Kegg Pathway: Biosynthesis of Secondary Metabolites

PLoS ONE. 2008; 3(7): e2724
Mehra S, Charaniya S, Takano E, Hu WS

Many microorganisms, including bacteria of the class Streptomycetes, produce various Secondary Metabolites including antibiotics to gain a competitive advantage in their natural habitat. The production of these compounds is highly coordinated in a population to expedite accumulation to an effective concentration. Furthermore, as antibiotics are often toxic even to their producers, a coordinated production allows microbes to first arm themselves with a defense mechanism to resist their own antibiotics before production commences. One possible mechanism of coordination among individuals is through the production of signaling molecules. The gamma-butyrolactone system in Streptomyces coelicolor is a model of such a signaling system for Secondary metabolite production. The accumulation of these signaling molecules triggers antibiotic production in the population. A pair of repressor-amplifier proteins encoded by scbA and scbR mediates the production and action of one particular gamma-butyrolactone, SCB1. Based on the proposed interactions of scbA and scbR, a mathematical model was constructed and used to explore the ability of this system to act as a robust genetic switch. Stability analysis shows that the butyrolactone system exhibits bistability and, in response to a threshold SCB1 concentration, can switch from an OFF state to an ON state corresponding to the activation of genes in the cryptic type I polyketide synthase gene cluster, which are responsible for production of the hypothetical polyketide. The switching time is inversely related to the inducer concentration above the threshold, such that short pulses of low inducer concentration cannot switch on the system, suggesting its possible role in noise filtering. In contrast, Secondary metabolite production can be triggered rapidly in a population of cells producing the butyrolactone signal due to the presence of an amplification loop in the system. S. coelicolor was perturbed experimentally by varying concentrations of SCB1, and the model simulations match the experimental data well. Deciphering the complexity of this butyrolactone switch will provide valuable insights into how robust and efficient systems can be designed using "simple" two-protein networks.

Biotechnol Bioeng. 1996 Jul 5; 51(1): 23-32
Beck HC, Lauritsen FR, Patrick JS, Cooks RG

Membrane inlet mass spectrometry has been used for the characterization of halogenated organic compounds produced by the fungus Bjerkandera adusta. Using this technique we obtained electron impact-, chemical ionization-, electron capture negative chemical ionization-mass spectra and tandem mass spectra directly from the growth medium. Through this direct analysis of the samples we identified novel bioconversion products and confirmed recently published data on the production of both chlorinated and brominated methoxybenzaldehyde Metabolites. Growth profiles of the culture grown on a defined medium showed that the production of Secondary Metabolites starts after approximately 6 days and reaches maximal concentrations of 25-250 muM after 15-20 days. Although delayed, the production of Secondary Metabolites paralleled a depletion of glucose from the medium and stopped shortly after all glucose had been consumed. Experiments in which fluoro- and bromo-labeled 4-methoxybenzaldehydes were added to the medium at day 8 showed biotransformation of these compounds into chloro-3-fluoro-4-methoxy-benzaldehyde and chloro-3-bromo-4-methoxybenzaldehyde, respectively. No dichlorinated products were observed, suggesting that halogenation takes place only at the meta position on the 4-methoxybenzaldehydes. These experiments are the first to bring direct evidence of a halogenation mechanism, where the enzymatic attack takes place directly on the 4-methoxybenzaldehyde intermediates. (c) John Wiley & Sons, Inc.

