Isatropolone/isarubrolone Cm from Streptomyces with biological activity of inducing incomplete autophagy.

Isatropolones/isarubrolones are Streptomyces secondary metabolites featuring a tropolone ring in the pentacyclic scaffolds of these molecules. They are able to induce complete autophagy in human HepG2 cells. Here, methyl isatropolones (1-2) and isarubrolone (3) are identified from Streptomyces CPCC 204095. They all have a methyl tropolone ring in the pentacyclic scaffold of these molecules resolved by MS and NMR spectra. Biological activity assay indicates that isatropolone Cm (1) and isarubrolone Cm (3) induce incomplete autophagy in human HepG2 cells.

Discovery of Tropolone Stipitaldehyde as a Potential Agent for Controlling Phytophthora Blight and Its Action Mechanism Research.

The fermentation of endophytic Nigrospora chinensis GGY-3 resulted in the isolation of tropolone stipitaldehyde (1), which exhibited broad-spectrum inhibition activity against fungi and bacteria, especially against Phytophthora capsici, with an EC50 value of 0.83 µg/mL and Xanthomonas oryzae pv. oryzicola, with a minimum inhibitory concentration value of 4.0 µg/mL. The in vitro and in vivo assays demonstrated that 1 had a significant protective effect on P. capsici. Furthermore, 1 inhibited the spore germination of P. capsici and damaged the plasma membrane structure. As observed by SEM and TEM, after exposure to 1, mycelia exhibited swelling, shrunken, branch-increasing phenomena, cell wall and membrane damage, and disordered content. Transcriptome analysis revealed that 1 might affect starch and sucrose metabolism and fatty acid biosynthesis by suppressing the expression of genes relevant to cell wall synthetases and cell membrane-associated genes. These findings indicate that 1 may be a potential agrochemical fungicide for controlling phytophthora blight.

Tropolone derivative hinokitiol ameliorates cerulein-induced acute pancreatitis in mice.

Hinokitiol is a natural bio-active tropolone derivative with promising antioxidant and anti-inflammatory properties. This study was conducted to evaluate the ameliorative effects of hinokitiol against acute pancreatitis induced by cerulein. Mice were pre-treated with hinokitiol intraperitoneally for 7 days (50 and 100 mg/kg), and on the final day of study, cerulein (6 × 50 µg/kg) was injected every hour for six times. Six hours after the last dose of cerulein, blood was collected from the mice through retro-orbital plexus for biochemical analysis. After blood collection, mice were euthanized and the pancreas was harvested for studying effects on oxidative stress, pro-inflammatory cytokines, immunohistochemistry and histopathology of tissue sections. Hinokitiol treatment significantly reduced edema of the pancreas and reduced the plasma levels of lipase and amylase in mice with cerulein-induced acute pancreatitis. It also attenuated the oxidative and nitrosative stress related damage as evident from the reduced malondialdehyde (MDA) and nitrite levels, which were significantly increased in the mice with acute pancreatitis. Furthermore, hinokitiol administration significantly reduced the pancreatitis-evoked decrease in the activity of catalase, glutathione (GSH) and superoxide dismutase (SOD) in the pancreatic tissue. Pre-treatment with hinokitiol significantly reduced the elevated levels of pro-inflammatory cytokines like interleukin-6 (IL-6), interleukin-1ß (IL-1ß), tumor necrosis factor-alpha (TNF-α) as well as increased the levels of anti-inflammatory cytokine interleukin-10 (IL-10) in the pancreatic tissue of mice with acute pancreatitis. The immunohistochemical expression of nuclear factor kappa light chain enhancer of activated B cells (NF-κB), cyclooxygenase (COX-2) and TNF-α were significantly decreased by hinokitiol in mice with cerulein-induced acute pancreatitis. In conclusion, the results of the present study demonstrate that hinokitiol has significant potential to prevent cerulein-induced acute pancreatitis.

A small molecule redistributes iron in ferroportin-deficient mice and patient-derived primary macrophages.

