Additional Secondary Metabolites Research Articles

Genomic and Transcriptome Analysis Reveals the Biosynthesis Network of Cordycepin in Cordyceps militaris.

Cordycepin is the primary active compound of Cordyceps militaris. However, the definitive genetic mechanism governing cordycepin synthesis in fruiting body growth and development remains elusive, necessitating further investigation. This study consists of 64 C. militaris strains collected from northeast China. The high-yielding cordycepin strain CMS19 was selected for the analysis of cordycepin production and the genetic basis of cordycepin anabolism. First, the whole-genome sequencing of CMS19 yielded a final size of 30.96 Mb with 8 contigs and 9781 protein-coding genes. The genome component revealed the presence of four additional secondary metabolite gene clusters compared with other published genomes, suggesting the potential for the production of new natural products. The analyses of evolutionary and genetic differentiation revealed a close relationship between C. militaris and Beauveria bassiana. The population of strains distributed in northeast China exhibited the significant genetic variation. Finally, functional genes associated with cordycepin synthesis were identified using a combination of genomic and transcriptomic analyses. A large number of functional genes associated with energy and purine metabolism were significantly enriched, facilitating the reconstruction of a hypothetical cordycepin metabolic pathway. Therefore, our speculation of the cordycepin metabolism pathway involved 24 genes initiating from the glycolysis and pentose phosphate pathways, progressing through purine metabolism, and culminating in the core region of cordycepin synthesis. These findings could offer fundamental support for scientific utilizations of C. militaris germplasm resources and standardized cultivation for cordycepin production.

Penicillium chrysogenum: Beyond the penicillin.

Almost one century after the Sir Alexander Fleming's fortuitous discovery of penicillin and the identification of the fungal producer as Penicillium notatum, later Penicillium chrysogenum (currently reidentified as Penicillium rubens), the molecular mechanisms behind the massive production of penicillin titers by industrial strains could be considered almost fully characterized. However, this filamentous fungus is not only circumscribed to penicillin, and instead, it seems to be full of surprises, thereby producing important metabolites and providing expanded biotechnological applications. This review, in addition to summarizing the classical role of P. chrysogenum as penicillin producer, highlights its ability to generate an array of additional bioactive secondary metabolites and enzymes, together with the use of this microorganism in relevant biotechnological processes, such as bioremediation, biocontrol, production of bioactive nanoparticles and compounds with pharmaceutical interest, revalorization of agricultural and food-derived wastes or the enhancement of food industrial processes and the agricultural production.

Tracking a broad inventory of cyanotoxins and related secondary metabolites using UHPLC-HRMS

Rising temperatures caused by global warming and elevated nutrient influx promote the growth of harmful cyanobacteria in freshwater ecosystems affecting human and environmental health. In this work, UHPLC-HRMS was used to simultaneously determine 7 common microcystins and 18 additional cyanobacterial metabolites in natural freshwater samples. Therefore, the particulate material of the water from Lake Stechlin in northern Brandenburg (Germany) was collected, extracted, and analyzed. The chromatographic method for the separation of cyanotoxins was optimized to enable the quantification of common toxins. By investigating additional cyanotoxins for which standards are unavailable, two more microcystins, three cyanopeptolins, five anabaenopeptins, one aeruginosin, and three additional secondary metabolites could be identified. In addition to these molecules, five unknown metabolites were detected and could be linked to Planktothrix rubescens, a native species in Lake Stechlin. Applying the proposed method yielded insights on cyanotoxin prevalence in northern German lowland Lake Stechlin and will enable further studies to elucidate additional coherences. Furthermore, by correlating additional biological, hydrodynamic, and ecological data with information on the distribution and productivity of cyanobacteria, the proposed method with the expanded toxin inventory can provide important information in risk assessment about the quality and toxicity of the ecosystem in and around the water.

