The Discovery of Weddellamycin, a Tricyclic Polyene Macrolactam Antibiotic from an Antarctic Deep-Sea-Derived Streptomyces sp. DSS69, by Heterologous Expression.

Polyene macrolactams are a special group of natural products with great diversity, unique structural features, and a wide range of biological activities. Herein, a cryptic gene cluster for the biosynthesis of putative macrolactams was disclosed from a sponge-associated bacterium, Streptomyces sp. DSS69, by genome mining. Cloning and heterologous expression of the whole biosynthetic gene cluster led to the discovery of weddellamycin, a polyene macrolactam bearing a 23/5/6 ring skeleton. A negative regulator, WdlO, and two positive regulators, WdlA and WdlB, involved in the regulation of weddellamycin production were unraveled. The fermentation titer of weddellamycin was significantly improved by overexpression of wdlA and wdlB and deletion of wdlO. Notably, weddellamycin showed remarkable antibacterial activity against various Gram-positive bacteria including MRSA, with MIC values of 0.10-0.83 µg/mL, and antifungal activity against Candida albicans, with an MIC value of 3.33 µg/mL. Weddellamycin also displayed cytotoxicity against several cancer cell lines, with IC50 values ranging from 2.07 to 11.50 µM.

Fusarins G-L with Inhibition of NO in RAW264.7 from Marine-Derived Fungus Fusarium solani 7227.

Six new fusarin derivatives, fusarins G-L (1-6), together with five known compounds (5-11) were isolated from the marine-derived fungus Fusarium solani 7227. The structures of the new compounds were elucidated by means of comprehensive spectroscopic methods (1D and 2D NMR, HRESIMS, ECD, and ORC) and X-ray crystallography. Compounds 5-11 exhibited potent anti-inflammatory activity by inhibiting the production of NO in RAW264.7 cells activated by lipopolysaccharide, with IC50 values ranging from 3.6 to 32.2 µM. The structure-activity relationships of the fusarins are discussed herein.

Isolation of manumycin-type derivatives and genome characterization of a marine Streptomyces sp. C1-2.

A marine actinomycete strain C1-2 was taxonomically characterized as the genus Streptomyces, based on whole-genome sequence analysis. The highest average nucleotide identity (ANI) value (98.96%) and digital DNA-DNA hybridization (DDH) value (90.4%) were observed between Streptomyces sp. C1-2 and Streptomyces griseoaurantiacus. Thus, Streptomyces sp. C1-2 could be identified as S. griseoaurantiacus. Genome analysis revealed that Streptomyces sp. C1-2 contained 22 biosynthetic gene clusters (BGCs) for secondary metabolites, where among them, 54% have low similarities with known BGCs. The chemical investigation led to the isolation of three new manumycin-type derivatives and two known analog antibiotics named SW-B and cornifronin B. All compounds showed antioxidant activity with the half-maximal inhibitory concentration (IC50) values in a range of 50.82 ± 0.8-112.04 ± 1.0 µg/mL with no cytotoxicity against Vero cells. This is the first report of the antioxidant property of manumycin-type derivatives. Moreover, two known compounds exhibited antifungal activity against Phytophthora capsici, Fusarium oxysporum f. sp. cucumerinum, and Magnaporthe grisea, with the minimum inhibitory concentration (MIC) values in a range of 125-500 µg/mL.

Identification of the Female-Produced Sex Pheromone of the Dotted White Geometrid Naxa seriaria (Lepidoptera: Geometridae).

The dotted white geometrid moth, Naxa seriaria Motschulsky (Lepidoptera: Geometridae), is a pest of Oleaceae in Korea, Japan, and China. In this study, we identified (3Z,6Z,9Z,12Z,15Z)-heneicosapentaene (C-21 pentaene) as the only compound in extracts of the pheromone glands of female N. seriaria causing a response from receptors on the antennae of males in analyses by gas chromatography with electroantennographic detection. The synthetic sex pheromone elicited dose-dependent electrophysiological responses from antennae of male N. seriaria. In field tests, more male moths were captured in traps baited with synthetic C-21 pentaene than in unbaited traps, and increasing the loading of C-21 pentaene in the lure increased catches of male moths. Significantly more male N. seriaria moths were caught in delta traps than in bucket traps. Based on these results, C-21 pentaene is proposed to be the major, if not the only, component of the sex pheromone of N. seriaria.

The Laetiporus polyketide synthase LpaA produces a series of antifungal polyenes.