Deficiencies of the transmembrane iron-transporting protein ferroportin (FPN1) cause the iron misdistribution that underlies ferroportin disease, anemia of inflammation, and several other human diseases and conditions. A small molecule natural product, hinokitiol, was recently shown to serve as a surrogate transmembrane iron transporter that can restore hemoglobinization in zebrafish deficient in other iron transporting proteins and can increase gut iron absorption in FPN1-deficient flatiron mice. However, whether hinokitiol can restore normal iron physiology in FPN1-deficient animals or primary cells from patients and the mechanisms underlying such targeted activities remain unknown. Here, we show that hinokitiol redistributes iron from the liver to red blood cells in flatiron mice, thereby increasing hemoglobin and hematocrit. Mechanistic studies confirm that hinokitiol functions as a surrogate transmembrane iron transporter to release iron trapped within liver macrophages, that hinokitiol-Fe complexes transfer iron to transferrin, and that the resulting transferrin-Fe complexes drive red blood cell maturation in a transferrin-receptor-dependent manner. We also show in FPN1-deficient primary macrophages derived from patients with ferroportin disease that hinokitiol moves labile iron from inside to outside cells and decreases intracellular ferritin levels. The mobilization of nonlabile iron is accompanied by reductions in intracellular ferritin, consistent with the activation of regulated ferritin proteolysis. These findings collectively provide foundational support for the translation of small molecule iron transporters into therapies for human diseases caused by iron misdistribution.

Biosynthesis of Ditropolonyl Sulfide, an Antibacterial Compound Produced by Burkholderia cepacia Complex Strain R-12632.

Burkholderia cepacia complex strain R-12632 produces ditropolonyl sulfide, an unusual sulfur-containing tropone, via a yet-unknown biosynthetic pathway. Ditropolonyl sulfide purified from a culture of strain R-12632 inhibits the growth of various Gram-positive and Gram-negative resistant bacteria, with MIC values as low as 16 µg/ml. In the present study, we used a transposon mutagenesis approach combined with metabolite analyses to identify the genetic basis for antibacterial activity of strain R-12632 against Gram-negative bacterial pathogens. Fifteen of the 8304 transposon mutants investigated completely lost antibacterial activity against Klebsiella pneumoniae LMG 2095. In these loss-of-activity mutants, nine genes were interrupted. Four of those genes were involved in assimilatory sulfate reduction, two were involved in phenylacetic acid (PAA) catabolism, and one was involved in glutathione metabolism. Via semipreparative fractionation and metabolite identification, it was confirmed that inactivation of the PAA degradation pathway or glutathione metabolism led to loss of ditropolonyl sulfide production. Based on earlier studies on the biosynthesis of tropolone compounds, the requirement for a functional PAA catabolic pathway for antibacterial activity in strain R-12632 indicated that this pathway likely provides the tropolone backbone for ditropolonyl sulfide. Loss of activity observed in mutants defective in assimilatory sulfate reduction and glutathione biosynthesis suggested that cysteine and glutathione are potential sources of the sulfur atom linking the two tropolone moieties. The demonstrated antibacterial activity of the unusual antibacterial compound ditropolonyl sulfide warrants further studies into its biosynthesis and biological role. IMPORTANCEBurkholderia bacteria are historically known for their biocontrol properties and have been proposed as a promising and underexplored source of bioactive specialized metabolites. Burkholderia cepacia complex strain R-12632 inhibits various Gram-positive and Gram-negative resistant pathogens and produces numerous specialized metabolites, among which is ditropolonyl sulfide. This unusual antimicrobial has been poorly studied and its biosynthetic pathway remains unknown. In the present study, we performed transposon mutagenesis of strain R-12632 and performed genome and metabolite analyses of loss-of-activity mutants to study the genetic basis for antibacterial activity. Our results indicate that phenylacetic acid catabolism, assimilatory sulfate reduction, and glutathione metabolism are necessary for ditropolonyl sulfide production. These findings contribute to understanding of the biosynthesis and biological role of this unusual antimicrobial.

A Flavoprotein Dioxygenase Steers Bacterial Tropone Biosynthesis via Coenzyme A-Ester Oxygenolysis and Ring Epoxidation.

