A naturally occurring polyacetylene isolated from carrots promotes health and delays signatures of aging.

To ameliorate or even prevent signatures of aging in ultimately humans, we here report the identification of a previously undescribed polyacetylene contained in the root of carrots (Daucus carota), hereafter named isofalcarintriol, which we reveal as potent promoter of longevity in the nematode C. elegans. We assign the absolute configuration of the compound as (3 S,8 R,9 R,E)-heptadeca-10-en-4,6-diyne-3,8,9-triol, and develop a modular asymmetric synthesis route for all E-isofalcarintriol stereoisomers. At the molecular level, isofalcarintriol affects cellular respiration in mammalian cells, C. elegans, and mice, and interacts with the α-subunit of the mitochondrial ATP synthase to promote mitochondrial biogenesis. Phenotypically, this also results in decreased mammalian cancer cell growth, as well as improved motility and stress resistance in C. elegans, paralleled by reduced protein accumulation in nematodal models of neurodegeneration. In addition, isofalcarintriol supplementation to both wild-type C57BL/6NRj mice on high-fat diet, and aged mice on chow diet results in improved glucose metabolism, increased exercise endurance, and attenuated parameters of frailty at an advanced age. Given these diverse effects on health parameters in both nematodes and mice, isofalcarintriol might become a promising mitohormesis-inducing compound to delay, ameliorate, or prevent aging-associated diseases in humans.

Structures and Biosynthesis of Caryoynencins, Unstable Bacterial Polyynes from Pseudomonas protegens Recombinant Expressing the cayG Gene.

Bacteria in certain genera can produce "bacterial polyynes" that contain a conjugated C≡C bond starting from a terminal alkyne. Protegenin A is a derivative of octadecanoic acid that contains an ene-tetrayne moiety. It was discovered in Pseudomonas protegens Cab57 and exhibits strong antioomycete and moderate antifungal activity. By introducing cayG, a cytochrome P450 gene from Burkholderia caryophylli, into P. protegens Cab57, protegenin A was converted into more complex polyynes, caryoynencins A-E. A purification method that minimized the degradation and isomerization of caryoynencins was established. For the first time, as far as we know, the 1H and 13C{1H} NMR signals of caryoynencins were completely assigned by analyzing the NMR data of the isolated compounds and protegenin A enriched with [1-13C]- or [2-13C]-acetate. Through the structural analysis of caryoynencins D/E and bioconversion experiments, we observed that CayG constructs the allyl alcohol moiety of caryoynencins A-C through sequential hydroxylation, dehydration, and hydroxylation. The recombinant strain exhibited a stronger antioomycete activity compared to the wild-type strain. This paper proposes a stable purification and structural determination method for various bacterial polyynes, and P. protegens Cab57 holds promise as an engineering host for the production of biologically active polyynes.

Cloning and expression of Burkholderia polyyne biosynthetic gene clusters in Paraburkholderia hosts provides a strategy for biopesticide development.

Burkholderia have potential as biocontrol agents because they encode diverse biosynthetic gene clusters (BGCs) for a range of antimicrobial metabolites. Given the opportunistic pathogenicity associated with Burkholderia species, heterologous BGC expression within non-pathogenic hosts is a strategy to construct safe biocontrol strains. We constructed a yeast-adapted Burkholderia-Escherichia shuttle vector (pMLBAD_yeast) with a yeast replication origin 2 µ and URA3 selection marker and optimised it for cloning BGCs using the in vivo recombination ability of Saccharomyces cerevisiae. Two Burkholderia polyyne BGCs, cepacin (13 kb) and caryoynencin (11 kb), were PCR-amplified as three overlapping fragments, cloned downstream of the pBAD arabinose promoter in pMLBAD_yeast and mobilised into Burkholderia and Paraburkholderia heterologous hosts. Paraburkholderia phytofirmans carrying the heterologous polyyne constructs displayed in vitro bioactivity against a variety of fungal and bacterial plant pathogens similar to the native polyyne producers. Thirteen Paraburkholderia strains with preferential growth at 30°C compared with 37°C were also identified, and four of these were amenable to genetic manipulation and heterologous expression of the caryoynencin construct. The cloning and successful heterologous expression of Burkholderia biosynthetic gene clusters within Paraburkholderia with restricted growth at 37°C opens avenues for engineering non-pathogenic biocontrol strains.

