, Lia V, Troglia C, Maringolo C, Norma P, Escande A, Esteban Hopp H, Lytovchenko A, Fernie AR, Heinz R, Carrari F

We report a comprehensive primary metabolite profiling of sunflower (Helianthus annuus) genotypes displaying contrasting behavior to Sclerotinia sclerotiorum infection. Applying a GC-MS-based metabolite profiling approach, we were able to identify differential patterns involving a total of 63 Metabolites including major and minor sugars and sugar alcohols, organic acids, amino acids, fatty acids and few soluble Secondary Metabolites in the sunflower capitulum, the main target organ of pathogen attack. Metabolic changes and disease incidence of the two contrasting genotypes were determined throughout the main infection period (R5.2-R6). Both point-by-point and non-parametric statistical analyses showed metabolic differences between genotypes as well as interaction effects between genotype and time after inoculation. Network correlation analyses suggested that these metabolic changes were synchronized in a time-dependent manner in response to the pathogen. Concerted differential metabolic changes were detected to a higher extent in the susceptible, rather than the resistant genotype, thereby allowing differentiation of modules composed by intermediates of the same pathway which are highly interconnected in the susceptible line but not in the resistant one. Evaluation of these data also demonstrated a genotype specific regulation of distinct metabolic pathways, suggesting the importance of detection of metabolic patterns rather than specific metabolite changes when looking for metabolic markers differentially responding to pathogen infection. In summary, the GC-MS strategy developed in this study was suitable for detection of differences in carbon primary metabolism in sunflower capitulum, a tissue which is the main entry point for this and other pathogens which cause great detrimental impact on crop yield.

Blood. 2009 Oct 20;
Ben-Ami R, Lewis RE, Leventakos K, Kontoyiannis DP

In susceptible hosts, angioinvasion by Aspergillus fumigatus triggers thrombosis, hypoxia and proinflammatory cytokine release, all of which are stimuli for angiogenesis. We sought to determine whether A. fumigatus directly modulates angiogenesis. A. fumigatus culture filtrates profoundly inhibited the differentiation, migration and capillary tube formation of human umbilical vein endothelial cells in vitro. To measure angiogenesis at the site of infection, we devised an in vivo matrigel assay in cyclophosphamide-treated Balb/c mice with cutaneous invasive aspergillosis. Angiogenesis was significantly suppressed in matrigel plugs implanted in A. fumigatus-infected mice compared with plugs from uninfected control mice. The antiangiogenic effect of A. fumigatus was completely abolished by deletion of the global regulator of Secondary metabolism, laeA, and to a lesser extent by deletion of gliP, which controls gliotoxin production. Moreover, pure gliotoxin potently inhibited angiogenesis in vitro in a dose-dependent manner. Finally, overexpression of multiple angiogenesis-mediator encoding genes was observed in the lungs of cortisone-treated mice during early invasive aspergillosis, whereas gene-expression returned rapidly to baseline levels in cyclophosphamide/cortisone-treated mice. Taken together, these results indicate that suppression of angiogenesis by A. fumigatus both in vitro and in a neutropenic mouse model is mediated through Secondary metabolite production.

Gastroenterology. 2009 Oct 17;
Chavarria L, Oria M, Romero J, Alonso J, Lope-Piedrafita S, Cordoba J

BACKGROUND & AIMS:: Brain edema is a severe complication of acute liver failure (ALF) that has been related to ammonia levels. Two mechanisms have been proposed in the pathogenesis: vasogenic edema that is Secondary to the breakdown of the blood-brain barrier and cytotoxic edema caused by ammonia Metabolites in astrocytes. METHODS:: We applied magnetic resonance techniques to assess the intra or extracellular distribution of brain water and Metabolites in rat a model of devascularized ALF. The brain water content was assessed by gravimetry and blood-brain-barrier permeability was determined from the transfer constant of 14C-labeled sucrose. RESULTS:: Rats with ALF had a progressive decrease in the apparent diffusion coefficient (ADC) in all brain regions. The average decrease in ADC was significant in precoma (-14%) and coma stages (-20%). These changes, which indicate an increase of the intracellular water compartment, were followed by a significant increase in total brain water (coma 82.4+/-0.3% vs sham 81.6+/-0.3%; p=0.0001). Brain levels of glutamine (6 hours: 540%, precoma: 851%, coma: 1086%) and lactate (6 hours: 166%, precoma: 998%, coma: 3293%) showed a marked increase in ALF that paralleled the decrease in ADC and neurological outcome. In contrast, the transfer constant of 14C-sucrose was unaltered. CONCLUSION:: The pathogenesis of brain edema in an experimental model of ALF involves a cytotoxic mechanism: the metabolism of ammonia in astrocytes induces an increase of glutamine and lactate that appears to mediate cellular swelling. Therapeutic measures should focus on removing ammonia and improving brain energy metabolism.