Chem Biol Interact. 2008 Jun 20;
Overk CR, Guo J, Chadwick LR, Lantvit DD, Minassi A, Appendino G, Chen SN, Lankin DC, Farnsworth NR, Pauli GF, van Breemen RB, Bolton JL

The lack of a safe and reliable alternative to hormone therapy (HT) for treating menopausal symptoms underscores the need for alternative therapies. OBJECTIVE: The purpose of this study was to assess the in vivo estrogenic effects of the botanical dietary supplements Trifolium pratense (red clover) and Humulus lupulus (hops), and two compounds obtained from H. lupulus, isoxanthohumol and 8-prenylnaringenin (8-PN) using the ovariectomized uterotrophic adult rat model. A H. lupulus extract and a 30% isoflavone extract of T. pratense were tested at three escalating doses as was one dose of isoxanthohumol for 21d. 8-Prenylnaringenin, the major estrogen in H. lupulus, was also tested at three relevant escalating doses. In order to determine the in vivo metabolism of 8-PN, the major phases I and II Metabolites were also identified. The primary outcome measure, uterus weight gain, indicated that H. lupulus and T. pratense did not have an estrogenic effect on the uterus, and none of the Secondary outcome measures were positive. In contrast, there was a clear dose response when 8-PN was evaluated where the middle and high doses of 8-PN were active. 8-Prenylnaringenin in rat plasma, liver, and mammary gland was measured and the major phases I and II 8-PN Metabolites were detected. Our findings suggest that while both the H. lupulus and T. pratense extracts do not have an effect on the rat uterus, 8-PN at equivalent doses to those previously used in humans did have an effect, and may therefore have a deleterious effect in women.

PLoS Biol. 2008 Jul 8; 6(7): e163
Chopra T, Banerjee S, Gupta S, Yadav G, Anand S, Surolia A, Roy RP, Mohanty D, Gokhale RS

In recent years, remarkable versatility of polyketide synthases (PKSs) has been recognized; both in terms of their structural and functional organization as well as their ability to produce compounds other than typical Secondary Metabolites. Multifunctional Type I PKSs catalyze the Biosynthesis of polyketide products by either using the same active sites repetitively (iterative) or by using these catalytic domains only once (modular) during the entire biosynthetic process. The largest open reading frame in Mycobacterium tuberculosis, pks12, was recently proposed to be involved in the Biosynthesis of mannosyl-beta-1-phosphomycoketide (MPM). The PKS12 protein contains two complete sets of modules and has been suggested to synthesize mycoketide by five alternating condensations of methylmalonyl and malonyl units by using an iterative mode of catalysis. The bimodular iterative catalysis would require transfer of intermediate chains from acyl carrier protein domain of module 2 to ketosynthase domain of module 1. Such bimodular iterations during PKS Biosynthesis have not been characterized and appear unlikely based on recent understanding of the three-dimensional organization of these proteins. Moreover, all known examples of iterative PKSs so far characterized involve unimodular iterations. Based on cell-free reconstitution of PKS12 enzymatic machinery, in this study, we provide the first evidence for a novel "modularly iterative" mechanism of Biosynthesis. By combination of biochemical, computational, mutagenic, analytical ultracentrifugation and atomic force microscopy studies, we propose that PKS12 protein is organized as a large supramolecular assembly mediated through specific interactions between the C- and N-terminus linkers. PKS12 protein thus forms a modular assembly to perform repetitive condensations analogous to iterative proteins. This novel intermolecular iterative biosynthetic mechanism provides new perspective to our understanding of polyketide biosynthetic machinery and also suggests new ways to engineer polyketide Metabolites. The characterization of novel molecular mechanisms involved in Biosynthesis of mycobacterial virulent lipids has opened new avenues for drug discovery.

J Biol Chem. 2008 Jul 7;
Borodina I, Siebring J, Zhang J, Smith CP, van Keulen G, Dijkhuizen L, Nielsen J

