Halogenation-Guided Chemical Screening Provides Insight into Tjipanazole Biosynthesis by the Cyanobacterium Fischerella ambigua

Halogenated natural products (HNPs) show a wide range of interesting biological activities. Chemistry-guided screening with a software tool dedicated to identifying halogenated compounds in HPLC-MS data indicated the presence of several uncharacterised HNPs in an extract of the cyanobacterium Fischerella ambigua (Näg.) Gomont 108b. Three new natural products, tjipanazoles K, L, and M, were isolated from this strain together with the known tjipanazoles D and I. Taking into account the structures of all tjipanazole derivatives detected in this strain, reanalysis of the tjipanazole biosynthetic gene cluster allowed us to propose a biosynthetic pathway for the tjipanazoles. As the isolated tjipanazoles show structural similarity to arcyriaflavin A, an inhibitor of the clinically relevant multidrug-transporter ABCG2 overexpressed by different cancer cell lines, the isolated compounds were tested for ABCG2 inhibitory activity. Only tjipanazole K showed appreciable transporter inhibition, whereas the compounds lacking the pyrrolo[3,4-c] ring or featuring additional chloro substituents were found to be much less active.     

Flower Scent of Floral Oil-Producing Lysimachia punctata as Attractant for the Oil-Bee Macropis fulvipes

Most flowers offer nectar and/or pollen as a reward for pollinators. However, some plants are known to produce mostly fatty oil in the flowers, instead of nectar. This oil is exclusively collected by specialized oil-bees, the pollinators of the oil-plants. Little is known about chemical communication in this pollination system, especially how the bees find their hosts. We collected the floral and vegetative scent emitted by oil-producing Lysimachia punctata by dynamic headspace, and identified the compounds by gas chromatography coupled to mass spectrometry. Thirty-six compounds were detected in the scent samples, several of which were flower-specific. Pentane extracts of flowers and floral oil were tested on Macropis fulvipes in a biotest. Flower and oil extracts attracted the bees, and some of the compounds identified are seldom found in the floral scent of other plants; these may have been responsible for the attraction of the bees.     

Epoxyquinone formation catalyzed by a two-component flavin-dependent monooxygenase involved in biosynthesis of the antibiotic actinorhodin

The biosynthetic gene cluster of the aromatic polyketide antibiotic actinorhodin (ACT) in Streptomyces coelicolor A3(2) carries a pair of genes, actVA-ORF5 and actVB, that encode a two-component flavin-dependent monooxygenase (FMO). Our previous studies have demonstrated that the ActVA-ORF5/ActVB system functions as a quinone-forming C-6 oxygenase in ACT biosynthesis. Furthermore, we found that this enzyme system exhibits an additional oxygenation activity with dihydrokalafungin (DHK), a proposed intermediate in the ACT biosynthetic pathway, and generates two reaction products. These compounds were revealed to be monooxygenated derivatives of kalafungin, which is spontaneously formed through oxidative lactonization of DHK. Their absolute structures were elucidated from their NMR spectroscopic data and by computer modeling and X-ray crystallography as (5S,14R)-epoxykalafungin and (5R,14S)-epoxykalafungin, demonstrating an additional epoxyquinone-forming activity of the ActVA-ORF5/ActVB system in vitro.     

A type I/type III polyketide synthase hybrid biosynthetic pathway for the structurally unique ansa compound kendomycin

Kendomycin is a bioactive polyketide that is produced by various Streptomyces strains. It displays strong antibiotic activities against a wide range of bacteria and exhibits remarkable cytotoxic effects on the growth of several human cancer cell lines. In this study we cloned the corresponding biosynthetic locus from the producer Streptomyces violaceoruber (strain 3844-33C). Our analysis shows that a mixed type I/type III polyketide synthase pathway is responsible for the formation of the fully carbogenic macrocyclic scaffold of kendomycin, which is unprecedented among all of the ansa compounds that have been isolated so far. Heterologous expression of a gene set in Streptomyces coelicolor shows that 3,5-dihydroxybenzoic acid is an intermediate in the starter unit biosynthesis that is initiated by the type III polyketide synthase. The identification of the kendomycin biosynthetic gene cluster sets the stage to study a novel chain termination mechanism by a type I PKS that leads to carbocycle formation and provides the starting material for the heterologous expression of the entire pathway, and the production of novel derivatives by genetic engineering.     