The conspicuous bright golden to orange-reddish coloration of species of the basidiomycete genus Laetiporus is a hallmark feature of their fruiting bodies, known among mushroom hunters as the "chicken of the woods". This report describes the identification of an eight-domain mono-modular highly reducing polyketide synthase as sole enzyme necessary for laetiporic acid biosynthesis. Heterologous pathway reconstitution in both Aspergillus nidulans and Aspergillus niger verified that LpaA functions as a multi-chain length polyene synthase, which produces a cocktail of laetiporic acids with a methyl-branched C26-C32 main chain. Laetiporic acids show a marked antifungal activity on Aspergillus protoplasts. Given the multiple products of a single biosynthesis enzyme, our work underscores the diversity-oriented character of basidiomycete natural product biosynthesis.

Insights into Streptomyces spp. isolated from the rhizospheric soil of Panax notoginseng: isolation, antimicrobial activity and biosynthetic potential for polyketides and non-ribosomal peptides.

BACKGROUND: Streptomycetes from the rhizospheric soils are a rich resource of novel secondary metabolites with various biological activities. However, there is still little information related to the isolation, antimicrobial activity and biosynthetic potential for polyketide and non-ribosomal peptide discovery associated with the rhizospheric streptomycetes of Panax notoginseng. Thus, the aims of the present study are to (i) identify culturable streptomycetes from the rhizospheric soil of P. notoginseng by 16S rRNA gene, (ii) evaluate the antimicrobial activities of isolates and analyze the biosynthetic gene encoding polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) of isolates, (iii) detect the bioactive secondary metabolites from selected streptomycetes, (iv) study the influence of the selected isolate on the growth of P. notoginseng in the continuous cropping field. This study would provide a preliminary basis for the further discovery of the secondary metabolites from streptomycetes isolated from the rhizospheric soil of P. notoginseng and their further utilization for biocontrol of plants. RESULTS: A total of 42 strains representing 42 species of the genus Streptomyces were isolated from 12 rhizospheric soil samples in the cultivation field of P. notoginseng and were analyzed by 16S rRNA gene sequencing. Overall, 40 crude cell extracts out of 42 under two culture conditions showed antibacterial and antifungal activities. Also, the presence of biosynthesis genes encoding type I and II polyketide synthase (PKS I and PKS II) and nonribosomal peptide synthetases (NRPSs) in 42 strains were established. Based on characteristic chemical profiles screening by High Performance Liquid Chromatography-Diode Array Detector (HPLC-DAD), the secondary metabolite profiles of strain SYP-A7257 were evaluated by High Performance Liquid Chromatography-High Resolution Mass Spectrometry (HPLC-HRMS). Finally, four compounds actinomycin X2 (F1), fungichromin (F2), thailandin B (F7) and antifungalmycin (F8) were isolated from strain SYP-A7257 by using chromatography techniques, UV, HR-ESI-MS and NMR, and their antimicrobial activities against the test bacteria and fungus were also evaluated. In the farm experiments, Streptomyces sp. SYP-A7257 showed healthy growth promotion and survival rate improvement of P. notoginseng in the continuous cropping field. CONCLUSIONS: We demonstrated the P. notoginseng rhizospheric soil-derived Streptomyces spp. distribution and diversity with respect to their metabolic potential for polyketides and non-ribosomal peptides, as well as the presence of biosynthesis genes PKS I, PKS II and NRPSs. Our results showed that cultivatable Streptomyces isolates from the rhizospheric soils of P. notoginseng have the ability to produce bioactive secondary metabolites. The farm experiments suggested that the rhizospheric soil Streptomyces sp. SYP-A7257 may be a potential biological control agent for healthy growth promotion and survival rate improvement of P. notoginseng in the continuous cropping field.

Raman spectroscopic signatures of carotenoids and polyenes enable label-free visualization of microbial distributions within pink biofilms.

Pink biofilms are multispecies microbial communities that are commonly found in moist household environments. The development of this pink stain is problematic from an aesthetic point of view, but more importantly, it raises hygienic concerns because they may serve as a potential reservoir of opportunistic pathogens. Although there have been several studies of pink biofilms using molecular analysis and confocal laser scanning microscopy, little is known about the spatial distributions of constituent microorganisms within pink biofilms, a crucial factor associated with the characteristics of pink biofilms. Here we show that Raman spectroscopic signatures of intracellular carotenoids and polyenes enable us to visualize pigmented microorganisms within pink biofilms in a label-free manner. We measured space-resolved Raman spectra of a pink biofilm collected from a bathroom, which clearly show resonance Raman bands of carotenoids. Multivariate analysis of the Raman hyperspectral imaging data revealed the presence of typical carotenoids and structurally similar but different polyenes, whose spatial distributions within the pink biofilm were found to be mutually exclusive. Raman measurements on individual microbial cells isolated from the pink biofilm confirmed that these distributions probed by carotenoid/polyene Raman signatures are attributable to different pigmented microorganisms. The present results suggest that Raman microspectroscopy with a focus on microbial pigments such as carotenoids is a powerful nondestructive method for studying multispecies biofilms in various environments.