Streptomycetes are exploited for production of a wide range of Secondary Metabolites and there is much interest in enhancing the level of production of these Metabolites. Secondary Metabolites are synthesized in dedicated biosynthetic routes, but precursors and co-factors are derived from the primary metabolism. High level production of antibiotics in streptomycetes therefore requires engineering of primary metabolism. Here we demonstrate this by targeting a key enzyme in glycolysis, phosphofructokinase, leading to improved antibiotic production in Streptomyces coelicolor A3(2). Deletion of pfkA2 (SCO5426), one of three annotated pfkA homologues in S. coelicolor A3(2), resulted in a higher production of the pigmented antibiotics actinorhodin and undecylprodigiosin. The pfkA2 deletion strain had an increased carbon flux through the pentose phosphate pathway, as measured by (13)C metabolic flux analysis, establishing the ATP-dependent PfkA2 as a key player in determining the carbon flux distribution. The increased pentose phosphate pathway flux appeared largely due to accumulation of glucose-6-phosphate and fructose-6-phosphate, as experimentally observed in the mutant strain. Through genome-scale metabolic model simulations we predicted that decreased phosphofructokinase activity leads to an increase in pentose phosphate pathway flux and in flux to pigmented antibiotics and pyruvate supposedly through increased production of NADPH and acetyl-CoA. Integrated analysis of gene expression data using the genome-scale metabolic model further revealed transcriptional changes in genes encoding redox co-factor-dependent enzymes as well as those encoding pentose phosphate pathway enzymes and enzymes involved in storage carbohydrate Biosynthesis.

Biotechnol Bioeng. 1992 Jan 20; 39(2): 195-202
Green KD, Thomas NH, Callow JA

Transformed roots of Nicotiana glauce synthesize the alkaloids nicotine and anabasine at levels reflecting the parent plants. Media composition, strength, and pH were evaluated with respect to biomass yield and productivity. Full-strength Gamborg's B5 medium proved the best for biomass yield while half-strength, or low-salt, medium enhanced alkaloid accumulation. A detailed investigation of media nitrate levels demonstrated how these may be manipulated to promote growth and intracellular or extracellular alkaloid levels. High nitrate concentrations were found to significantly enhance media alkaloid levels at the end of the growth phase. Media pH is also important, although transformed roots will grow in Gamborg's B5 medium between pH 3 and 9, root biomass is favored by an increase in medium alkalinity, while alkaloid release is encouraged by mildly acidic pH.Transformed roots release a proportion of their Secondary Metabolites into the growth medium. By continually removing root products, any feedback inhibition on enzymatic reactions is reduced, as are the toxic effects resulting from product accumulation. In this article we describe the use of Amberlite resins (XAD-2 and XAD-4) to enhance alkaloid levels (nicotine and anabasine) of hairy root cultures of Nicotiana glauca by a factor of 10 with no adverse effect on root growth. The performance of the Amberlite columns was subsequently investigated with respect to alkaloid adsorption and desorption, including an evaluation of the effects of pH and loading capacity. The resins also adsorb media constituents which are identified and quantified as part of this work. Resulting nutritional stresses are thought to be partly responsible for enhancing Secondary metabolism at the expense of biomass yield. However, the net effects of using Amberlite resins as a means of product removal significantly increases the overall product yield and the extent to which products are released into the growth medium.

Neurology. 2008 Jul 1; 71(1): 44-9
Debray FG, Boulanger Y, Khiat A, Decarie JC, Orquin J, Roy MS, Lortie A, Ramos F, Verhoeven NM, Struys E, Blom HJ, Jakobs C, Levy E, Mitchell GA, Lambert M