Integrated omics approach to unveil antifungal bacterial polyynes as acetyl-CoA acetyltransferase inhibitors.

Bacterial polyynes are highly active natural products with a broad spectrum of antimicrobial activities. However, their detailed mechanism of action remains unclear. By integrating comparative genomics, transcriptomics, functional genetics, and metabolomics analysis, we identified a unique polyyne resistance gene, masL (encoding acetyl-CoA acetyltransferase), in the biosynthesis gene cluster of antifungal polyynes (massilin A 1, massilin B 2, collimonin C 3, and collimonin D 4) of Massilia sp. YMA4. Crystallographic analysis indicated that bacterial polyynes serve as covalent inhibitors of acetyl-CoA acetyltransferase. Moreover, we confirmed that the bacterial polyynes disrupted cell membrane integrity and inhibited the cell viability of Candida albicans by targeting ERG10, the homolog of MasL. Thus, this study demonstrated that acetyl-CoA acetyltransferase is a potential target for developing antifungal agents.

Comparative analysis of carbon and nitrogen metabolism, antioxidant indexes, polysaccharides and lobetyolin changes of different tissues from Codonopsis pilosula co-inoculated with Trichoderma.

Codonopsis pilosula is a traditional Chinese herbal medicinal plant and contains various bioactive components, such as C. pilosula polysaccharides (CPPs) and lobetyolin (Lob). Hydrogen peroxide (H2O2) and nitric oxide (NO) are gaseous molecule and have been well known for their ability to relieve some adverse influences on plant from abiotic stress. Endophytic fungus is non-pathogenic plant-associated fungus that could play a significant role in improving plant tolerance by signal molecule. In this work, we determined how inoculation of Trichoderma strain RHTA01 with C. pilosula changed the plant's growth, metabolite accumulation, and related enzyme activity. Results demonstrated that application of Trichoderma strain RHTA01 significantly improved the growth of C. pilosula. Moreover, it noticeably decreased antioxidant enzyme superoxide dismutase (SOD) and catalase (CAT) activity in C. pilosula leaves, reduced the content of H2O2 and malondialdehyde (MDA), and weakened the peroxidation of cell membrane lipids, which reduced the damage of abiotic stress to C. pilosula. Research has shown that it had obvious effects on levels of nitrogen and carbon metabolic enzymes. For example, sucrose synthase (SS) and acid invertase (AI) levels in C. pilosula roots were nearly 1.43 and 1.7 times higher, respectively, than those in the control (CK) group. In addition, it was notable that the production of CPPs and Lob, the most significant secondary metabolites in C. pilosula, were influenced by Trichoderma strain RHTA01. The obtained results indicate that inoculating C. pilosula with Trichoderma stimulates the carbon and nitrogen metabolism of the plant, and helps to increase the content of CPPs and Lob in the root of the plant.

Discovery of the Pseudomonas Polyyne Protegencin by a Phylogeny-Guided Study of Polyyne Biosynthetic Gene Cluster Diversity.

Natural products that possess alkyne or polyyne moieties have been isolated from a variety of biological sources and possess a broad a range of bioactivities. In bacteria, the basic biosynthesis of polyynes is known, but their biosynthetic gene cluster (BGC) distribution and evolutionary relationship to alkyne biosynthesis have not been addressed. Through comprehensive genomic and phylogenetic analyses, the distribution of alkyne biosynthesis gene cassettes throughout bacteria was explored, revealing evidence of multiple horizontal gene transfer events. After investigation of the evolutionary connection between alkyne and polyyne biosynthesis, a monophyletic clade was identified that possessed a conserved seven-gene cassette for polyyne biosynthesis that built upon the conserved three-gene cassette for alkyne biosynthesis. Further diversity mapping of the conserved polyyne gene cassette revealed a phylogenetic subclade for an uncharacterized polyyne BGC present in several Pseudomonas species, designated pgn. Pathway mutagenesis and high-resolution analytical chemistry showed the Pseudomonas protegens pgn BGC directed the biosynthesis of a novel polyyne, protegencin. Exploration of the biosynthetic logic behind polyyne production, through BGC mutagenesis and analytical chemistry, highlighted the essentiality of a triad of desaturase proteins and a thioesterase in both the P. protegens pgn and Trinickia caryophylli (formerly Burkholderia caryophylli) caryoynencin pathways. We have unified and expanded knowledge of polyyne diversity and uniquely demonstrated that alkyne and polyyne biosynthetic gene clusters are evolutionarily related and widely distributed within bacteria. The systematic mapping of conserved biosynthetic genes across the available bacterial genomic diversity proved to be a fruitful method for discovering new natural products and better understanding polyyne biosynthesis. IMPORTANCE Natural products bearing alkyne (triple carbon bond) or polyyne (multiple alternating single and triple carbon bonds) moieties exhibit a broad range of important biological activities. Polyyne metabolites have been implicated in important ecological roles such as cepacin mediating biological control of plant pathogens and caryoynencin protecting Lagriinae beetle eggs against pathogenic fungi. After further phylogenetic exploration of polyyne diversity, we identified a novel gene cluster in Pseudomonas bacteria with known biological control abilities and proved it was responsible for synthesizing a new polyyne metabolite, protegencin. The evolutionary analysis of polyyne pathways showed that multiple biosynthetic genes were conserved, and using mutagenesis, their essentiality was demonstrated. Our research provides a foundation for the future modification of polyyne metabolites and has identified a novel polyyne, protegencin, with potential bioactive roles of ecological and agricultural importance.