Bacterial tropone natural products such as tropolone, tropodithietic acid, or the roseobacticides play crucial roles in various terrestrial and marine symbiotic interactions as virulence factors, antibiotics, algaecides, or quorum sensing signals. We now show that their poorly understood biosynthesis depends on a shunt product from aerobic CoA-dependent phenylacetic acid catabolism that is salvaged by the dedicated acyl-CoA dehydrogenase-like flavoenzyme TdaE. Further characterization of TdaE revealed an unanticipated complex catalysis, comprising substrate dehydrogenation, noncanonical CoA-ester oxygenolysis, and final ring epoxidation. The enzyme thereby functions as an archetypal flavoprotein dioxygenase that incorporates both oxygen atoms from O2 into the substrate, most likely involving flavin-N5-peroxide and flavin-N5-oxide species for consecutive CoA-ester cleavage and epoxidation, respectively. The subsequent spontaneous decarboxylation of the reactive enzyme product yields tropolone, which serves as a key virulence factor in rice panicle blight caused by pathogenic edaphic Burkholderia plantarii. Alternatively, the TdaE product is most likely converted to more complex sulfur-containing secondary metabolites such as tropodithietic acid from predominant marine Rhodobacteraceae (e.g., Phaeobacter inhibens).

Genomic Evolution of the Marine Bacterium Phaeobacter inhibens during Biofilm Growth.

Phaeobacter inhibens 2.10 is an effective biofilm former on marine surfaces and has the ability to outcompete other microorganisms, possibly due to the production of the plasmid-encoded secondary metabolite tropodithietic acid (TDA). P. inhibens 2.10 biofilms produce phenotypic variants with reduced competitiveness compared to the wild type. In the present study, we used longitudinal, genome-wide deep sequencing to uncover the genetic foundation that contributes to the emergent phenotypic diversity in P. inhibens 2.10 biofilm dispersants. Our results show that phenotypic variation is not due to the loss of the plasmid that carries the genes for TDA synthesis but instead show that P. inhibens 2.10 biofilm populations become rapidly enriched in single nucleotide variations in genes involved in the synthesis of TDA. While variants in genes previously linked to other phenotypes, such as lipopolysaccharide production (i.e., rfbA) and cellular persistence (i.e., metG), also appear to be selected for during biofilm dispersal, the number and consistency of variations found for genes involved in TDA production suggest that this metabolite imposes a burden on P. inhibens 2.10 cells. Our results indicate a strong selection pressure for the loss of TDA in monospecies biofilm populations and provide insight into how competition (or a lack thereof) in biofilms might shape genome evolution in bacteria. IMPORTANCE Biofilm formation and dispersal are important survival strategies for environmental bacteria. During biofilm dispersal, cells often display stable and heritable variants from the parental biofilm. Phaeobacter inhibens is an effective colonizer of marine surfaces, in which a subpopulation of its biofilm dispersal cells displays a noncompetitive phenotype. This study aimed to elucidate the genetic basis of these phenotypic changes. Despite the progress made to date in characterizing the dispersal variants in P. inhibens, little is understood about the underlying genetic changes that result in the development of the specific variants. Here, P. inhibens phenotypic variation was linked to single nucleotide polymorphisms (SNPs), in particular in genes affecting the competitive ability of P. inhibens, including genes related to the production of the antibiotic tropodithietic acid (TDA) and bacterial cell-cell communication (e.g., quorum sensing). This work is significant as it reveals how the biofilm lifestyle might shape genome evolution in a cosmopolitan bacterium.

Pseudomonas donghuensis HYS 7-hydroxytropolone contributes to pathogenicity toward Caenorhabditis elegans and is influenced by pantothenic acid.

Pseudomonas donghuensis HYS, a bacterial strain identified from Donghu Lake, has tremendous toxicity toward Caenorhabditis elegans and is characterized by high 7-hydroxytropolone siderophore production. Here, the relationship between pathogenic siderophore production and pantothenic acid was evaluated. The pathogenicity of P. donghuensis HYS was illustrated using C. elegans as a host. Based on slow-killing assay findings, a 7-hydroxytropolone deficiency-causing mutation attenuated P. donghuensis HYS pathogenicity, which was restored by the addition of extracted 7-hydroxytropolone. Moreover, data from real-time qPCR analysis and characteristic absorption assays indicated that pantothenic acid deficiency repressed transcriptional levels of orf9, which further reduced 7-hydroxytropolone production. Furthermore, slow-killing assays indicated that panB and pantothenic acid affected the virulence of P. donghuensis. These results indicate that a 7-hydroxytropolone siderophore-producing strain is virulent toward C. elegans. Our findings demonstrate that pantothenic acid is associated with P. donghuensis siderophore production-related pathogenicity.

Bacterial Tropone Natural Products and Derivatives: Overview of their Biosynthesis, Bioactivities, Ecological Role and Biotechnological Potential.

