Biosynthesis of the Tetramic Acids Sch210971 and Sch210972

A biosynthetic pathway to fungal polyketide–nonribosomal peptide natural products, Sch210971 (<b>1a</b>) and Sch210972 (<b>1b</b>) from <i>Hapsidospora irregularis</i>, was characterized by reconstitution and heterologous expression in <i>Fusarium heterosporum</i>. Using genetic, biochemical, and feeding experiments, we show that the incorporated amino acid 4-hydroxyl-4-methyl glutamate (HMG) is synthesized by an aldolase, probably using pyruvate as the precursor

Biosynthesis of the Tetramic Acids Sch210971 and Sch210972

A biosynthetic pathway to fungal polyketide–nonribosomal peptide natural products, Sch210971 (<b>1a</b>) and Sch210972 (<b>1b</b>) from <i>Hapsidospora irregularis</i>, was characterized by reconstitution and heterologous expression in <i>Fusarium heterosporum</i>. Using genetic, biochemical, and feeding experiments, we show that the incorporated amino acid 4-hydroxyl-4-methyl glutamate (HMG) is synthesized by an aldolase, probably using pyruvate as the precursor

Additional file 1: of Manipulation of two regulatory genes for efficient production of chromomycins in Streptomyces reseiscleroticus

Figure S1. UV and ESI-MS spectra of SEK15b (3, a), chromomycin A3 (1, b) and chromomycin A2 (2, c). Table S1. The 1H (300 MHz) and 13C NMR (75 MHz) data for SEK15b (3) (DMSO-d 6 , δ in ppm, J in Hz). Table S2. The 1H NMR (300 MHz) data for chromomycins A3 (1) and A2 (2) (CDCl3, δ in ppm, J in Hz). (DOCX 200 kb

Insights into the Biosynthesis of 12-Membered Resorcylic Acid Lactones from Heterologous Production in <i>Saccharomyces cerevisiae</i>

The phytotoxic fungal polyketides lasiodiplodin and resorcylide inhibit human blood coagulation factor XIIIa, mineralocorticoid receptors, and prostaglandin biosynthesis. These secondary metabolites belong to the 12-membered resorcylic acid lactone (RAL₁₂) subclass of the benzenediol lactone (BDL) family. Identification of genomic loci for the biosynthesis of lasiodiplodin from <i>Lasiodiplodia theobromae</i> and resorcylide from <i>Acremonium zeae</i> revealed collaborating iterative polyketide synthase (iPKS) pairs whose efficient heterologous expression in <i>Saccharomyces cerevisiae</i> provided a convenient access to the RAL₁₂ scaffolds desmethyl-lasiodiplodin and <i>trans</i>-resorcylide, respectively. Lasiodiplodin production was reconstituted in the heterologous host by co-expressing an <i>O</i>-methyltransferase also encoded in the lasiodiplodin cluster, while a glutathione-<i>S</i>-transferase was found not to be necessary for heterologous production. Clarification of the biogenesis of known resorcylide congeners in the heterologous host helped to disentangle the roles that biosynthetic irregularities and chemical interconversions play in generating chemical diversity. Observation of 14-membered RAL homologues during <i>in vivo</i> heterologous biosynthesis of RAL₁₂ metabolites revealed “stuttering” by fungal iPKSs. The close global and domain-level sequence similarities of the orthologous BDL synthases across different structural subclasses implicate repeated horizontal gene transfers and/or cluster losses in different fungal lineages. The absence of straightforward correlations between enzyme sequences and product structural features (the size of the macrocycle, the conformation of the exocyclic methyl group, or the extent of reduction by the hrPKS) suggest that BDL structural variety is the result of a select few mutations in key active site cavity positions

Image3_Formononetin isolated from Sophorae flavescentis inhibits B cell-IgE production by regulating ER-stress transcription factor XBP-1.tif