Metabolomics analysis reveals metabolite changes during freeze-drying and oven-drying of Angelica dahurica

Angelica dahurica (Angelica dahurica Fisch. ex Hoffm.) is widely used as a traditional Chinese medicine and the secondary metabolites have significant pharmacological activities. Drying has been shown to be a key factor affecting the coumarin content of Angelica dahurica. However, the underlying mechanism of metabolism is unclear. This study sought to determine the key differential metabolites and metabolic pathways related to this phenomenon. Liquid chromatography with tandem mass spectrometry (LC–MS/MS) based targeted metabolomics analysis was performed on Angelica dahurica that were freeze-drying (− 80 °C/9 h) and oven-drying (60 °C/10 h). Furthermore, the common metabolic pathways of paired comparison groups were performed based on KEEG enrichment analysis. The results showed that 193 metabolites were identified as key differential metabolites, most of which were upregulated under oven drying. It also displayed that many significant contents of PAL pathways were changed. This study revealed the large-scale recombination events of metabolites in Angelica dahurica. First, we identified additional active secondary metabolites apart from coumarins, and volatile oil were significantly accumulated in Angelica dahurica. We further explored the specific metabolite changes and mechanism of the phenomenon of coumarin upregulation caused by temperature rise. These results provide a theoretical reference for future research on the composition and processing method of Angelica dahurica.

Light and Temperature Shape the Phenylpropanoid Profile of Azolla filiculoides Fronds.

Azolla is a genus of floating freshwater ferns. By their high growth and N2 fixation rates, Azolla species have been exploited for centuries by populations of South-east Asia as biofertilizers in rice paddies. The use of Azolla species as a sustainable plant material for diverse applications, such as feeding, biofuel production, and bioremediation, has encountered a growing interest over the last few years. However, high levels of feed deterrent flavonoids in their fronds have discouraged the use of these ferns as a sustainable protein source for animal consumption. Additionally, information on how and to what extent environmental determinants affect the accumulation of secondary metabolites in these organisms remains poorly understood. Moving from these considerations, here, we investigated by an untargeted metabolomics approach the profiles of phenylpropanoid compounds in the fronds of Azolla filiculoides sampled under control and pigment-inducing stress conditions. In parallel, we assayed the expression of essential structural genes of the phenylpropanoid pathway by quantitative RT-PCR. This study provides novel information concerning A. filiculoides phenylpropanoid compounds and their temporal profiling in response to environmental stimuli. In particular, we show that besides the already known 3-deoxyanthocyanidins, anthocyanidins, and proanthocyanidins, this fern can accumulate additional secondary metabolites of outstanding importance, such as chemoattractants, defense compounds, and reactive oxygen species (ROS) scavengers, and crucial as dietary components for humans, such as dihydrochalcones, stilbenes, isoflavones, and phlobaphenes. The findings of this study open an opportunity for future research studies to unveil the interplay between genetic and environmental determinants underlying the elicitation of the secondary metabolites in ferns and exploit these organisms as sustainable sources of beneficial metabolites for human health.

The bacterium Pseudomonas protegens antagonizes the microalga Chlamydomonas reinhardtii using a blend of toxins.

The unicellular alga Chlamydomonas reinhardtii and the bacterium Pseudomonas protegens serve as a model to study the interactions between photosynthetic and heterotrophic microorganisms. P. protegens secretes the cyclic lipopeptide orfamide A that interferes with cytosolic Ca2+ homeostasis in C. reinhardtii resulting in deflagellation of the algal cells. Here, we studied the roles of additional secondary metabolites secreted by P. protegens using individual compounds and co-cultivation of algae with bacterial mutants. Rhizoxin S2, pyrrolnitrin, pyoluteorin, 2,4-diacetylphloroglucinol (DAPG) and orfamide A all induce changes in cell morphology and inhibit the growth of C. reinhardtii. Rhizoxin S2 exerts the strongest growth inhibition, and its action depends on the spatial structure of the environment (agar versus liquid culture). Algal motility is unaffected by rhizoxin S2 and is most potently inhibited by orfamide A (IC50 = 4.1μM). Pyrrolnitrin and pyoluteorin both interfere with algal cytosolic Ca2+ homeostasis and motility whereas high concentrations of DAPG immobilize C. reinhardtii without deflagellation or disturbance of Ca2+ homeostasis. Co-cultivation with a regulatory mutant of bacterial secondary metabolism (ΔgacA) promotes algal growth under spatially structured conditions. Our results reveal how a single soil bacterium uses an arsenal of secreted antialgal compounds with complementary and partially overlapping activities.

Does the Future of Antibiotics Lie in Secondary Metabolites Produced by Xenorhabdus spp.? A Review.