Thailandenes, Cryptic Polyene Natural Products Isolated from Burkholderia thailandensis Using Phenotype-Guided Transposon Mutagenesis.

Burkholderia thailandensis has emerged as a model organism for investigating the production and regulation of diverse secondary metabolites. Most of the biosynthetic gene clusters encoded in B. thailandensis are silent, motivating the development of new methods for accessing their products. In the current work, we add to the canon of available approaches using phenotype-guided transposon mutagenesis to characterize a silent biosynthetic gene cluster. Because secondary metabolite biosynthesis is often associated with phenotypic changes, we carried out random transposon mutagenesis followed by phenotypic inspection of the resulting colonies. Several mutants exhibited intense pigmentation and enhanced expression of an iterative type I polyketide synthase cluster that we term org. Disruptions of orgA, orgB, and orgC abolished the biosynthesis of the diffusible pigment, thus linking it to the org operon. Isolation and structural elucidation by HR-MS and 1D/2D NMR spectroscopy revealed three novel, cryptic metabolites, thailandene A-C. Thailandenes are linear formylated or acidic polyenes containing a combination of cis and trans double bonds. Variants A and B exhibited potent antibiotic activity against Staphylococcus aureus and Saccharomyces cerevisiae but not against Escherichia coli. One of the transposon mutants that exhibited an enhanced expression of org contained an insertion upstream of a σ54-dependent transcription factor. Closer inspection of the org operon uncovered a σ54 promoter consensus sequence upstream of orgA, providing clues regarding its regulation. Our results showcase the utility of phenotype-guided transposon mutagenesis in uncovering cryptic metabolites encoded in bacterial genomes.

Discovery, biosynthesis and antifungal mechanism of the polyene-polyol meijiemycin.

Produced by a newly isolated Streptomycetes strain, meijiemycin is a gigantic linear polyene-polyol that exhibits structural features not seen in other members of the polyene-polyol family. We propose a biosynthetic mechanism and demonstrate that meijiemycin inhibits hyphal growth by inducing the aggregation of ergosterol and restructuring of the fungal plasma membrane.

The Discovery of Fungal Polyene Macrolides via a Postgenomic Approach Reveals a Polyketide Macrocyclization by trans-Acting Thioesterase in Fungi.

Heterologous expression of a unique biosynthetic gene cluster (BGC) comprising a highly reducing polyketide synthase and stand-alone thioesterase genes in Aspergillus oryzae enabled us to isolate a novel 34-membered polyene macrolide, phaeospelide A (1). This is the first isolation of a fungal polyene macrolide and the first demonstration of fungal aliphatic macrolide biosynthetic machinery. In addition, sequence similarity network analysis demonstrated the existence of a large number of BGCs for novel fungal macrolides.

Kenalactams A-E, Polyene Macrolactams Isolated from Nocardiopsis CG3.

In our screening program for new biologically active secondary metabolites, a new strain, Nocardiopsis CG3 (DSM 106572), isolated from the saltpan of Kenadsa, was found to produce five new polyene macrolactams, the kenalactams A-E (1-5). Their structures were elucidated by spectral methods (NMR and HRESIMS), and the absolute configuration was derived by chemical derivatization (Mosher's method). Through a feeding experiment, alanine was proven to be the nitrogen-bearing starter unit involved in biosynthesis of the polyketide kenalactam A (1). Kenalactam E (5) was cytotoxic against human prostate cancer PC-3 cells with an IC50 value of 2.1 µM.

Asperyellone pretreatment protects HaCaT cells from UVB irradiation induced oxidative damages: Assessment under in vitro and in vivo conditions and at molecular level.