Human intestinal microbiota derived metabolism signature from a North American native botanical Oplopanax horridus with UPLC/Q-TOF-MS analysis.

Oplopanax horridus, widely distributed in North America, is an herbal medicine traditionally used by Pacific indigenous peoples for various medical conditions. After oral ingestion, constituents in O. horridus extract (OhE) could be converted to their metabolites by the enteric microbiome before absorption. In this study, in order to mimic gut environment, the OhE was biotransformed using the enteric microbiome of healthy human subjects. For accurate and reliable data collection with optimized approaches in sample preparation and analytical conditions, ultra-performance liquid chromatography and quadrupole time-of-flight mass spectrometry were used to characterize parent constituents and their metabolites. In the extract, 20 parent compounds were identified including polyynes, sesquiterpenes, monoterpeondids, phenylpropanoids and phenolic acids. After the biotransformation, a total of 78 metabolites were identified, of which 37 belonged to polyynes metabolites. The common biotransformation pathways are hydroxylation, acetylization, methylation and demethylation. Based on the pathway distributions, the metabolism signature of OhE has been explored. The metabolism pathways of OhE compounds are dependent on their structural classifications and hydrophilic/hydrophobic properties. In summary, with comprehensive analysis, we systematically investigated human microbiome-derived OhE metabolites. The enteric microbial metabolism signature provides novel information for future effective use of O. horridus.

Biosynthesis of the widely distributed hydrocarbon (Z,Z)-6,9-heptadecadiene in astigmatid mites.

Using a crude enzyme solution prepared from astigmatid mites, the conversion reaction to (Z,Z)-6,9-heptadecadiene (6,9-C17) using linoleyl aldehyde (LAld) as a substrate was successful. The mass spectrum of the reaction product using 13C-labeled LAld as a substrate could be assigned as 13C-labeled 6,9-C17. Unlike the findings in other species, the decarbonylase derived from mites did not require a coenzyme.

Response of Bioactive Metabolite and Biosynthesis Related Genes to Methyl Jasmonate Elicitation in Codonopsis pilosula.

Bioactive metabolites in Codonopsis pilosula are of particular interest as an immunostimulant. Methyl jasmonate (MeJA) plays an important role in the elicitation of metabolite biosynthesis. Here, we explored the response of metabolites to MeJA elicitation in C. pilosula adventitious roots and multiple shoots. The results showed that the biomass, polysaccharide, and lobetyolin content of adventitious roots exhibited the highest increases with 100 µmol·L-1 MeJA at the 16th day of subculture, whereas the atractylenolide III (a terpenoid) content increased extremely with 50 µmol·L-1 MeJA treatment at the 7th day of subculture. In addition, the biomass and lobetyolin content significantly increased at the 4th day after treatment. Similarly, the polysaccharide and lobetyolin content increased in multiple shoots. Further identification of different metabolites responding to MeJA by ¹H-NMR showed an extremely significant increase of the lobetyolinin level, which coincided with lobetyolin. Accordingly, the precursor, fatty acids, showed a highly significant decrease in their levels. Furthermore, a significant increase in ß-d-fructose-butanol glycoside was detected, which was accompanied by a decrease in the sucrose level. Accordingly, the enzyme genes responsible for terpenoid and carbohydrate biosynthesis, CpUGPase, and CpPMK, were up regulated. In conclusion, MeJA promoted culture growth and accelerated bioactive metabolite accumulation by regulating the expression of the metabolite biosynthesis related genes, CpUGPase and CpPMK in C. pilosula.