OBJECTIVE: To investigate whether Secondary impairment of the transmethylation pathway is a mechanism underlying the neurologic involvement in homocystinuria due to remethylation defects. METHODS: Twelve patients with neurologic disease due to remethylation defects were examined by brain magnetic resonance spectroscopic imaging ((1)H MRSI). Brain N-acetylaspartate, choline-containing compounds (Cho), and creatine (Cr) were quantified and compared to with controls. Metabolites of remethylation cycle and creatine Biosynthesis pathway were measured in plasma and urine. RESULTS: MRSI revealed isolated Cho deficiency in all regions examined (mean concentration units +/- SD, patients vs controls): frontal white matter (0.051 +/- 0.010 vs 0.064 +/- 0.010; p = 0.001), lenticular nucleus (0.056 +/- 0.011 vs 0.069 +/- 0.009; p < 0.001), and thalamus (0.063 +/- 0.010 vs 0.071 +/- 0.007; p = 0.006). In contrast to controls, the Cho/Cr ratio decreased with age in patients in the three brain regions examined. Low creatine urinary excretion (p < 0.005), normal urine and plasma guanidinoacetate, and a paradoxical increase in plasma S-adenosylmethionine (p < 0.005) concentrations were observed. CONCLUSION: Patients with homocystinuria due to remethylation defects have an isolated brain choline deficiency, probably Secondary to depletion of labile methyl groups produced by the transmethylation pathway. Although biochemical studies suggest mild peripheral creatine deficiency, brain creatine is in the reference range, indicating a possible compartmentation phenomenon. Paradoxical increase of S-adenosylmethionine suggests that Secondary inhibition of methylases contributes to the transmethylation defect in these conditions.

Talanta. 2008 Jul 15; 76(2): 276-81
Liu HM, Zhang XH, Huang XQ, Cao CX, Xu YQ

Natural phenazines in Secondary Metabolites of bacteria have been receiving increasing attention in recent years due to their potential usage as antibiotics. In the present study, a rapid and reliable capillary zone electrophoresis (CZE) method was developed and validated for monitoring for the first time dynamic phenazine-1-carboxylic acid (PCA) and the 2-hydroxyphenazine (2-OH-PHZ) production of Pseudomonas chlororaphis GP72 during the entire fermentation cycle. The paper begins with the optimization of separate conditions for 2-OH-PHZ and PCA together with phenazine (PHZ), which is used as internal standard. The optimized conditions are: 10mM, pH 7.3 phosphate buffer, a fused-silica capillary with a total length of 49 cm x 75 microm ID, 375 microm OD with an effective length of 40 cm, 25 kV, 13 mbar 10s pressure sample injection and 25 degrees C air-cooling. The three compounds could be separated within 2 min under optimized conditions. The validation of the newly developed study shows the linear response of 2-OH-PHZ and PCA ranging from 10 to 250 microg mL(-1) with high correlation coefficient (r=0.9997 and 0.9993, n=7), low limits of detection (0.47 and 0.38 microg mL(-1)) and quantification (1.56 and 1.28 microg mL(-1)), respectively. Good precision values for intra- and inter-day detection and acceptable individual recovery ranges for 2-OH-PHZ and PCA are indicated. The newly developed method was also validated through monitoring dynamic PCA and 2-OH-PHZ production of P. chlororaphis GP72 during an 84 h growth cycle.

Fungal Genet Biol. 2008 May 21;
Bölker M, Basse CW, Schirawski J

The dimorphic phytopathogenic fungus Ustilago maydis encounters different environments during its life cycle. As free-living unicellular haploid cell, the fungus must compete with other microorganisms for space and nutrients. As a pathogen, it also has to withstand the defense reactions of its host plant corn and to subvert the plant metabolism for its own purposes. During these interactions small molecules produced by the fungus serve important functions in the communication with its host and other organisms. The genome sequence of U. maydis makes it possible to deduce the full inventory of enzymatic functions that are involved in the production of these Secondary Metabolites. Although the fungus is known to secrete interesting small molecules the genome contains surprisingly few genes involved in the Biosynthesis of polyketides (PKS) and non-ribosomal peptide synthetases (NRPS). Additional genes predicted to be part of Secondary metabolism are located in subtelomeric regions suggesting that they are subject to high genetic and genomic variation. Here we review the pathways for the production of extracellular glycolipids that serve as biosurfactants, iron-chelating siderophores, tryptophan-derived indole pigments and indole acetic acid, the elucidation of which has greatly profited from the availability of the U. maydis genome sequence.