Tropone natural products are non-benzene aromatic compounds of significant ecological and pharmaceutical interest. Herein, we highlight current knowledge on bacterial tropones and their derivatives such as tropolones, tropodithietic acid, and roseobacticides. Their unusual biosynthesis depends on a universal CoA-bound precursor featuring a seven-membered carbon ring as backbone, which is generated by a side reaction of the phenylacetic acid catabolic pathway. Enzymes encoded by separate gene clusters then further modify this key intermediate by oxidation, CoA-release, or incorporation of sulfur among other reactions. Tropones play important roles in the terrestrial and marine environment where they act as antibiotics, algaecides, or quorum sensing signals, while their bacterial producers are often involved in symbiotic interactions with plants and marine invertebrates (e. g., algae, corals, sponges, or mollusks). Because of their potent bioactivities and of slowly developing bacterial resistance, tropones and their derivatives hold great promise for biomedical or biotechnological applications, for instance as antibiotics in (shell)fish aquaculture.

7-hydroxytropolone is the main metabolite responsible for the fungal antagonism of Pseudomonas donghuensis strain SVBP6.

Pseudomonas donghuensis strain SVBP6, an isolate from an agricultural plot in Argentina, displays a broad-spectrum and diffusible antifungal activity, which requires a functional gacS gene but could not be ascribed yet to known secondary metabolites typical of Pseudomonas biocontrol species. Here, we report that Tn5 mutagenesis allowed the identification of a gene cluster involved in both the fungal antagonism and the production of a soluble tropolonoid compound. The ethyl acetate extract from culture supernatant showed a dose-dependent inhibitory effect against the phytopathogenic fungus Macrophomina phaseolina. The main compound present in the organic extract was identified by spectroscopic and X-ray analyses as 7-hydroxytropolone (7HT). Its structure and tautomerism was confirmed by preparing the two key derivatives 2,3-dimethoxy- and 2,7-dimethoxy-tropone. 7HT, but not 2,3- or 2,7-dimethoxy-tropone, mimicked the fungal inhibitory activity of the ethyl acetate extract from culture supernatant. The activity of 7HT, as well as its production, was barely affected by the presence of up to 50 µM added iron (Fe+2 ). To summarize, P. donghuensis SVBP6 produces 7HT under the positive control of the Gac-Rsm cascade and is the main active metabolite responsible for the broad-spectrum inhibition of different phytopathogenic fungi.

A direct comparison of divalent metal-ion transporter (DMT1) and hinokitiol, a potential small molecule replacement.

Hinokitiol, a natural lipophilic chelator, appears capable of replacing several iron transporters after they have been genetically ablated. Divalent metal-ion transporter (DMT1) is the major iron importer in enterocytes and erythroblasts. We have compared DMT1 and hinokitiol in multiple fashions to learn if the smaller molecule is a suitable substitute using two HEK293 cell lines engineered to overexpress different isoforms of DMT1. Both the macromolecule and the lipophilic chelator enable import of ferrous ions into HEK293 cells. Hinokitiol also mediates ferric ion import but DMT1 cannot do so. While DMT1 can also import Mn2+ ions, hinokitiol lacks this ability. The Michaelis-Menten analysis for kinetics of macromolecular catalysis is also suitable for hinokitiol-supported iron import. To compare hinokitiol to DMT1 relative to other metal ions that DMT1 can transport, we employed an organic extraction procedure with which we initially matched the results obtained for Fe2+, Fe3+ and Mn2+, and then showed that multiple other cations were unlikely to enter via hinokitiol. The small chelator thus shares some functional properties with DMT1, but distinct difference were also noted.

Transcriptome analysis to understand the effects of the toxoflavin and tropolone produced by phytopathogenic Burkholderia on Escherichia coli.