Metabolite amyloid-like fibrils interact with model membranes.

Amyloid-like structure formation by various metabolites represents a significant extension of the amyloidogenic building block family. Similar to protein amyloids, metabolite amyloids induce apoptotic toxicity, a process that was linked to membrane association. Here, we demonstrate that metabolite amyloids interact with model membranes and study the mechanism by molecular dynamics.

Biotin-functionalized targeted polydiacetylene micelles.

Polydiacetylene micelles were functionalized with controlled amounts of biotin using bioorthogonal click chemistry. The biotinylated micelles were evaluated in the selective targeting of the MCF-7 cancerous cell line and were shown to be readily internalized. The efficiency of the cellular uptake was correlated to the density of grafted biotin.

Dietary polyacetylenes, falcarinol and falcarindiol, isolated from carrots prevents the formation of neoplastic lesions in the colon of azoxymethane-induced rats.

Falcarinol (FaOH) and falcarindiol (FaDOH) are found in many food plants of the Apiaceae family. Carrots are a major dietary source of these polyacetylenes. Feeding azoxymethane (AOM)-induced rats with carrots and purified FaOH have previously been shown to inhibit neoplastic transformations in the colon. FaOH and FaDOH have also shown to have a synergistic effect in vitro, resulting in a significant increased cytotoxic activity. Based on these findings the antineoplastic effect of FaOH and FaDOH (purity > 99%) was investigated in the AOM-induced rat model. Twenty rats received rat diet containing 7 µg FaOH per g feed and 7 µg FaDOH per g feed and 20 rats were controls receiving only rat diet. Then carcinogenesis was induced in all 40 rats with the carcinogen AOM. All animals received the designated diet for 2 weeks before AOM induction and continued on the designated diet throughout the experiment. Rats were euthanized 18 weeks after the first AOM injection and macroscopic polyp/cancers were measured, harvested and stained for histology. The difference in sizes of aberrant crypt foci (ACF) were analysed in a Wilcoxon rank sum test, in which the median number of small ACF was 218 in controls and 145 in polyacetylene treated rats (P < 0.001). Fifteen control rats and 8 treated rats had macroscopic tumors (P = 0.027). The number of tumors larger than 3 mm were 6 and 1 in control and treated rats, respectively (P = 0.032). In conclusion dietary supplements with FaOH and FaDOH reduced the number of neoplastic lesions as well as the growth rate of the polyps suggesting a preventive effect of FaOH and FaDOH on the development of colorectal cancer.

Stereoselective Total Syntheses of Polyacetylene Plant Metabolites via Ester-Tethered Ring Closing Metathesis.

Total syntheses of five naturally occurring polyacetylenes from three different plants are described. These natural products have in common an E,Z-configured conjugated diene linked to a di- or triyne chain. As the key method to stereoselectively establish the E,Z-diene part, an ester-tethered ring-closing metathesis/base-induced eliminative ring opening sequence was used. The results presented herein do not only showcase the utility of this tethered RCM variant but have also prompted us to suggest that the originally assigned absolute configurations of chiral polyacetylenes from Atractylodes macrocephala should be revised or at least reconsidered.

Polydiacetylene sensor interaction with food sanitizers and surfactants.

Polydiacetylene (PDA) vesicles are of interest as biosensors, particularly for pathogenic bacteria. As part of a food monitoring system, interaction with food sanitizers/surfactants was investigated. PDA vesicles were prepared by inkjet-printing, photopolymerized and characterized by dynamic light scattering (DLS) and UV/Vis spectroscopy. The optical response of PDA vesicles at various concentrations verses a fixed sanitizer/surfactant concentration was determined using a two variable factorial design. Sanitizer/surfactant response at various concentrations over time was also measured. Results indicated that only Vigilquat and TritonX-100 interacted with PDA vesicles giving visible colour change out of 8 sanitizers/surfactants tested. PDA vesicle concentration, sanitizer/surfactant concentration, and time all had a significant (P<0.0001) effect on colour change. As they are highly sensitive to the presence of Vigilquat and TritonX-100, PDA sensors could be used to detect chemical residues as well as for detection of various contaminants in the food industry.