Plant Cell. 2008 Jun 20;
Ringli C, Bigler L, Kuhn BM, Leiber RM, Diet A, Santelia D, Frey B, Pollmann S, Klein M

Flavonoids are Secondary Metabolites known to modulate plant growth and development. A primary function of flavonols, a subgroup of flavonoids, is thought to be the modification of auxin fluxes in the plant. Flavonols in the cell are glycosylated, and the repressor of lrx1 (rol1) mutants of Arabidopsis thaliana, affected in rhamnose Biosynthesis, have a modified flavonol glycosylation profile. A detailed analysis of the rol1-2 allele revealed hyponastic growth, aberrant pavement cell and stomatal morphology in cotyledons, and defective trichome formation. Blocking flavonoid Biosynthesis suppresses the rol1-2 shoot phenotype, suggesting that it is induced by the modified flavonol profile. The hyponastic cotyledons of rol1-2 are likely to be the result of a flavonol-induced increase in auxin concentration. By contrast, the pavement cell, stomata, and trichome formation phenotypes appear not to be induced by the modified auxin distribution. Together, these results suggest that changes in the composition of flavonols can have a tremendous impact on plant development through both auxin-induced and auxin-independent processes.

J Anim Sci. 2008 Jun 20;
Lourenço M, Cardozo PW, Calsamiglia S, Fievez V

Four different plant Secondary Metabolites were screened for their effect on rumen biohydrogenation of forage long chain fatty acids, using dual-flow continuous culture fermenters. Treatments were: control (no additive), positive control (12 mg/L of monensin), and plant extracts (500 and 1,000 mg/L triterpene saponin; 250 and 500 mg/L quercetin; 250 mg/L eugenol; 500 mg/L cinnamaldehyde). Monensin increased propionate, decreased acetate and butyrate proportions, and inhibited the complete biohydrogenation of fatty acids resulting in the accumulation of intermediates of the biohydrogenation process (C18:2 trans-11, cis-15 rather than C18:1 trans-11). Cinnamaldehyde reduced total VFA concentration and proportions of odd and branched chain fatty acids in total fat effluent. Apparent biohydrogenation of C18:2n-6 and C18:3n-3 was also lower, and a shift from the major known biohydrogenation pathway to a Secondary pathway of C18:2n-6 was observed, as evidenced by an accumulation of C18:1 trans-10 and trans-10, cis-12 CLA. Quercetin (500 mg/L) increased total VFA concentration, but no shifts in the pathways or extent of biohydrogenation were observed. Eugenol resulted in the accumulation of C18:1 trans-15 and C18:1 cis-15, end products of an alternative biohydrogenation pathway of C18:3n-3. Triterpene saponins did not affect the fermentation pattern, the biohydrogenation pathways, or the extent of biohydrogenation. At the doses tested in this study, we could only show a direct relation between changes in the rumen fatty acid metabolism and the presence of cinnamaldehyde, but not for eugenol, quercetin, or triterpene saponins.

Planta. 2008 Jun 18;
Lanot A, Hodge D, Lim EK, Vaistij FE, Bowles DJ

The phenylpropanoid pathway is used in Biosynthesis of a wide range of soluble Secondary Metabolites including hydroxycinnamic acid esters, flavonoids and the precursors of lignin and lignans. In Arabidopsis thaliana a small cluster of three closely related genes, UGT72E1-E3, encode glycosyltransferases (GTs) that glucosylate phenylpropanoids in vitro. This study explores the effect of constitutively over-expressing two of these GTs (UGT72E1 and E3) in planta using the CaMV-35S promoter to determine whether phenylpropanoid homeostasis can be altered in a similar manner to that achieved by over-expression of UGT72E2 as previously reported. The data show that impact of over-expressing UGT72E3 in leaves is highly similar to that of UGT72E2 in that the production of massive levels of coniferyl and sinapyl alcohol 4-O-glucosides and a substantial loss in sinapoyl malate. In contrast, the over-expression of UGT72E1 in leaves led only to minimal changes in coniferyl alcohol 4-O-glucoside and no effect was observed on sinapoyl malate levels. In roots, over-expression of both UGTs led to some increase in the accumulation of the two glucosides. The cell specificity expression of the whole UGT72E gene cluster was investigated and interestingly only UGT72E3 was found to be wound and touch responsive.