The over-prescription of antibiotics for treatment of infections is primarily to blame for the increase in bacterial resistance. Added to the problem is the slow rate at which novel antibiotics are discovered and the many processes that need to be followed to classify antimicrobials safe for medical use. Xenorhabdus spp. of the family Enterobacteriaceae, mutualistically associated with entomopathogenic nematodes of the genus Steinernema, produce a variety of antibacterial peptides, including bacteriocins, depsipeptides, xenocoumacins and PAX (peptide antimicrobial-Xenorhabdus) peptides, plus additional secondary metabolites with antibacterial and antifungal activity. The secondary metabolites of some strains are active against protozoa and a few have anti-carcinogenic properties. It is thus not surprising that nematodes invaded by a single strain of a Xenorhabdus species are not infected by other microorganisms. In this review, the antimicrobial compounds produced by Xenorhabdus spp. are listed and the gene clusters involved in synthesis of these secondary metabolites are discussed. We also review growth conditions required for increased production of antimicrobial compounds.

A genomic view of trophic and metabolic diversity in clade-specific Lamellodysidea sponge microbiomes

BackgroundMarine sponges and their microbiomes contribute significantly to carbon and nutrient cycling in global reefs, processing and remineralizing dissolved and particulate organic matter. Lamellodysidea herbacea sponges obtain additional energy from abundant photosynthetic Hormoscilla cyanobacterial symbionts, which also produce polybrominated diphenyl ethers (PBDEs) chemically similar to anthropogenic pollutants of environmental concern. Potential contributions of non-Hormoscilla bacteria to Lamellodysidea microbiome metabolism and the synthesis and degradation of additional secondary metabolites are currently unknown.ResultsThis study has determined relative abundance, taxonomic novelty, metabolic capacities, and secondary metabolite potential in 21 previously uncharacterized, uncultured Lamellodysidea-associated microbial populations by reconstructing near-complete metagenome-assembled genomes (MAGs) to complement 16S rRNA gene amplicon studies. Microbial community compositions aligned with sponge host subgroup phylogeny in 16 samples from four host clades collected from multiple sites in Guam over a 3-year period, including representatives of Alphaproteobacteria, Gammaproteobacteria, Oligoflexia, and Bacteroidetes as well as Cyanobacteria (Hormoscilla). Unexpectedly, microbiomes from one host clade also included Cyanobacteria from the prolific secondary metabolite-producer genus Prochloron, a common tunicate symbiont.Two novel Alphaproteobacteria MAGs encoded pathways diagnostic for methylotrophic metabolism as well as type III secretion systems, and have been provisionally assigned to a new order, designated Candidatus Methylospongiales. MAGs from other taxonomic groups encoded light-driven energy production pathways using not only chlorophyll, but also bacteriochlorophyll and proteorhodopsin. Diverse heterotrophic capabilities favoring aerobic versus anaerobic conditions included pathways for degrading chitin, eukaryotic extracellular matrix polymers, phosphonates, dimethylsulfoniopropionate, trimethylamine, and benzoate. Genetic evidence identified an aerobic catabolic pathway for halogenated aromatics that may enable endogenous PBDEs to be used as a carbon and energy source.ConclusionsThe reconstruction of high-quality MAGs from all microbial taxa comprising greater than 0.1% of the sponge microbiome enabled species-specific assignment of unique metabolic features that could not have been predicted from taxonomic data alone. This information will promote more representative models of marine invertebrate microbiome contributions to host bioenergetics, the identification of potential new sponge parasites and pathogens based on conserved metabolic and physiological markers, and a better understanding of biosynthetic and degradative pathways for secondary metabolites and halogenated compounds in sponge-associated microbiota.Dn7znYuJ-m6v4UERxLrGDHVideo

Reconstitution of polythioamide antibiotic backbone formation reveals unusual thiotemplated assembly strategy

Closthioamide (CTA) is a rare example of a thioamide-containing nonribosomal peptide and is one of only a handful of secondary metabolites described from obligately anaerobic bacteria. Although the biosynthetic gene cluster responsible for CTA production and the thioamide synthetase that catalyzes sulfur incorporation were recently discovered, the logic for peptide backbone assembly has remained a mystery. Here, through the use of in vitro biochemical assays, we demonstrate that the amide backbone of CTA is assembled in an unusual thiotemplated pathway involving the cooperation of a transacylating member of the papain-like cysteine protease family and an iteratively acting ATP-grasp protein. Using the ATP-grasp protein as a bioinformatic handle, we identified hundreds of such thiotemplated yet nonribosomal peptide synthetase (NRPS)-independent biosynthetic gene clusters across diverse bacterial phyla. The data presented herein not only clarify the pathway for the biosynthesis of CTA, but also provide a foundation for the discovery of additional secondary metabolites produced by noncanonical biosynthetic pathways.

