Hypothemycin is a macrolide protein kinase inhibitor from the fungus Hypomyces subiculosus. During biosynthesis, its carbon framework is assembled by two iterative polyketide synthases (PKSs), Hpm8 ...(highly reducing) and Hpm3 (nonreducing). These were heterologously expressed in Saccharomyces cerevisiae BJ5464-NpgA, purified to near homogeneity, and reconstituted in vitro to produce (6′S,10′S)-trans-7′,8′-dehydrozearalenol (1) from malonyl-CoA and NADPH. The structure of 1 was determined by X-ray crystallographic analysis. In the absence of functional Hpm3, the reducing PKS Hpm8 produces and offloads truncated pyrone products instead of the expected hexaketide. The nonreducing Hpm3 is able to accept an N-acetylcysteamine thioester of a correctly functionalized hexaketide to form 1, but it is unable to initiate polyketide formation from malonyl-CoA. We show that the starter-unit:ACP transacylase (SAT) of Hpm3 is critical for crosstalk between the two enzymes and that the rate of biosynthesis of 1 is determined by the rate of hexaketide formation by Hpm8.
Abstract
The corrosion of aluminum alloys (AA) caused by fungi has been considered as the typical reason causing the failure of aviation materials, but its corrosion mechanism is still unclear. In ...this work, AA 7075 corrosion induced by Fungus
Aspergillus terreus
with continued organic carbon starvation was investigated in artificial seawater. Results indicate that
A. terreus
can survive with organic carbon starvation and form a biofilm on AA surface, then accelerate AA corrosion. The pitting corrosion is more severe in biotic environments, and it is closely related to the initial spore concentration. The higher initial spore concentration, the more
A. terreus
survivors, leading to the more severe pitting corrosion. Acid corrosion caused by organic acids of
A. terreus
has no contribution to the corrosion acceleration of aluminum alloys.
A. terreus
can help to destroy the passive film of aluminum alloys, leading to more corrosion pits compared with the control.
Ilicicolin H is a broad-spectrum antifungal agent targeting mitochondrial cytochrome bc1 reductase. Unfortunately, ilicicolin H shows reduced activities in vivo. Here, we report our effort on the ...identification of ilicicolin H biosynthetic gene cluster (BGC) by genomic sequencing a producing strain,
sp. DH2, and its heterologous production in
. In addition, a shunt product with similar antifungal activities, ilicicolin J, was uncovered. This effort would provide a base for future combinatorial biosynthesis of ilicicolin H analogues. Bioinformatics analysis suggests that the backbone of ilicicolin H is assembled by a polyketide-nonribosomal peptide synthethase (IliA), and then offloaded with a tetramic acid moiety. Similar to tenellin biosynthesis, the tetramic acid is then converted to pyridone by a putative P450, IliC. The decalin portion is most possibly constructed by a
-adenosyl-l-methionine (SAM)-dependent Diels-Alderase (IliD).
Highly reducing polyketide synthases (HR-PKSs) from fungi synthesize complex natural products using a single set of domains in a highly programmed, iterative fashion. The most enigmatic feature of ...HR-PKSs is how tailoring domains function selectively during different iterations of chain elongation to afford structural diversity. Using the lovastatin nonaketide synthase LovB as a model system and a variety of acyl substrates, we characterized the substrate specificity of the LovB methyltransferase (MT) domain. We showed that, while the MT domain displays methylation activity toward different β-ketoacyl groups, it is exceptionally selective toward its naturally programmed β-keto-dienyltetraketide substrate with respect to both chain length and functionalization. Accompanying characterization of the ketoreductase (KR) domain displays broader substrate specificity toward different β-ketoacyl groups. Our studies indicate that selective modifications by tailoring domains, such as the MTs, are achieved by higher kinetic efficiency on a particular substrate relative to the rate of transformation by other competing domains.