J Theor Biol. 2008 Apr 25;
Bastian P, Chavarría-Krauser A, Engwer C, Jäger W, Marnach S, Ptashnyk M

Hairy roots are plants genetically transformed by Agrobacterium rhizogenes, which do not produce shoots and are composed mainly by roots. Hairy roots of Ophiorrhiza mungos Linn. are currently gaining interest of pharmacologists, since a Secondary product of their metabolism, camptothecin, is used in chemotherapy. To optimize the production of valuable Secondary Metabolites it is necessary to understand the metabolism and growth of these roots systems. In this work, a mathematical model for description of apical growth of a dense root network (e.g. hairy roots) is derived. A continuous approach is used to define densities of root tips and root volume. Equations are posed to describe the evolution of these and are coupled to the distribution of nutrient concentration in the medium and inside the network. Following the principles of irreversible thermodynamics, growth velocity is defined as the sum over three different driving forces: nutrient concentration gradients, space gradients and root tip diffusion. A finite volume scheme was used for the simulation and parameters were chosen to fit experimental data from O. mungos Linn. hairy roots. Internal nutrient concentration determines short-term growth. Long-term behavior is limited by the total nutrient amount in the medium. Therefore, mass yield could be increased by guaranteeing a constant supply of nutrients. Increasing the initial mass of inoculation did not result in higher mass yields, since nutrient consumption due to metabolism also rose. Four different growth strategies are compared and their properties discussed. This allowed to understand which strategy might be the best to increase mass production optimally. The model is able to describe very well the temporal evolution of mass increase and nutrient uptake. Our results provide further understanding of growth and density distribution of hairy root network and therefore it is a sound base for future applications to describe, e.g., Secondary metabolite production.

Exp Dermatol. 2008 Jul; 17(7): 625
Fischer TW, Naumann V, Bodo E, Paus R

Based on extensive investigation of melatonin in human cell models (keratinocytes, fibroblasts), a great variety of melatonin actions, mechanisms, intracellularly localized synthesis and metabolism as well as receptor expression has been unravelled, identifying melatonin as a key player in cutaneous biology and dermato-endocrinology. Melatonin was reported to act directly protective as a radical scavenger in many conditions of skin-related stress (ultraviolet radiation, ionizing radiation, thermal injury, ulcer formation, skin flap necrosis) and to modulate cell growth via membrane (MT1, MT2), cytosolic (MT3/NQO2) and nuclear receptors (RORalpha). Membrane and nuclear melatonin receptor expression is hair cycle dependent in murine hair follicles, whereas estradiol receptor (ERalpha) expression is down-regulated by melatonin. Furthermore, the modulation of Secondary endocrine signalling (e.g. prolactin release, estrogen receptor-mediated signalling) in the skin is mediated by melatonin. Melatonin levels of magnitudes higher than in the plasma have been detected in human keratinocytes and murine and human hair follicles, whereby noradrenalin, the classical stimulator of melatonin synthesis, increased melatonin production in hair follicles. Additionally, a melatoninergic antioxidative system (MAS) of the skin has been identified with UV-enhanced formation of melatonin Metabolites, 2-OH-melatonin and AFMK, the latter being a strong antioxidant itself. Alternatively, melatonin indirectly protects cells by up-regulating gene expression and activity of non-melatonin autonomous protective systems, the intracellular antioxidative enzyme network which is composed of Mn-SOD, Cu-Zn-SOD, CAT and GPx. To further drive melatonin research towards the human organism level, two human organ culture models have been choosen or newly established i) the human hair organ culture model and ii) the human full thickness skin model, respectively. Two crucial stressors of human skin/hair biology are used to investigate their morphology and functional effect expression in these models: ultraviolet radiation and chemotherapy-induced cytotoxicity. Standardized stress conditions in both models serve to investigate differential expression of stress levels, their time-dependent dynamics and effect quality. Finally, reduction of UV-damaged keratinocytes (sun-burn cells) by melatonin in the full-skin organ model and anti-apoptotic effects in chemotherapy-induced hair follicle damage have been observed.