Whole-genome comparisons of Penicillium spp. reveals secondary metabolic gene clusters and candidate genes associated with fungal aggressiveness during apple fruit decay.

Blue mold is a postharvest rot of pomaceous fruits caused by Penicillium expansum and a number of other Penicillium species. The genome of the highly aggressive P. expansum strain R19 was re-sequenced and analyzed together with the genome of the less aggressive P. solitum strain RS1. Whole genome scale similarities and differences were examined. A phylogenetic analysis of P. expansum, P. solitum, and several closely related Penicillium species revealed that the two pathogens isolated from decayed apple with blue mold symptoms are not each other’s closest relatives. Among a total of 10,560 and 10,672 protein coding sequences respectively, a comparative genomics analysis revealed 41 genes in P. expansum R19 and 43 genes in P. solitum RS1 that are unique to these two species. These genes may be associated with pome fruit–fungal interactions, subsequent decay processes, and mycotoxin accumulation. An intact patulin gene cluster consisting of 15 biosynthetic genes was identified in the patulin producing P. expansum strain R19, while only a remnant, seven-gene cluster was identified in the patulin-deficient P. solitum strain. However, P. solitum contained a large number of additional secondary metabolite gene clusters, indicating that this species has the potential capacity to produce an array of known as well as not-yet-identified products of possible toxicological or biotechnological interest.

Analysis of the global regulator Lae1 uncovers a connection between Lae1 and the histone acetyltransferase HAT1 in Fusarium fujikuroi.

The fungus Fusarium fujikuroi causes "bakanae" disease of rice due to its ability to produce gibberellins (GAs), a family of plant hormones. Recent genome sequencing revealed the genetic capacity for the biosynthesis of 46 additional secondary metabolites besides the industrially produced GAs. Among them are the pigments bikaverin and fusarubins, as well as mycotoxins, such as fumonisins, fusarin C, beauvericin, and fusaric acid. However, half of the potential secondary metabolite gene clusters are silent. In recent years, it has been shown that the fungal specific velvet complex is involved in global regulation of secondary metabolism in several filamentous fungi. We have previously shown that deletion of the three components of the F. fujikuroi velvet complex, vel1, vel2, and lae1, almost totally abolished biosynthesis of GAs, fumonisins and fusarin C. Here, we present a deeper insight into the genome-wide regulatory impact of Lae1 on secondary metabolism. Over-expression of lae1 resulted in de-repression of GA biosynthetic genes under otherwise repressing high nitrogen conditions demonstrating that the nitrogen repression is overcome. In addition, over-expression of one of five tested histone acetyltransferase genes, HAT1, was capable of returning GA gene expression and GA production to the GA-deficient Δlae1 mutant. Deletion and over-expression of HAT1 in the wild type resulted in downregulation and upregulation of GA gene expression, respectively, indicating that HAT1 together with Lae1 plays an essential role in the regulation of GA biosynthesis.

Breath-Based Diagnosis of Invasive Mucormycosis (IM)