RationaleIgE plays an important pathologic role in most, if not all, allergic conditions. We previously showed that ASHMI (anti-asthma herbal medicine intervention) suppressed IgE production in murine models of asthma and in asthma subjects. However, the active compounds in ASHMI responsible for the IgE suppression are still unknown.ObjectiveWe sought to identify the compound(s) in ASHMI that are responsible for IgE inhibition as well as investigate the mechanisms by which the identified compound(s) decreases IgE production.MethodsThe compounds in Sophorae Flavescentis were separated using Column chromatography and preparative-HPLC. The separated compounds were identified using LC-MS and 1H-NMR. U266 cells, an IgE-producing plasma cell line, were cultured with various concentrations of identified compounds. The levels of IgE production by the U266 cell were measured by ELISA. Trypan blue exclusion was used to determine the cell viability. The gene expression of XBP-1 and IgE-heavy chain was determined by RT-PCR.ResultsA single compound identified as formononetin was isolated from Sophorae Flavescentis. Formononetin significantly and dose dependently decreased the IgE production in U266 cells across a concentration range of 2–20 µg/ml (p ConclusionFormononetin decreased IgE production in human B cell line U266 cells in a dose-dependent fashion through the regulation of XBP-1 ER transcription. Formononetin may be a potential therapy for allergic asthma and other IgE-mediated diseases.</p

Image4_Formononetin isolated from Sophorae flavescentis inhibits B cell-IgE production by regulating ER-stress transcription factor XBP-1.tif

RationaleIgE plays an important pathologic role in most, if not all, allergic conditions. We previously showed that ASHMI (anti-asthma herbal medicine intervention) suppressed IgE production in murine models of asthma and in asthma subjects. However, the active compounds in ASHMI responsible for the IgE suppression are still unknown.ObjectiveWe sought to identify the compound(s) in ASHMI that are responsible for IgE inhibition as well as investigate the mechanisms by which the identified compound(s) decreases IgE production.MethodsThe compounds in Sophorae Flavescentis were separated using Column chromatography and preparative-HPLC. The separated compounds were identified using LC-MS and 1H-NMR. U266 cells, an IgE-producing plasma cell line, were cultured with various concentrations of identified compounds. The levels of IgE production by the U266 cell were measured by ELISA. Trypan blue exclusion was used to determine the cell viability. The gene expression of XBP-1 and IgE-heavy chain was determined by RT-PCR.ResultsA single compound identified as formononetin was isolated from Sophorae Flavescentis. Formononetin significantly and dose dependently decreased the IgE production in U266 cells across a concentration range of 2–20 µg/ml (p ConclusionFormononetin decreased IgE production in human B cell line U266 cells in a dose-dependent fashion through the regulation of XBP-1 ER transcription. Formononetin may be a potential therapy for allergic asthma and other IgE-mediated diseases.</p

Image5_Formononetin isolated from Sophorae flavescentis inhibits B cell-IgE production by regulating ER-stress transcription factor XBP-1.tif

RationaleIgE plays an important pathologic role in most, if not all, allergic conditions. We previously showed that ASHMI (anti-asthma herbal medicine intervention) suppressed IgE production in murine models of asthma and in asthma subjects. However, the active compounds in ASHMI responsible for the IgE suppression are still unknown.ObjectiveWe sought to identify the compound(s) in ASHMI that are responsible for IgE inhibition as well as investigate the mechanisms by which the identified compound(s) decreases IgE production.MethodsThe compounds in Sophorae Flavescentis were separated using Column chromatography and preparative-HPLC. The separated compounds were identified using LC-MS and 1H-NMR. U266 cells, an IgE-producing plasma cell line, were cultured with various concentrations of identified compounds. The levels of IgE production by the U266 cell were measured by ELISA. Trypan blue exclusion was used to determine the cell viability. The gene expression of XBP-1 and IgE-heavy chain was determined by RT-PCR.ResultsA single compound identified as formononetin was isolated from Sophorae Flavescentis. Formononetin significantly and dose dependently decreased the IgE production in U266 cells across a concentration range of 2–20 µg/ml (p ConclusionFormononetin decreased IgE production in human B cell line U266 cells in a dose-dependent fashion through the regulation of XBP-1 ER transcription. Formononetin may be a potential therapy for allergic asthma and other IgE-mediated diseases.</p

Characterization of Three Tailoring Enzymes in Dutomycin Biosynthesis and Generation of a Potent Antibacterial Analogue