ANTENNAL AND BEHAVIORAL RESPONSES OF GRAPEVINE MOTH <Emphasis Type="Italic">Lobesia botrana</Emphasis> FEMALES TO VOLATILES FROM GRAPEVINE

Grapevine moth Lobesia botrana is the economically most important insect of grapevine Vitis vinifera in Europe. Flower buds, flowers, and green berries of Chardonnay grapevine are known to attract L. botrana for oviposition. The volatile compounds collected from these phenological stages were studied by gas chromatography—mass spectrometry, and the antennal response of L. botrana females to these headspace collections was recorded by gas chromatography—electroantennography. The compounds found in all phenological stages, which consistently elicited a strong antennal response, were pentadecane, nonanal, and -farnesene. In a wind tunnel, gravid L. botrana females flew upwind to green grapes, as well as to headspace collections from these berries released by a piezoelectric sprayer release device. However, no females landed at the source of headspace volatiles, possibly due to inappropriate concentrations or biased ratios of compounds in the headspace extracts.     

Automated Identification of Depsipeptide Natural Products by an Informatic Search Algorithm

Nonribosomal depsipeptides are a class of potent microbial natural products, which include several clinically approved pharmaceutical agents. Genome sequencing has revealed a large number of uninvestigated natural‐product biosynthetic gene clusters. However, while novel informatic search methods to access these gene clusters have been developed to identify peptide natural products, depsipeptide detection has proven challenging. Herein, we present an improved version of our informatic search algorithm for natural products (iSNAP), which facilitates the detection of known and genetically predicted depsipeptides in complex microbial culture extracts. We validated this technology by identifying several depsipeptides from novel producers, and located a large number of novel depsipeptide gene clusters for future study. This approach highlights the value of chemoinformatic search methods for the discovery of genetically encoded metabolites by targeting specific areas of chemical space.     

Secondary Metabolites Released by The Burying Beetle <Emphasis Type="Italic">Nicrophorus vespilloides</Emphasis>: Chemical Analyses and Possible Ecological Functions

Burying beetles of the genus Nicrophorus localize small vertebrate carcasses by emitted volatiles. The carcass that serves as reproduction and breeding site is buried in the soil by the beetles. Biparental care for offspring includes both preservation of the carrion and its preparation as diet and nursery. Buried carcasses show no signs of microbial decay, and those experimentally treated with Nicrophorus secretions are known to grow fewer bacteria and fungi. In order to investigate the chemical composition of these secretions, we used GC-MS for analysis of methanolic extracts of anal and oral secretions released by adult N. vespilloides. Furthermore, we analyzed the headspace of adult N. vespilloides by SPME-GC-MS and searched for compounds with known antimicrobial activity. We identified 34 compounds in the headspace, and anal and oral secretions, 26 of which occurred consistently. We discuss the ecological relevance of these compounds with respect to both their antimicrobial activity and ecological relevance.     

Direct Cloning, Genetic Engineering, and Heterologous Expression of the Syringolin Biosynthetic Gene Cluster in E. coli through Red/ET Recombineering

The reconstruction of a natural product biosynthetic pathway from bacteria in a vector and subsequent heterologous expression in a technically amenable microbial system represents an efficient alternative to empirical traditional methods for functional discovery, yield improvement, and genetic engineering to produce "unnatural" derivatives. However, the traditional cloning procedure based on genomic library construction and screening are complicated due to the large size (>10 kb) of most biosynthetic pathways. Here, we describe the direct cloning of a partial syringolin biosynthetic gene cluster (sylCDE, 19 kb) from a digested genomic DNA mixture of Pseudomonas syringae into a plasmid in which sylCDE is under the control of an inducible promoter by one step linear-plus-linear homologous recombination (LLHR) in Escherichia coli. After expression in E. coli GB05-MtaA, two new syringolin derivatives were discovered. The complete syringolin gene cluster was assembled by addition of sylAB and exchange of a synthetic bidirectional promoter against the native promoter to drive sylB and sylC expression by using Red/ET recombineering. The varying production distribution of syringolin derivatives showed the different efficiencies of native and synthetic promoters in E. coli. The successful reconstitution and expression of the syringolin biosynthetic pathway shows that Red/ET recombineering is an efficient tool to clone and engineer secondary metabolite biosynthetic pathways.     

Identification and Characterization of the Streptazone E Biosynthetic Gene Cluster in Streptomyces sp. MSC090213JE08

Streptazone derivatives isolated from Streptomyces species are piperidine alkaloids with a cyclopenta[b]pyridine scaffold. Previous studies indicated that these compounds are polyketides, but the biosynthetic enzymes responsible for their synthesis are unknown. Here, we have identified the streptazone E biosynthetic gene cluster in Streptomyces sp. MSC090213JE08, which encodes a modular type I PKS and tailoring enzymes that include an aminotransferase, three oxidoreductases, and two putative cyclases. The functions of the six tailoring enzymes were analyzed by gene disruption, and two putative biosynthetic intermediates that accumulated in particular mutants were structurally elucidated. On the basis of these results, we propose a pathway for the biosynthesis of streptazone E in which the two putative cyclases of the nuclear transport factor 2–like superfamily are responsible for C?C bond formation coupled with epoxide ring opening to give the five‐membered ring of streptazone E.     