BackgroundTimely diagnosis of IM remains a major challenge in clinical mycology. Because of the lack of specific diagnostic methods for IM and the frequently fulminant nature of this infection, IM-associated mortality remains high.MethodsWe examined breath volatile metabolite profiles in a neutropenic murine model of IM, using the 3 Mucorales species that most commonly cause human IM - Rhizopus arrhizus var. arrhizus, R. arrhizus var. delemar, and R. microsporus - and for comparison, Aspergillus fumigatus. We infected female balb/c mice (N = 4 per group) treated with cyclophosphamide and cortisone followed by intranasal administration of 106 conidia of each species. 3 days post-infection, we collected breath samples from each mouse via tracheostomy using a flexiVent murine ventilator, examining breath volatile metabolites using thermal desorption gas chromatography/tandem mass spectrometry (GC-MS/MS). We also sampled breath prospectively from five patients eventually diagnosed with proven IM caused by R. microsporus, analyzing breath volatile metabolites using thermal desorption GC-MS/MS.ResultsEach Mucorales species produced a consistent profile of breath sesquiterpene secondary metabolite VOCs in our murine models, which distinguished these species from each other and from murine invasive aspergillosis (Figure A). These fungi shifted their secondary metabolism significantly in vivo, compared with their previously characterized in vitro metabolism. We found overlapping VOC sesquiterpene metabolites between breath samples from the murine model of R. microsporus infection and 5 of 5 patients with R. microsporus IM, with additional sesquiterpene secondary metabolites detected in patient breath, compared with the murine IM model (Figure B). In one patient with serial breath samples, these sesquiterpenes declined in abundance and disappeared with antifungal therapy, in parallel with clinical improvement (Figure C).ConclusionThe three Mucorales species that cause most human IM have distinct breath sesquiterpene profiles that can be used to identify these infections in vivo noninvasively. These profiles distinguish these infections from each other and from aspergillosis, and may be useful in monitoring clinical response to treatment.Disclosures F. M. Marty, Astellas Pharma US: Consultant and Grant Investigator, Consulting fee and Grant recipient; Chimerix: Consultant and Grant Investigator, Consulting fee and Grant recipient; Fate Therapeutics: Scientific Advisor, Consulting fee; Gilead Sciences: Consultant and Grant Investigator, Consulting fee and Grant recipient; LFB: Consultant, Consulting fee; Merck: Consultant, Grant Investigator and Scientific Advisor, Consulting fee and Grant recipient; Roche Molecular Systems: Consultant, Consulting fee; Shire: Consultant and Grant Investigator, Consulting fee and Grant recipient; D. P. Kontoyiannis, Pfizer: Research Contractor, Research support and Speaker honorarium; Astellas: Research Contractor, Research support and Speaker honorarium; Merck: Honorarium, Speaker honorarium; Cidara: Honorarium, Speaker honorarium; Amplyx: Honorarium, Speaker honorarium; F2G: Honorarium, Speaker honorarium

Secondary metabolites from Streptococcus mutans and their ecological roles in dental biofilm

Dental biofilms are composed of hundreds of bacterial species, among which Streptococcus mutans is widely recognized as the major pathogen of dental caries. The cariogenic potential of S. mutans is related to its ability to form a robust biofilm on the tooth surface and its acidogenic and acid-tolerant properties. Co-evolution of S. mutans with the host has resulted in the diversity of secondary metabolism of S. mutans in strain level. A variety of secondary metabolites, including 10 bacteriocins (mutacins) and one hybrid Polyketide/Non-Ribosomal Peptide type compound, have been characterized. Studies on these secondary metabolites indicate that they play a significant role either in interspecies or in inter-kingdom interactions in the dental biofilm. As more S. mutans strains are isolated and sequenced, additional secondary metabolites with novel functions will be discovered. The study of secondary metabolites in S. mutans is anticipated to be helpful for oral disease treatment and prevention by providing new strategies.

Bacillus velezensis RC 218 as a biocontrol agent to reduce Fusarium head blight and deoxynivalenol accumulation: Genome sequencing and secondary metabolite cluster profiles

Bacillus subtilis RC 218 was originally isolated from wheat anthers as a potential antagonist of Fusarium graminearum, the causal agent of Fusarium head blight (FHB). It was demonstrated to have antagonist activity against the plant pathogen under in vitro and greenhouse assays. The current study extends characterizing B. subtilis RC 218 with a field study and genome sequencing. The field study demonstrated that B. subtilis RC 218 could reduce disease severity and the associated mycotoxin (deoxynivalenol) accumulation, under field conditions. The genome sequencing allowed us to accurately determine the taxonomy of the strain using a phylogenomic approach, which places it in the Bacillus velezensis clade. In addition, the draft genome allowed us to use bioinformatics to mine the genome for potential metabolites. The genome mining allowed us to identify 9 active secondary metabolites conserved by all B. velezensis strains and one additional secondary metabolite, the lantibiotic ericin, which is unique to this strain. This study represents the first confirmed production of ericin by a B. velezensis strain. The genome also allowed us to do a comparative genomics with its closest relatives and compare the secondary metabolite production of the publically available B. velezensis genomes. The results showed that the diversity in secondary metabolites of strains in the B. velezensis clade is driven by strains making different antibacterials.