Improved Growth and Metabolite Accumulation in Codonopsis pilosula (Franch.) Nannf. by Inoculation of Bacillus amyloliquefaciens GB03.

Codonopsis pilosula (Franch.) Nannf. is a traditional Chinese herbal medicinal plant and a low-cost succedaneum for Panax ginseng and contains various bioactivity components. In this work, we first evaluated the effects of the inoculation of the plant growth-promoting rhizobacteria Bacillus amyloliquefaciens strain GB03 on growth and metabolite accumulation of C. pilosula. The results demonstrated that application of B. amyloliquefaciens GB03 significantly improved the growth of C. pilosula compared to DH5α, Luria broth medium, and water treatment, respectively. On the other hand, we observed that the content of lobetyolin, one of the most important secondary metabolites in C. pilosula, was obviously improved by inoculation of GB03 and almost reached twice that compared to the other three treatments. In addition, some amino acids of roots were elevated by GB03, although not significantly. In conclusion, B. amyloliquefaciens GB03 could induce positive effects on the growth and further stimulate accumulation of secondary metabolites in C. pilosula.

Quantifying biochemical quality parameters in carrots (Daucus carota L.) - FT-Raman spectroscopy as efficient tool for rapid metabolite profiling.

Application of FT-Raman spectroscopy for simultaneous quantification of carotenoids, carbohydrates, polyacetylenes and phenylpropanoids with high bioactive potential was investigated in storage roots of Daucus carota. Within single FT-Raman experiment carbohydrates, carotenoids, and polyacetylenes could be reliably quantified with high coefficients of determination of R(2)>0.91. The most abundant individual representatives of each compound class could be quantified with comparably high quality resulting in R(2)=0.97 and 0.96 for α-carotene and ß-carotene, in R(2)=0.90 for falcarindiol (FaDOH), R(2)=0.99, 0.98 and 0.96 for fructose, glucose and sucrose. In contrast, application of FT-Raman spectroscopy for quantification of two laserine-type phenylpropanoids was investigated but failed due to low concentration and Raman response. Furthermore, evaluation of metabolic profiles by principle component analysis (PCA) revealed metabolic variety of carrot root composition depending on root color and botanical relationship.

Chemoproteomic Evaluation of the Polyacetylene Callyspongynic Acid.

Polyacetylenes are a class of alkyne-containing natural products. Although potent bioactivities and thus possible applications as chemical probes have already been reported for some polyacetylenes, insights into the biological activities or molecular mode of action are still rather limited in most cases. To overcome this limitation, we describe the application of the polyacetylene callyspongynic acid in the development of an experimental roadmap for characterizing potential protein targets of alkyne-containing natural products. To this end, we undertook the first chemical synthesis of callyspongynic acid. We then used in situ chemical proteomics methods to demonstrate extensive callyspongynic acid-mediated chemical tagging of endoplasmic reticulum-associated lipid-metabolizing and modifying enzymes. We anticipate that an elucidation of protein targets of natural products may serve as an effective guide to the development of subsequent biological assays that aim to identify chemical phenotypes and bioactivities.

Chemical and pharmacological progress on polyacetylenes isolated from the family apiaceae.

This review presents an up-to-date survey of natural polyacetylenes isolated from the family Apiaceae, and their biosynthesis and biological activities up to May 2013, with 107 references. A total of 103 polyacetylenes from 72 species of 41 genera of Apiaceae have been isolated so far, among which falcarinol-type polyacetylenes are most widely distributed.

The molecular basis of conjugated polyyne biosynthesis in phytopathogenic bacteria.

Polyynes (polyacetylenes), which are produced by a variety of organisms, play important roles in ecology. Whereas alkyne biosynthesis in plants, fungi, and insects has been studied, the biogenetic origin of highly unstable bacterial polyynes has remained a riddle. Transposon mutagenesis and genome sequencing unveiled the caryoynencin (cay) biosynthesis gene cluster in the plant pathogen B. caryophylli, and homologous gene clusters were found in various other bacteria by comparative genomics. Gene inactivation and phylogenetic analyses revealed that novel desaturase/acetylenase genes mediate bacterial polyyne assembly. A cytochrome P450 monooxygenase is involved in the formation of the allylic alcohol moiety, as evidenced by analysis of a fragile intermediate, which was stabilized by an in situ click reaction. This work not only grants first insight into bacterial polyyne biosynthesis but also demonstrates that the click reaction can be employed to trap fragile polyynes from crude mixtures.