Structural Diversification of Lyngbyatoxin A by Host‐Dependent Heterologous Expression of the tleABC Biosynthetic Gene Cluster

Natural products have enormous structural diversity, yet little is known about how such diversity is achieved in nature. Here we report the structural diversification of a cyanotoxin—lyngbyatoxin A—and its biosynthetic intermediates by heterologous expression of the Streptomyces‐derived tleABC biosynthetic gene cluster in three different Streptomyces hosts: S. lividans, S. albus, and S. avermitilis. Notably, the isolated lyngbyatoxin derivatives, including four new natural products, were biosynthesized by crosstalk between the heterologous tleABC gene cluster and the endogenous host enzymes. The simple strategy described here has expanded the structural diversity of lyngbyatoxin A and its biosynthetic intermediates, and provides opportunities for investigation of the currently underestimated hidden biosynthetic crosstalk.     

Armeniaspirol Antibiotic Biosynthesis: Chlorination and Oxidative Dechlorination Steps Affording Spiro[4.4]non-8-ene

Armeniaspirols are potent antibiotics containing an unusual spiro[4.4]non-8-ene moiety. Herein, we describe the cloning and functional analysis of the armeniaspirol biosynthetic gene cluster. Gene-inactivation studies and subsequent isolation of previously unknown biosynthetic intermediates shed light on intriguing biosynthetic details. Remarkably, deletion of ams15, which encodes a protein bearing a flavin-binding domain, led to the accumulation of several non-spiro intermediates with various numbers of chlorine substitutions on the pyrrole moiety. The di- and trichloropyrrole species were converted by Streptomyces albus expressing Ams15 into mono- and dichlorinated spiro derivatives, respectively. In addition, in vitro conversion of these non-spiro intermediates into des-N-methyl spiro intermediates by the cell lysate of the same recombinant strain proved Ams15 to be responsible for spiro formation through oxidative dehalogenation.     

Generation of new landomycins with altered saccharide patterns through over-expression of the glycosyltransferase gene lanGT3 in the biosynthetic gene cluster of landomycin A in Streptomyces cyanogenus S-136

Two novel landomycin compounds, landomycins I and J, were generated with a new mutant strain of Streptomyces cyanogenus in which the glycosyltransferase that is encoded by lanGT3 was over-expressed. This mutant also produced the known landomycins A, B, and D. All these compounds consist of the same polyketide-derived aglycon but differ in their sugar moieties, which are chains of different lengths. The major new metabolite, landomycin J, was found to consist of landomycinone with a tetrasaccharide chain attached. Combined with previous results of the production of landomycin E (which contains three sugars) by the LanGT3- mutant strain (obtained by targeted gene deletion of lanGT3), it was verified that LanGT3 is a D-olivosyltransferase responsible for the transfer of the fourth sugar required for landomycin A biosynthesis. The experiments also showed that gene over-expression is a powerful method for unbalancing biosynthetic pathways in order to generate new metabolites. The cytotoxicity of the new landomycins--compared to known ones--was assessed by using three different tumor cell lines, and their structure-activity relationship (SAR) with respect to the length of the deoxysugar side chain was deduced from the results.     

Levels of Isoflavonoids as Indicators of Resistance of Subterranean Clover Trifoliates to Redlegged Earth Mite Halotydeus destructor

The extracts, obtained with methanol from crushed trifoliates and from the surface layer of the trifoliates, contained isoflavones in both the free and bound forms as the major components, compounds known to exhibit feeding deterrence towards the redlegged earth mite. The levels of these compounds were quantified and showed considerable variation in extracts of one susceptible variety and four varieties with differing levels of resistance. The relative resistance of the five varieties established from field observations was compared with the amounts of isoflavone derivatives obtained in the two extracts. A better ranking of resistance was found with levels of isoflavones on the leaf surface than with the total amount of isoflavones in the trifoliates. These results suggest a simple method for determining the relative resistance of subterranean clover to the mite.     