FunGeneClusterS: Predicting fungal gene clusters from genome and transcriptome data

IntroductionSecondary metabolites of fungi are receiving an increasing amount of interest due to their prolific bioactivities and the fact that fungal biosynthesis of secondary metabolites often occurs from co-regulated and co-located gene clusters. This makes the gene clusters attractive for synthetic biology and industrial biotechnology applications. We have previously published a method for accurate prediction of clusters from genome and transcriptome data, which could also suggest cross-chemistry, however, this method was limited both in the number of parameters which could be adjusted as well as in user-friendliness. Furthermore, sensitivity to the transcriptome data required manual curation of the predictions. In the present work, we have aimed at improving these features. ResultsFunGeneClusterS is an improved implementation of our previous method with a graphical user interface for off- and on-line use. The new method adds options to adjust the size of the gene cluster(s) being sought as well as an option for the algorithm to be flexible with genes in the cluster which may not seem to be co-regulated with the remainder of the cluster. We have benchmarked the method using data from the well-studied Aspergillus nidulans and found that the method is an improvement over the previous one. In particular, it makes it possible to predict clusters with more than 10 genes more accurately, and allows identification of co-regulated gene clusters irrespective of the function of the genes. It also greatly reduces the need for manual curation of the prediction results. We furthermore applied the method to transcriptome data from A. niger. Using the identified best set of parameters, we were able to identify clusters for 31 out of 76 previously predicted secondary metabolite synthases/synthetases. Furthermore, we identified additional putative secondary metabolite gene clusters. In total, we predicted 432 co-transcribed gene clusters in A. niger (spanning 1.323 genes, 12% of the genome). Some of these had functions related to primary metabolism, e.g. we have identified a cluster for biosynthesis of biotin, as well as several for degradation of aromatic compounds. The data identifies that suggests that larger parts of the fungal genome than previously anticipated operates as gene clusters. This includes both primary and secondary metabolism as well as other cellular maintenance functions. ConclusionWe have developed FunGeneClusterS in a graphical implementation and made the method capable of adjustments to different datasets and target clusters. The method is versatile in that it can predict co-regulated clusters not limited to secondary metabolism. Our analysis of data has shown not only the validity of the method, but also strongly suggests that large parts of fungal primary metabolism and cellular functions are both co-regulated and co-located.

Complete genome sequence of producer of the glycopeptide antibiotic Aculeximycin Kutzneria albida DSM 43870T, a representative of minor genus of Pseudonocardiaceae.

BackgroundKutzneria is a representative of a rarely observed genus of the family Pseudonocardiaceae. Kutzneria species were initially placed in the Streptosporangiaceae genus and later reconsidered to be an independent genus of the Pseudonocardiaceae. Kutzneria albida is one of the eight known members of the genus. This strain is a unique producer of the glycosylated polyole macrolide aculeximycin which is active against both bacteria and fungi. Kutzneria albida genome sequencing and analysis allow a deeper understanding of evolution of this genus of Pseudonocardiaceae, provide new insight in the phylogeny of the genus, as well as decipher the hidden secondary metabolic potential of these rare actinobacteria.ResultsTo explore the biosynthetic potential of Kutzneria albida to its full extent, the complete genome was sequenced. With a size of 9,874,926 bp, coding for 8,822 genes, it stands alongside other Pseudonocardiaceae with large circular genomes. Genome analysis revealed 46 gene clusters potentially encoding secondary metabolite biosynthesis pathways. Two large genomic islands were identified, containing regions most enriched with secondary metabolism gene clusters. Large parts of this secondary metabolism “clustome” are dedicated to siderophores production.ConclusionsKutzneria albida is the first species of the genus Kutzneria with a completely sequenced genome. Genome sequencing allowed identifying the gene cluster responsible for the biosynthesis of aculeximycin, one of the largest known oligosaccharide-macrolide antibiotics. Moreover, the genome revealed 45 additional putative secondary metabolite gene clusters, suggesting a huge biosynthetic potential, which makes Kutzneria albida a very rich source of natural products. Comparison of the Kutzneria albida genome to genomes of other actinobacteria clearly shows its close relations with Pseudonocardiaceae in line with the taxonomic position of the genus.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-885) contains supplementary material, which is available to authorized users.