The present study explores the UVB protective role of Asperyellone (AY), a secondary metabolite of Aspergillus niger strain AN01. The in vitro UVB protective efficacy of AY was studied using the Human Epidermal keratinocytes cells (HaCaT) cell line. The results suggest the appreciable scavenging of UVB-induced reactive oxygen species in the AY-pretreated cells compared with UVB control. Experimental results on the antioxidant enzymes (Catalase, SOD, LPO, and GPx) profile, histochemical, and molecular analyses support the UVB protective effect of AY in HaCaT cells. Further, the in vivo UVB protective efficacy of AY was studied using animal models and compared with that of commercially available UVB protective agents. Physical, biochemical, and molecular analyses of skin samples emphasized the UVB protective role of AY. Thus, the important beneficial effects of AY have been explored in the present study.

Secondary metabolites from Antarctic marine-derived fungus Penicillium crustosum HDN153086.

A new polyene compound (1) and a new diketopiperazine (2), as well as three known compounds (3-5), were isolated from the Antarctic marine-derived fungus Penicillium crustosum HDN153086. The structures of 1-5 were deduced based on MS, NMR and TD-DFT calculations of specific ECD spectra. These compounds were evaluated for their cytotoxic activities against K562 cell line and only compound 2 exhibited cytotoxicity against K562 cell, with IC50 value of 12.7 µM.

Discovery and Elucidation of the Biosynthesis of Aspernidgulenes: Novel Polyenes from Aspergillus Nidulans by Using Serial Promoter Replacement.

Through serial promoter exchanges, we isolated several novel polyenes, the aspernidgulenes, from Aspergillus nidulans and uncovered their succinct biosynthetic pathway involving only four enzymes. An enoyl reductase (ER)-less highly reducing polyketide synthase (HR-PKS) putatively produces a 5,6-dihydro-α-pyrone polyene, which undergoes bisepoxidation, epoxide ring opening, cyclization, and hydrolytic cleavage by three tailoring enzymes to generate aspernidgulene A1 and A2. Our findings demonstrate the prowess of fungal-tailoring enzymes to transform a polyketide scaffold concisely and efficiently into complex structures. Moreover, comparison with citreoviridin and aurovertin biosynthesis suggests that methylation of the α-pyrone hydroxy group by methyltransferase (CtvB or AurB) is the branching point at which the biosynthesis of these two classes of compounds diverge. Therefore, scanning for the presence or absence of the gatekeeping α-pyrone methyltransferase gene in homologous clusters might be a potential way to classify the product bioinformatically as belonging to methylated α-pyrone polyenes or polyenes containing rings derived from the cyclization of the unmethylated 5,6-dihydro-α-pyrone, such as 2,3-dimethyl-γ-lactone and oxabicyclo[2.2.1]heptane.

Two Types of Volatile Polyenes in the Brown Alga Sargassum thunbergii.

An essential oil from the brown alga Sargassum thunbergii, prepared by a simultaneous distillation extraction method, contained in two types of volatile polyenes with a terminal double bond such as (6Z,9Z,12Z,15Z,18Z)-1,6,9,12,15,18-henicosahexaene and (6Z,9Z,12Z,15Z)-1,6,9,12,15-henicosapentaene and with their saturated terminal structures such as (3Z,6Z,9Z,12Z,15Z,18Z)-3,6,9,12,15,18-henicosahexaene and (3Z,6Z,9Z,12Z,15Z)-3,6,9,12,15-henicosapentaene. These volatile polyenes were identified by comparison with the GC-MS and NMR spectra of synthetics. The polyenes with the saturated terminal structures were found in the brown algae for the first time.

Characteristic Aroma Components from Dried "Wakame" Undaria pinnatifida.

An essential oil from dried "wakame" (Undaria pinnatifida), prepared by a simultaneous distillation extraction method, was analyzed by GC-MS, indicating the presence of one major component of volatiles. The volatile component was identified as (6Z,9Z,12Z,15Z,18Z)-1,6,9,12,15,18-henicosahexaene by comparison with the GC-MS and NMR spectra of synthetic. The henicosahexaene showed a subtly marine aroma. (6Z,9Z,12Z,15Z)-1,6,9,12,15-Henicosapentaene was also detected as a minor polyene in the essential oils. It was suggested that these polyenes contribute to the characteristic aroma of the dried wakame.

Nystatin-like Pseudonocardia polyene B1, a novel disaccharide-containing antifungal heptaene antibiotic.