Science. 2008 Jun 13; 320(5882): 1504-6
Bayram O, Krappmann S, Ni M, Bok JW, Helmstaedt K, Valerius O, Braus-Stromeyer S, Kwon NJ, Keller NP, Yu JH, Braus GH

Differentiation and Secondary metabolism are correlated processes in fungi that respond to light. In Aspergillus nidulans, light inhibits sexual reproduction as well as Secondary metabolism. We identified the heterotrimeric velvet complex VelB/VeA/LaeA connecting light-responding developmental regulation and control of Secondary metabolism. VeA, which is primarily expressed in the dark, physically interacts with VelB, which is expressed during sexual development. VeA bridges VelB to the nuclear master regulator of Secondary metabolism, LaeA. Deletion of either velB or veA results in defects in both sexual fruiting-body formation and the production of Secondary Metabolites.

J Biotechnol. 2008 Jun 30; 135(3): 255-61
Sandmann A, Frank B, Müller R

Myxobacteria are proficient producers of biologically active Secondary Metabolites. However, efforts to exploit these natural products for the development of new therapeutics and agrochemicals are frequently hampered by low production levels. We describe here a transposon-based strategy to identify genes encoding regulators of Secondary metabolite Biosynthesis in the myxobacterium Angiococcus disciformis An d48, which produces the highly efficient electron transport inhibitor myxothiazol. Extracts from 1200 transposon mutants were analyzed by HPLC, leading to the identification of six mutants in which myxothiazol production was increased by as much as 30-fold. Identifying the sites of integration coupled with sequencing of flanking regions, showed that some of the inactivated genes encode proteins with similarity to known bacterial regulators such as two-component systems and serine-threonine protein kinases. However, other gene products do not resemble any characterized proteins. Taken together, these data show that this transposon-based strategy is a valuable tool to identify regulatory genes of Secondary metabolism, including gene loci which cannot be detected using current in silico approaches.

Plant Physiol. 2008 Jun 11;
Hagel JM, Weljie AM, Vogel HJ, Facchini PJ

Opium poppy (Papaver somniferum) produces a diverse array of bioactive benzylisoquinoline alkaloids and has emerged as a versatile model system to study plant alkaloid metabolism. The plant is widely cultivated as the only commercial source of the narcotic analgesics morphine and codeine. Variations in plant Secondary metabolism as a result of genetic diversity are often associated with perturbations in other metabolic pathways. As part of a functional genomics platform, we have used (1)H NMR metabolite profiling for the analysis of primary and Secondary metabolism in opium poppy. Aqueous and chloroform extracts of six different opium poppy cultivars were subjected to chemometric analysis. Principle component analysis of the (1)H NMR spectra for latex extracts clearly distinguished two varieties, including a low-alkaloid variety, and a high-thebaine, low-morphine cultivar. Distinction was also made between pharmaceutical-grade opium poppy cultivars and a condiment variety. Such phenotypic differences were not observed in root extracts. Loadings plots confirmed that morphinan alkaloids contributed predominantly to the variance in latex extracts. Quantification of 34 root and 21 latex Metabolites, performed using Chenomx NMR Suite v. 4.6, showed major differences in the accumulation of specific alkaloids in the latex of the low-alkaloid and high-thebaine, low-morphine varieties. Relatively few differences were found in the levels of other Metabolites indicating that the variation was specific for alkaloid metabolism. Exceptions in the low-alkaloid cultivar included an increased accumulation of the alkaloid precursor tyramine, and reduced levels of sucrose, some amino acids and malate. Real-time PCR analysis of 42 genes involved in primary and Secondary metabolism showed differential gene expression mainly associated with alkaloid Biosynthesis. Reduced alkaloid levels in the condiment variety were associated the reduced abundance of transcripts encoding several alkaloid biosynthetic enzymes.