Plant Biotechnol J. 2009 Dec; 7(9): 939-51
Cloutier M, Chen J, De Dobbeleer C, Perrier M, Jolicoeur M

A dynamic model for plant cell metabolism was used as a basis for a rational analysis of plant production potential in in vitro cultures. The model was calibrated with data from 3-L bioreactor cultures. A dynamic sensitivity analysis framework was developed to analyse the response curves of Secondary metabolite production to metabolic and medium perturbations. Simulation results suggest that a straightforward engineering of cell metabolism or medium composition might only have a limited effect on productivity. To circumvent the problem of the dynamic allocation of resources between growth and production pathways, the sensitivity analysis framework was used to assess the effect of stabilizing intracellular nutrient concentrations. Simulations showed that a stabilization of intracellular glucose and nitrogen reserves could lead to a 116% increase in the specific production of Secondary Metabolites compared with standard culture protocol. This culture strategy was implemented experimentally using a perfusion bioreactor. To stabilize intracellular concentrations, adaptive medium feeding was performed using model mass balances and estimations. This allowed for a completely automated culture, with controlled conditions and pre-defined decision making algorithm. The proposed culture strategy leads to a 73% increase in specific production and a 129% increase in total production, as compared with a standard batch culture protocol. The sensitivity analysis on a mathematical model of plant metabolism thus allowed producing new insights on the links between intracellular nutritional management and cell productivity. The experimental implementation was also a significant improvement on current plant bioprocess strategies.

J Biol Chem. 2009 Oct 19;
Takeuchi K, Kiefer P, Reimmann C, Keel C, Dubuis C, Rolli J, Vorholt JA, Haas D

Pseudomonas fluorescens CHA0, an antagonist of phytopathogenic fungi in the rhizosphere of crop plants, elaborates and excretes several Secondary Metabolites with antibiotic properties. Their synthesis depends on three small RNAs (RsmX, RsmY, RsmZ) whose expression is positively controlled by the GacS-GacA two-component system at high cell population densities. To find regulatory links between primary and Secondary metabolism in P. fluorescens and in the related species Pseudomonas aeruginosa, we searched for null mutations that affected central carbon metabolism as well as the expression of rsmY-gfp and rsmZ-gfp reporter constructs, however, without slowing down the growth rate in rich media. Mutation in the pycAB genes (for pyruvate carboxylase) led to down-regulation of rsmXYZ and Secondary metabolism, whereas mutation in fumA (for a fumarase isoenzyme) resulted in up-regulation of the three small RNAs and Secondary metabolism, in the absence of detectable nutrient limitation. These effects required the GacS sensor kinase, but not the accessory sensors RetS and LadS. An analysis of intracellular Metabolites in P. fluorescens revealed a strong positive correlation between small RNA expression and the pools of 2-oxoglutarate, succinate and fumarate. We conclude that Krebs cycle intermediates (already known to control GacA-dependent virulence factors in P. aeruginosa) exert a critical trigger function in Secondary metabolism via the expression of GacA-dependent small RNAs.

Gene. 2009 Oct 17;
Legrand S, Valot N, Nicolé F, Moja S, Baudino S, Jullien F, Magnard JL, Caissard JC, Legendre L

The outermost floral whorl, composed of sepals, is generally thought to function in the protection of reproductive tissues. In the plant family Lamiaceae, sepals are fused into a tube that is densely covered by hairs for mechanical defence and contains Secondary Metabolites for chemical defence against insects and abiotic stresses. Despite the importance of this tissue in plant fitness, virtually no study has addressed the basic aspects of sepal development and functioning. Because of its large size and its impressive metabolic activity (both in terms of quantity and diversity of Secondary Metabolites), we have used clary sage calyx as a model system to generate the first high throughput sequencing of the transcriptome of an angiosperm calyx. We applied massive parallel 454 pyrosequencing technology to a normalized cDNA extract and unveiled potential candidate genes for all steps of Secondary metabolite pathways (phenylpropanoids and terpenoids). It also proved efficient in predicting the expression of large numbers of transcription factors and, with the use of bioinformatics tools, it predicted in the same sequencing run the presence of a novel class of gene transcription regulatory elements, miRNAs, without the need to generate a separate miRNA library. In our clary sage EST library, 18 conserved miRNAs were predicted. Among them, 15 were present in most studied plant species while the others were only shared with limited or discrete plant lineages. A separate data mining of the same clary sage EST library suggested the presence of 19 potential target genes to the 18 predicted conserved miRNAs. These coded for only 6 transcription factors or F-box proteins, 11 metabolism or abiotic stress response related proteins and 2 products with no known predicted function. All in all, this study provides novel genomic information on an angiosperm calyx and an experimental framework to predict in a single step metabolic pathway enzymes and regulator genes including miRNAs.

Appl Microbiol Biotechnol. 2009 Oct 17;
Rodríguez-Ortiz R, Mehta BJ, Avalos J, Limón MC

The fungus Fusarium fujikuroi (Gibberella fujikuroi mating group C) exhibits a rich Secondary metabolism that includes the synthesis of compounds of biotechnological interest, such as gibberellins, bikaverin, and carotenoids. The effect of the carbon source on their production was checked using a two-phase incubation protocol, in which nine different sugars were added upon transfer of the fungus from repressed to appropriate inducing conditions, i.e., nitrogen starvation for gibberellins and bikaverin and illumination for carotenoids production. Most of the carbon sources allowed the synthesis of these Metabolites in significant amounts. However, bikaverin production was strongly increased by the presence of sucrose in comparison to other carbon sources, an effect not exhibited for the production of gibberellins and carotenoids. The bikaverin inducing effect was enhanced in the absence of phosphate and/or sulfate. Similar results were also observed in carotenoid-overproducing strains known to be altered in bikaverin production. The induction by salt starvation, but not by sucrose, correlated with an increase in messenger RNA levels of gene bik1, encoding a polyketide synthase of the bikaverin pathway.