Production of Dehydrogingerdione Derivatives in Escherichia coli by Exploiting a Curcuminoid Synthase from Oryza sativa and a β‐Oxidation Pathway from Saccharomyces cerevisiae

Gingerol derivatives are bioactive compounds isolated from the rhizome of ginger. They possess various beneficial activities, such as anticancer and hepatoprotective activities, and are therefore attractive targets of bioengineering. However, the microbial production of gingerol derivatives has not yet been established, primarily because the biosynthetic pathway of gingerol is unknown. Here, we report the production of several dehydrogingerdione (a gingerol derivative) analogues from a recombinant Escherichia coli strain that has an “artificial” biosynthesis pathway for dehydrogingerdione that was not based on the original biosynthesis pathway of gingerol derivatives in plants. The system consists of a 4‐coumarate:CoA ligase from Lithospermum erythrorhizon, a fatty acid CoA ligase from Oryza sativa, a β‐oxidation system from Saccharomyces cerevisiae, and a curcuminoid synthase from O. sativa. To our knowledge, this is the first report of the microbial production of a plant metabolite the biosynthetic pathway of which has not yet been identified.     

Lipid Oxidation and Degradation Products in Raw Materials: Low‐Fat Topical Skin‐Care Formulations

Topical skin formulations with a lipid content below 15% were stored for 6 months at 5, 20, or 40 °C or for 2 weeks at 50 °C in darkness or at 20 °C with exposure to light for 6 months. The volatile lipid‐oxidation compounds formed during this storage period were compared to those formed in the raw materials during 3 months of accelerated stability storage at 40 °C. The volatile compounds were collected by dynamic headspace and analyzed using gas chromatography–mass spectrometry. It was possible to link eight out of nine volatile compounds detected during storage of topical skin formulations to the raw materials. In addition, a possible link between the appearance of butane nitrile and the decomposition of an initiator used for polyacrylate crosspolymer‐6 production was observed. The polymer may originate from texture modifiers added to the topical skin formulation or from plastics used for packaging of topical skin formulations. Furthermore, six well‐known lipid‐oxidation and nonenzymatic browning products were suggested to originate from the two raw materials, tricaprylin/tricaprin and coconut oil.     

A Syrphid Fly Uses Olfactory Cues to Find a Non-Yellow Flower

Syrphid flies are frequent flower visitors, but little is known about the cues they use to find flowers. We determined the importance of visual and olfactory cues in a flight cage bioassay using Cirsium arvense (Asteraceae) flower heads and experienced Episyrphus balteatus (Diptera, Syrphidae). We tested the response of antennae of the flies to headspace inflorescence scent samples by using gas chromatography coupled to electroantennography (GC-EAD). The bioassays revealed that both sexes of experienced flies rely on olfactory, not visual, cues to find C. arvense flower heads. The GC-EAD measurements demonstrated that male and female flies have olfactory receptors for several of the compounds emitted by the inflorescences. These electroantennographic-active compounds may be responsible for the attraction of flies to the C. arvense flower heads. Among the compounds eliciting an antennal response are methyl salicylate and 2-phenylethanol, which were previously described as syrphid attractants. Overall, our study demonstrates for the first time that a syrphid fly uses olfactory and not visual cues to find a pollen/nectar host-plant.     

Analysis of vegetable oil volatiles by multiple headspace extraction

Quantitative determination of the volatiles produced from oxidized vegetable oils is an important indicator of oil quality. Five vegetable oils, low-erucic acid rapeseed, corn, soybean, sunflower and high oleic sunflower, were stored at 60°C for four and eight days to yield oils with several levels of oxidation. Peroxide values of the fresh oils ranged from 0.6 to 1.8 while those of the oxidized oils were from 1.6 to 42. Volatile analysis by the multiple headspace extraction (MHE) technique, which includes a pressure and time controlled injection onto the gas chromatography (GC) column (a chemically bonded capillary column), was compared with that obtained by static headspace gas chromatography (SHS-GC). Several volatile compounds indicative of the oxidation of polyunsaturated fatty acids from the vegetable oils were identified and measured by MHE; pure compounds of twelve major volatiles also were measured by MHE, and peak area was determined. Multiple extractions of the oil headspace provided a more reproducible measure of volatile compounds than was obtained by SHS-GC. Concentration of all volatiles increased with increased oxidation as measured by peroxide value of the oil. Presented at the Annual American Oil Chemists' Society Meeting, May 8–12, 1988, Phoenix, AZ.     

咪唑化合物在铜表面的成膜机理

咪唑化合物是近年来研究较多的一类杂环化合物,在生物工程、医药、高性能复合材料等领域显示了重要的学术价值和突出的应用前景．尤其咪唑化合物与金属铜具有很好的配位反应活性,能瞬间吸附在铜表面,生成致密的防氧化、抗腐蚀薄膜(0．20～0．40μm),成为一类优良的金属表面处理剂,是制造高精密微型计算机的优良化学材料,但是有关咪唑化合物在金属表面成膜过程与机理方面的研究还不多见．