The phytopathogenic Burkholderia species B. glumae and B. plantarii are the causal agents of bacterial wilt, grain rot, and seedling blight, which threaten the rice industry globally. Toxoflavin and tropolone are produced by these phytopathogens and are considered the most hostile biohazards with a broad spectrum of target organisms. However, despite their nonspecific toxicity, the effects of toxoflavin and tropolone on bacteria remain unknown. RNA-seq based transcriptome analysis was employed to determine the genome-wide expression patterns under phytotoxin treatment. Expression of 2327 and 830 genes was differentially changed by toxoflavin and tropolone, respectively. Enriched biological pathways reflected the down-regulation of oxidative phosphorylation and ribosome function, beginning with the inhibition of membrane biosynthesis and nitrogen metabolism under oxidative stress or iron starvation. Conversely, several systems such as bacterial chemotaxis, flagellar assembly, biofilm formation, and sulfur/taurine transporters were highly expressed as countermeasures against the phytotoxins. In addition, our findings revealed that three hub genes commonly induced by both phytotoxins function as the siderophore enterobactin, an iron-chelator. Our study provides new insights into the effects of phytotoxins on bacteria for better understanding of the interactions between phytopathogens and other microorganisms. These data will also be applied as a valuable source in subsequent applications against phytotoxins, the major virulence factor.

Identification of the gene cluster for bistropolone-humulene meroterpenoid biosynthesis in Phoma sp.

Eupenifeldin, a bistropolone meroterpenoid, was first discovered as an antitumor agent from the fungus Eupenicillium brefeldianum. We also isolated this compound and a new congener from a strain of Phoma sp. (CGMCC 10481), and evaluated their antitumor effects. Eupenifeldin showed potent in vitro anti-glioma activity. This tropolone-humulene-tropolone meroterpenoid could be originated from two units of tropolone orthoquinone methides and a 10-hydroxyhumulene moiety via hetero-Diels-Alder reactions. To explore the biosynthesis of this class of tropolonic sesquiterpenes, the genome of a eupenifeldin-producing Phoma sp. was sequenced and analyzed. The biosynthetic gene cluster of eupenifeldin (eup) was identified and partially validated by genomic analysis, gene disruption, and product analysis. A nonreducing polyketide synthase EupA, a FAD-dependent monooxygenase EupB, and a non-heme Fe (II)-dependent dioxygenase EupC, were identified as the enzymes responsible for tropolone formation. While the terpene cyclase EupE of an unknown family was characterized to catalyze humulene formation, and a cytochrome P450 enzyme EupD was responsible for hydroxylation of humulene. This study sheds light on the biosynthesis of eupenifeldin, and paves the way to further decipher its biosynthetic pathway.

Shunting Phenylacetic Acid Catabolism for Tropone Biosynthesis.

Tropone is a seven-membered ring nonbenzenoid aromatic compound. It is the core structure of tropolonoids, which have various biological activities. In this study, a hybrid tropone biosynthetic pathway was designed by connecting phenylacetic acid (PAA) degradation with its biosynthesis and reconstituted in Escherichia coli. To simplify pathway construction and optimization, the use of E. coli endogenous genes was maximized and only three exogenous genes were employed. The entire pathway was divided into four modules: the endogenous shikimate pathway module, the hybrid PAA biosynthetic module, the endogenous PAA catabolic module and the heterogeneous tropone biosynthetic module. Efficiency of the PAA catabolic module was enhanced using PAA consumption rate as the indicator. Then, a single point mutation was introduced to inactivate the ALDH domain of PaaZ and the carbon flow was redirected toward tropone synthesis. Assembly of the full pathway led to de novo tropone production with the best titer of 65.2 ± 1.4 mg/L in shake flask experiment. This study provides a potential alternative for sustainable production of tropone and its derivatives.

Tropolone natural products.

Covering: 2008 to 2018 This review provides a comprehensive overview of newly discovered natural products containing a tropolonoid motif covering 2008 up to 2018 and depicts the ecological context in which they have been isolated. This review has a strong focus on describing the different analytical tools and molecular biological approaches used to identify the underlying biosynthetic pathways.

The marine bacterium Phaeobacter inhibens secures external ammonium by rapid buildup of intracellular nitrogen stocks.

Reduced nitrogen species are key nutrients for biological productivity in the oceans. Ammonium is often present in low and growth-limiting concentrations, albeit peaks occur during collapse of algal blooms or via input from deep sea upwelling and riverine inflow. Autotrophic phytoplankton exploit ammonium peaks by storing nitrogen intracellularly. In contrast, the strategy of heterotrophic bacterioplankton to acquire ammonium is less well understood. This study revealed the marine bacterium Phaeobacter inhibens DSM 17395, a Roseobacter group member, to have already depleted the external ammonium when only ∼⅓ of the ultimately attained biomass is formed. This was paralleled by a three-fold increase in cellular nitrogen levels and rapid buildup of various nitrogen-containing intracellular metabolites (and enzymes for their biosynthesis) and biopolymers (DNA, RNA and proteins). Moreover, nitrogen-rich cells secreted potential RTX proteins and the antibiotic tropodithietic acid, perhaps to competitively secure pulses of external ammonium and to protect themselves from predation. This complex response may ensure growing cells and their descendants exclusive provision with internal nitrogen stocks. This nutritional strategy appears prevalent also in other roseobacters from distant geographical provenances and could provide a new perspective on the distribution of reduced nitrogen in marine environments, i.e. temporary accumulation in bacterioplankton cells.