Polyene macrolides such as nystatin A1 and amphotericin B belong to a large family of very valuable antifungal polyketide compounds typically produced by soil actinomycetes. Recently, nystatin-like Pseudonocardia polyene (NPP) A1 has been identified as a unique disaccharide-containing tetraene antifungal macrolide produced by Pseudonocardia autotrophica. Despite its significantly increased water solubility and decreased hemolytic activity, its antifungal activity remains limited compared with that of nystatin A1. In this study, we developed NPP B1, a novel NPP A1 derivative harboring a heptaene core structure, by introducing two amino acid substitutions in the putative NADPH-binding motif of the enoyl reductase domain in module 5 of the NPP A1 polyketide synthase NppC. The low level NPP B1 production yield was successfully improved by eliminating the native plasmid encoding a polyketide biosynthetic gene cluster present in P. autotrophica. In vitro and in vivo antifungal activity and toxicity studies indicated that NPP B1 exhibited comparable antifungal activity against Candida albicans and was less toxic than the most potent heptaene antifungal, amphotericin B. Moreover, NPP B1 showed improved pharmacokinetic parameters compared to those of amphotericin B, suggesting that NPP B1 could be a promising candidate for development into a pharmacokinetically improved and less-toxic polyene antifungal antibiotic.

Structure elucidation and antitumour activity of a new macrolactam produced by marine-derived actinomycete Micromonospora sp. FIM05328.

Chemical investigation of a marine-derived actinomycete strain Micromonospora sp. FIM05328 isolated from a soil sample collected from the East China Sea, resulted in the discovery of a new 26-membered polyene macrolactam metabolite FW05328-1 (1), together with a known polyene with pyridone ring compound aurodox (2). The structures of compounds 1 and 2 were determined by the detailed analysis of 1D, 2D NMR and HR-TOF-MS data, along with literature data analysis. 1 and 2 exhibited excellent antiproliferative activities against KYSE30, KYSE180 and EC109 human tumour cell lines, but displayed no antibacterial activities against bacteria or fungi were tested.

Bacillus amyloliquefaciens ssp. plantarum F11 isolated from Algerian salty lake as a source of biosurfactants and bioactive lipopeptides.

In this study, we identified a new Bacillus strain isolated from an Algerian salty lake that produces metabolites that are active against Gram-positive and Gram-negative bacteria, as well as fungal pathogens. The draft genome sequence of the strain is presented herein. Genome sequence analysis identified the strain to be B. amyloliquefaciens subspecies plantarum F11, and showed that the strain carries the gene clusters for the production of a number of bioactive and surface-active compounds. These include the lipopeptides surfactin and fengycin, antibacterial polyketides macrolactin and bacillaene, and a putative novel lanthipeptide, among others. Through an activity-guided purification method using hydrophobic interaction chromatographic techniques, we confirmed the ability of the strain to produce fengycin lipopeptides. The identities of the isolated fengycin homologs were ascertained through tandem mass spectrometry.

Unsaturated Cuticular Hydrocarbons Enhance Responses to Sex Pheromone in Spruce Budworm, Choristoneura fumiferana.

The primary sex pheromone components of the female spruce budworm, Choristoneura fumiferana (Clem.) (Lepidoptera: Tortricidae), are (E)- and (Z)-11-tetradecenal, produced in 95:5 ratio. However, male flight responses to calling females in a wind tunnel were faster and maintained longer than responses to any synthetic aldehyde blend. Analyses of cuticular extracts from spruce budworm adults revealed series of n-alkanes and n-monoalkenes with predominantly odd numbers of carbon atoms from C23- C29 in both sexes. (Z,Z,Z)-3,6,9-tricosatriene and (Z,Z,Z)-3,6,9-pentacosatriene were identified only in cuticular extracts from females. Pheromonally naïve males showed wing fanning and circling responses to forewing scales from females but not to scales from males. Males also exhibited the same strong responses to scales excised from pharate females, indicating that the pheromone components are produced by females prior to emergence. (Z)-11-hexadecenal and (Z)-5-tricosene enhanced male responses to the primary sex pheromone aldehydes in wind tunnel bioassays, including higher proportions of in-flight and copulatory responses by males and increased time on the source. Addition of (Z,Z,Z)-3,6,9-tricosatriene to the 95/5 blend of (E)- and (Z)-11-tetradecenal released close-range copulatory responses including abdomen curling on treated septa. We propose that the sex pheromone blend of C. fumiferana is composed of the 95/5 blend of (E)- and (Z)-11-tetradecenal as primary components, with (Z)-11-hexadecenal, (Z)-5-tricosene and (Z,Z,Z)-3,6,9-tricosatriene fulfilling secondary roles in orientation and close-range courtship.