The anthracycline natural product dutomycin and its precursor POK-MD1 were isolated from <i>Streptomyces minoensis</i> NRRL B-5482. The dutomycin biosynthetic gene cluster was identified by genome sequencing and disruption of the ketosynthase gene. Two polyketide synthase (PKS) systems are present in the gene cluster, including a type II PKS and a rare highly reducing iterative type I PKS. The type I PKS DutG repeatedly uses its active sites to create a nine-carbon triketide chain that is subsequently transferred to the α-l-axenose moiety of POK-MD1 at 4″-OH to yield dutomycin. Using a heterologous recombination approach, we disrupted a putative methyltransferase gene (<i>dutMT1</i>) and two glycosyltransferase genes (<i>dutGT1</i> and <i>dutGT2</i>). Analysis of the metabolites of these mutants revealed the functions of these genes and yielded three dutomycin analogues SW140, SW91, and SW75. The major product SW91 in <i>Streptomyces minoensis</i> NRRL B-5482-ΔDutMT1 was identified as 12-desmethyl-dutomycin, suggesting that DutMT1 is the dedicated 12-methyltransferase. This was confirmed by the <i>in vitro</i> enzymatic assay. DutGT1 and DutGT2 were found to be responsible for the introduction of β-d-amicetose and α-l-axenose, respectively. Dutomycin and SW91 showed strong antibacterial activity against <i>Staphylococcus aureus</i> and methicillin-resistant <i>S. aureus</i>, whereas POK-MD1 and SW75 had no obvious inhibition, which revealed the essential role of the C-4″ triketide chain in antibacterial activity. The minimal inhibitory concentration of SW91 against the two strains was 0.125 μg mL^–1, lower than that of dutomycin (0.25 μg mL^–1), indicating that the antibacterial activity of dutomycin can be improved through biosynthetic structural modification

Image2_Formononetin isolated from Sophorae flavescentis inhibits B cell-IgE production by regulating ER-stress transcription factor XBP-1.tif

RationaleIgE plays an important pathologic role in most, if not all, allergic conditions. We previously showed that ASHMI (anti-asthma herbal medicine intervention) suppressed IgE production in murine models of asthma and in asthma subjects. However, the active compounds in ASHMI responsible for the IgE suppression are still unknown.ObjectiveWe sought to identify the compound(s) in ASHMI that are responsible for IgE inhibition as well as investigate the mechanisms by which the identified compound(s) decreases IgE production.MethodsThe compounds in Sophorae Flavescentis were separated using Column chromatography and preparative-HPLC. The separated compounds were identified using LC-MS and 1H-NMR. U266 cells, an IgE-producing plasma cell line, were cultured with various concentrations of identified compounds. The levels of IgE production by the U266 cell were measured by ELISA. Trypan blue exclusion was used to determine the cell viability. The gene expression of XBP-1 and IgE-heavy chain was determined by RT-PCR.ResultsA single compound identified as formononetin was isolated from Sophorae Flavescentis. Formononetin significantly and dose dependently decreased the IgE production in U266 cells across a concentration range of 2–20 µg/ml (p ConclusionFormononetin decreased IgE production in human B cell line U266 cells in a dose-dependent fashion through the regulation of XBP-1 ER transcription. Formononetin may be a potential therapy for allergic asthma and other IgE-mediated diseases.</p

Image1_Formononetin isolated from Sophorae flavescentis inhibits B cell-IgE production by regulating ER-stress transcription factor XBP-1.tif

RationaleIgE plays an important pathologic role in most, if not all, allergic conditions. We previously showed that ASHMI (anti-asthma herbal medicine intervention) suppressed IgE production in murine models of asthma and in asthma subjects. However, the active compounds in ASHMI responsible for the IgE suppression are still unknown.ObjectiveWe sought to identify the compound(s) in ASHMI that are responsible for IgE inhibition as well as investigate the mechanisms by which the identified compound(s) decreases IgE production.MethodsThe compounds in Sophorae Flavescentis were separated using Column chromatography and preparative-HPLC. The separated compounds were identified using LC-MS and 1H-NMR. U266 cells, an IgE-producing plasma cell line, were cultured with various concentrations of identified compounds. The levels of IgE production by the U266 cell were measured by ELISA. Trypan blue exclusion was used to determine the cell viability. The gene expression of XBP-1 and IgE-heavy chain was determined by RT-PCR.ResultsA single compound identified as formononetin was isolated from Sophorae Flavescentis. Formononetin significantly and dose dependently decreased the IgE production in U266 cells across a concentration range of 2–20 µg/ml (p ConclusionFormononetin decreased IgE production in human B cell line U266 cells in a dose-dependent fashion through the regulation of XBP-1 ER transcription. Formononetin may be a potential therapy for allergic asthma and other IgE-mediated diseases.</p