Effect of TDA-producing Phaeobacter inhibens on the fish pathogen Vibrio anguillarum in non-axenic algae and copepod systems.

The expanding aquaculture industry plays an important role in feeding the growing human population and with the expansion, sustainable bacterial disease control, such as probiotics, becomes increasingly important. Tropodithietic acid (TDA)-producing Phaeobacter spp. can protect live feed, for example rotifers and Artemia as well as larvae of turbot and cod against pathogenic vibrios. Here, we show that the emerging live feed, copepods, is unaffected by colonization of the fish pathogen Vibrio anguillarum, making them potential infection vectors. However, TDA-producing Phaeobacter inhibens was able to significantly inhibit V. anguillarum in non-axenic cultures of copepod Acartia tonsa and the copepod feed Rhodomonas salina. Vibrio grew to 106  CFU ml-1 and 107  CFU ml-1 in copepod and R. salina cultures, respectively. However, vibrio counts remained at the inoculum level (104  CFU ml-1 ) when P. inhibens was also added. We further developed a semi-strain-specific qPCR for V. anguillarum to detect and quantify the pathogen in non-axenic systems. In conclusion, P. inhibens efficiently inhibits the fish larval pathogen V. anguillarum in the emerging live feed, copepods, supporting its use as a probiotic in aquaculture. Furthermore, qPCR provides an effective method for detecting vibrio pathogens in complex non-axenic live feed systems.

A Complex Mechanism Involving LysR and TetR/AcrR That Regulates Iron Scavenger Biosynthesis in Pseudomonas donghuensis HYS.

7-Hydroxytropolone (7-HT) is a symmetrical seven-membered heteroatomic ring with a carboxyl group and two hydroxyl groups and was recently reported to be an iron scavenger of Pseudomonas donghuensis HYS. Cluster 1 includes 12 genes related to the synthesis of 7-HT; among these genes, those for two regulators, Orf1 and Orf12, were predicted to regulate 7-HT biosynthesis and to be LysR-type transcriptional regulators (LTTRs) and TetR/AcrR family transcriptional regulators, respectively. Data from real-time quantitative PCR and ß-galactosidase and classical siderophore assays indicated that the transcription levels of orf1 and orf12, as well as those of crucial genes orf6 to orf9, were repressed under high-iron conditions. The deletion of orf1 and orf12 led to an absence of 7-HT and a decrease in orf6-orf9 expression. Orf1 and Orf12 were essential for the production of 7-HT through orf6-orf9 These two regulators are regulated by the Gac/Rsm system; Orf1 facilitates the expression of Orf12, and Orf12 concomitantly stimulates the expression of orf6-orf9 to synthesize 7-HT. The overexpression of Orf12 decreased 7-HT yields, possibly through decreased orf6-orf9 expression. This work thus outlines a complex mechanism regulating the biosynthesis of the iron scavenger 7-HT in P. donghuensis HYS. The synergy between Orf1 and Orf12 ensures that 7-HT acts as an iron chelator despite being toxic to bacteria and provides new ideas for the novel regulation of dual-functional secondary metabolism and research on 7-HT and its derivates in other bacteria.IMPORTANCE A complex regulation mechanism including two regulators, LysR and TetR/AcrR, in the biosynthesis of the novel iron scavenger 7-hydroxytropolone (7-HT) was verified in Pseudomonas donghuensis HYS. The coaction of LysR Orf1 and TetR/AcrR Orf12 may balance the toxicity and iron chelation of 7-HT in P. donghuensis HYS to overcome iron deficiency, as well as improve the bacterial competitiveness under iron-scarce conditions because of the toxicity of 7-HT toward other bacteria, making the accurate regulation of 7-HT biosynthesis indispensable. This regulation mechanism may be ubiquitous in the Pseudomonas putida group but may better explain the group's strong adaptability.