Hawaiinolides E–G, cytotoxic cassane and cleistanthane diterpenoids from the entomogenous fungus Paraconiothyrium hawaiiense

Hawaiinolides E–G (1–3), three additional new secondary metabolites including two cassane (1 and 2) types of diterpene lactones and one cleistanthane (3) diterpenoid, were isolated from the scale-up fermentation extract of Paraconiothyrium hawaiiense, an entomogenous fungus isolated from the Septobasidium-infected insect Diaspidiotus sp. The structures of 1–3 were elucidated by nuclear magnetic resonance experiments, and 1 and 3 were further confirmed by X-ray crystallography. The absolute configurations of 1 and 3 were assigned by single-crystal X-ray diffraction analysis using Cu Kα radiation, whereas that of 2 was deduced via the circular dichroism data. Compound 1 showed significant cytotoxic effects against the human tumor cell line T24, with an IC50 value (9.32μM) comparable to that of the positive control cisplatin.

Molecular basis of nitrate uptake by the plant nitrate transporter NRT1.1

SummaryThe NRT1/PTR family of proton-coupled transporters are responsible for nitrogen assimilation in eukaryotes and bacteria through the uptake of peptides. However, in the majority of plant species members of this family have evolved to transport nitrate as well as additional secondary metabolites and hormones. In response to falling nitrate levels, NRT1.1 is phosphorylated on an intracellular threonine that switches the transporter from a low to high affinity state. Here we present both the apo and nitrate bound crystal structures of Arabidopsis thaliana NRT1.1, which together with in vitro binding and transport data identify a key role for His356 in nitrate binding. Our data support a model whereby phosphorylation increases structural flexibility and in turn the rate of transport. Comparison with peptide transporters further reveals how the NRT1/PTR family has evolved to recognize diverse nitrogenous ligands, whilst maintaining elements of a conserved coupling mechanism within this superfamily of nutrient transporters.

Insights into naturally minimised Streptomyces albus J1074 genome

BackgroundThe Streptomyces albus J1074 strain is one of the most widely used chassis for the heterologous production of bioactive natural products. The fast growth and an efficient genetic system make this strain an attractive model for expressing cryptic biosynthetic pathways to aid drug discovery.ResultsTo improve its capabilities for the heterologous expression of biosynthetic gene clusters, the complete genomic sequence of S. albus J1074 was obtained. With a size of 6,841,649 bp, coding for 5,832 genes, its genome is the smallest within the genus streptomycetes. Genome analysis revealed a strong tendency to reduce the number of genetic duplicates. The whole transcriptomes were sequenced at different time points to identify the early metabolic switch from the exponential to the stationary phase in S. albus J1074.ConclusionsS. albus J1074 carries the smallest genome among the completely sequenced species of the genus Streptomyces. The detailed genome and transcriptome analysis discloses its capability to serve as a premium host for the heterologous production of natural products. Moreover, the genome revealed 22 additional putative secondary metabolite gene clusters that reinforce the strain’s potential for natural product synthesis.

Fluorescence Excitation–Emission Features of Aflatoxin and Related Secondary Metabolites and Their Application for Rapid Detection of Mycotoxins

The persistent occurrence of aflatoxins in food and feed remains a problem for producers of commodities subject to colonization with toxigenic molds. Aflatoxins are secondary metabolites of fungi of the Aspergillus spp. associated with deleterious health effects. Because current screening methods for these toxins are lengthy, destructive, and costly, there is a continuous search for a more rapid, noninvasive, and cost-effective technology. The present study utilized a fluorescence excitation–emission matrix (EEM) of aflatoxin as well as two additional secondary metabolites (kojic acid and the bright greenish-yellow fluorescence (BGYF) compound) of Aspergillus flavus measured with a fluorescence spectrophotometer. The results were compared to image data acquired with a fluorescence hyperspectral sensor in order to evaluate the potential of image-based technology for detecting aflatoxin in grain. The excitation–emission matrix of aflatoxin B1 standard produced overlapping peaks in 340–400 nm of excitation range emitting in the blue range at around 450 nm. The spectral signature extracted from the hyperspectral image was also in the blue range, emitting blue fluorescence. Because the results from both systems were comparable, where all fluorescence peaks were in the blue range, the present study validates the feasibility of image-based technology for nondestructive detection of aflatoxin in corn. Additional peaks were revealed in the aflatoxin EEM in the 260-nm excitation range that were not present in the kojic acid and BGYF compound mixture. This new information allows for the separation of the aflatoxin signature from the potentially confounding overlap of other secondary metabolites occurring in the blue and blue-green spectral ranges.