J Biol Chem. 2008 Jun 10;
Shao Z, Blodgett JA, Circello BT, Eliot AC, Woodyer R, Li G, van der Donk WA, Metcalf WW, Zhao H

Phosphonic acids encompass a common, yet chemically diverse, class of natural products that often possess potent biological activities. Here we report that, despite the significant structural differences between many of these compounds, their biosynthetic routes contain an unexpected common intermediate, 2-hydroxyethyl-phosphonate (HEP), which is synthesized from phosphonoacetaldehyde (PnAA) by a distinct family of metal-dependent alcohol dehydrogenases (ADHs). Although the sequence identity of the ADH family members is relatively low (34-37%), in vitro biochemical characterization of the homologs involved in Biosynthesis of the antibiotics fosfomycin, phosphinothricin tripeptide (PTT), and dehydrophos (formerly A53868) unequivocally confirms their enzymatic activities. These unique ADHs have exquisite substrate specificity, unusual metal requirements, and an unprecedented monomeric quaternary structure. Further, sequence analysis shows that these ADHs form a monophyletic group along with additional family members encoded by putative phosphonate biosynthetic gene clusters. Thus, the reduction of phosphonoacetaldehyde to hydroxyethyl-phosphonate may represent a common step in the Biosynthesis of many phosphonate natural products, a finding that lends insight into the evolution of phosphonate biosynthetic pathways and the chemical structures of new C-P containing Secondary Metabolites.

Appl Environ Microbiol. 2008 Jun 6;
Große C, Heinekamp T, Kniemeyer O, Gehrke A, Brakhage AA

Aspergillus fumigatus is an opportunistic human pathogenic fungus, causing severe infections in immunocompromised patients. Cyclic AMP signal transduction plays an important role for virulence. A central component of this signaling cascade is the protein kinase A (PKA), which regulates cellular processes by phosphorylation of specific target proteins. Here, we describe the generation and analysis of A. fumigatus mutants expressing the gene encoding the catalytic subunit of PKA, pkaC1, under control of an inducible promoter. Strains overexpressing pkaC1 showed high PKA activity, reduced growth, sporulation deficiency and formation of a dark pigment in the mycelium. These data indicate that cAMP/PKA signaling is involved in the regulation of important processes like growth, asexual reproduction and Biosynthesis of Secondary Metabolites. Furthermore, elevated PKA activity led to increased expression of the pksP gene. The polyketide synthase PksP is an essential enzyme for production of dihydroxynaphthalene (DHN) melanin in A. fumigatus and contributes to virulence. Our results suggest, that increased pksP expression is responsible for pigment formation in the mycelium. Comparative proteome analysis of the pkaC1 overexpressing strain and the wild-type strain led to the identification of proteins regulated by the cAMP/PKA signal transduction pathway. We showed, that elevated PKA activity resulted in activation of stress-associated proteins and of enzymes involved in protein Biosynthesis and glucose catabolism. By contrast, proteins which were involved in nucleotide and amino acid Biosynthesis were downregulated as well as enzymes involved in catabolism of carbon sources other than glucose.

Curr Opin Pharmacol. 2008 Jun 3;
Baltz RH

The soil actinomycetes have been important sources of antibiotics, but were nearly abandoned in recent years in favor of high-throughput target-based screening of chemical libraries. The latter approach has not been productive, so it is time to reinvigorate the discovery of new antibiotics from a proven source. Recent progress has been made on antibiotic discovery from actinomycetes by using high-throughput fermentation, isolation of marine actinomycetes, mining genomes for cryptic pathways, and combinatorial Biosynthesis to generate new Secondary Metabolites related to existing pharmacophores.