Biosynthesis of Tropolones in Streptomyces spp.: Interweaving Biosynthesis and Degradation of Phenylacetic Acid and Hydroxylations on the Tropone Ring.

Tropolonoids are important natural products that contain a unique seven-membered aromatic tropolone core and exhibit remarkable biological activities. 3,7-Dihydroxytropolone (DHT) isolated from Streptomyces species is a multiply hydroxylated tropolone exhibiting antimicrobial, anticancer, and antiviral activities. In this study, we determined the DHT biosynthetic pathway by heterologous expression, gene deletion, and biotransformation. Nine trl genes and some of the aerobic phenylacetic acid degradation pathway genes (paa) located outside the trl biosynthetic gene cluster are required for the heterologous production of DHT. The trlA gene encodes a single-domain protein homologous to the C-terminal enoyl coenzyme A (enoyl-CoA) hydratase domain of PaaZ. TrlA truncates the phenylacetic acid catabolic pathway and redirects it toward the formation of heptacyclic intermediates. TrlB is a 3-deoxy-d-arabino-heptulosonic acid-7-phosphate (DAHP) synthase homolog. TrlH is an unusual bifunctional protein bearing an N-terminal prephenate dehydratase domain and a C-terminal chorismate mutase domain. TrlB and TrlH enhanced de novo biosynthesis of phenylpyruvate, thereby providing abundant precursor for the prolific production of DHT in Streptomyces spp. Six seven-membered carbocyclic compounds were identified from the trlC, trlD, trlE, and trlF deletion mutants. Four of these chemicals, including 1,4,6-cycloheptatriene-1-carboxylic acid, tropone, tropolone, and 7-hydroxytropolone, were verified as key biosynthetic intermediates. TrlF is required for the conversion of 1,4,6-cycloheptatriene-1-carboxylic acid into tropone. The monooxygenases TrlE and TrlCD catalyze the regioselective hydroxylations of tropone to produce DHT. This study reveals a natural association of anabolism of chorismate and phenylpyruvate, catabolism of phenylacetic acid, and biosynthesis of tropolones in Streptomyces spp.IMPORTANCE Tropolonoids are promising drug lead compounds because of the versatile bioactivities attributed to their highly oxidized seven-membered aromatic ring scaffolds. Our present study provides clear insight into the biosynthesis of 3,7-dihydroxytropolone (DHT) through the identification of key genes responsible for the formation and modification of the seven-membered aromatic core. We also reveal the intrinsic mechanism of elevated production of DHT and related tropolonoids in Streptomyces spp. The study on DHT biosynthesis in Streptomyces exhibits a good example of antibiotic production in which both anabolic and catabolic pathways of primary metabolism are interwoven into the biosynthesis of secondary metabolites. Furthermore, our study sets the stage for metabolic engineering of the biosynthetic pathway for natural tropolonoid products and provides alternative synthetic biology tools for engineering novel tropolonoids.

Yellow-Cedar, Callitropsis (Chamaecyparis) nootkatensis, Secondary Metabolites, Biological Activities, and Chemical Ecology.

Yellow-cedar, Callitropsis nootkatensis, is prevalent in coastal forests of southeast Alaska, western Canada, and inland forests along the Cascades to northern California, USA. These trees have few microbial or animal pests, attributable in part to the distinct groups of biologically active secondary metabolites their tissues store for chemical defense. Here we summarize the new yellow-cedar compounds identified and their biological activities, plus new or expanded activities for tissues, extracts, essential oils and previously known compounds since the last review more than 40 years ago. Monoterpene hydrocarbons are the most abundant compounds in foliage, while heartwood contains substantial quantities of oxygenated monoterpenes and oxygenated sesquiterpenes, with one or more tropolones. Diterpenes occur in foliage and bark, whereas condensed tannins have been isolated from inner bark. Biological activities expressed by one or more compounds in these groups include fungicide, bactericide, sporicide, acaricide, insecticide, general cytotoxicity, antioxidant and human anticancer. The diversity of organisms impacted by whole tissues, essential oils, extracts, or individual compounds now encompasses ticks, fleas, termites, ants, mosquitoes, bacteria, a water mold, fungi and browsing animals. Nootkatone, is a heartwood component with sufficient activity against arthropods to warrant research focused toward potential development as a commercial repellent and biopesticide for ticks, mosquitoes and possibly other arthropods that vector human and animal pathogens.