Resorcylic acid lactones are fungal polyketides that display diverse biological activities, with the potent Hsp90 inhibitor radicicol being an important representative member. Two fungal iterative ...polyketide synthases (IPKSs), Rdc5, the highly reducing IPKS, and Rdc1, the nonreducing IPKS, are required for the biosynthesis of radicicol in Pochonia chlamydosporia. In this study, the complete reconstitution of Rdc5 and Rdc1 activities both in vitro and in Saccharomyces cerevisiae uncovered the earliest resorcylic acid lactone intermediate of the radicicol biosynthetic pathway, (R)-monocillin II. The enzymatic synthesis of (R)-monocillin II confirmed the exquisite timing of the Rdc5 enoyl reductase domain. Using precursor-directed biosynthesis, the chemical modularity of the dual IPKS system was determined. Rdc1 readily accepted an N-acetylcysteamine thioester mimic of the reduced pentaketide product of Rdc5 to synthesize (R)-monocillin II with four additional iterations of polyketide elongation, indicating the C2′ ketone group found in (R)-monocillin II is incorporated via the functions of Rdc1 instead of Rdc5. The involvement of the thioesterase domain in Rdc1 in macrolactonization was confirmed through both site-directed mutagenesis and domain deletion. The Rdc1 thioesterase domain was also shown to be tolerant of the opposite stereochemistry of the terminal hydroxyl nucleophile, demonstrated in the precursor-directed synthesis of the enantiomeric (S)-monocillin II. Finally, reconstitution of the halogenase Rdc2 was demonstrated both in vivo and in vitro in the synthesis of pochonin D and a new halogenated analog 6-chloro, 7′,8′-dehydrozearalenol.
Culturing ascidian-derived fungus Amphichorda felina SYSU-MS7908 under standard laboratory conditions mainly yielded meroterpenoid, and nonribosomal peptide-type natural products. We sequenced the ...genome of Amphichorda felina SYSU-MS7908 and found 56 biosynthetic gene clusters (BGCs) after bioinformatics analysis, suggesting that the majority of those BGCSs are silent. Here we report our genome mining effort on one cryptic BGC by heterologous expression in Aspergillus oryzae NSAR1, and the identification of two new α-pyrone derivatives, amphichopyrone A (1) and B (2), along with a known compound, udagawanone A (3). Anti-inflammatory activities were performed, and amphichopyrone A (1) and B (2) displayed potent anti-inflammatory activity by inhibiting nitric oxide (NO) production in RAW264.7 cells with IC50 values 18.09 ± 4.83 and 7.18 ± 0.93 μM, respectively.
In the current study, we report the high-quality draft genome sequence of
Neonectria
sp. DH2, an endophytic fungus isolated from
Meconopsis grandis
Prain in Tibet. The whole genome is about 45.8 Mbp, ...with a GC content of 53%. A total of 14,163 genes are predicted to encode proteins, and 557 of them are considered as unique, as no matches are found in five gene databases. A neighbor-joining phylogenetic tree based on internal transcribed spacer (ITS) region sequences shows that
Neonectria
sp. DH2 was most closely related to
Neonectria ramulariae
. 47 biosynthetic gene clusters (BGC) were identified in
Neonectria
sp. DH2 genome, and only 5 BGCs shows significant similarities to previously reported BGCs. The presence of 42 unique BGCs in
Neonectria
sp. DH2 suggests that it has great potential to produce novel secondary metabolites.
Cure the patellar tendinopathy with arthroscopy Chen, Wenzhao; Gao, Zhizeng
Asia-Pacific journal of sports medicine, arthroscopy, rehabilitation and technology,
July 2017, 2017-07-00, 2017-07-01, Letnik:
9, Številka:
C
Journal Article
Valerian (Valeriana officinalis) is a popular medicinal plant in North America and Europe. Its root extract is commonly used as a mild sedative and anxiolytic. Among dozens of chemical constituents ...(e.g. alkaloids, iridoids, flavonoids, and terpenoids) found in valerian root, valerena‐4,7(11)‐diene and valerenic acid (C15 sesquiterpenoid) have been suggested as the active ingredients responsible for the sedative effect. However, the biosynthesis of the valerena‐4,7(11)‐diene hydrocarbon skeleton in valerian remains unknown to date. To identify the responsible terpene synthase, next‐generation sequencing (Roche 454 pyrosequencing) was used to generate ∼ 1 million transcript reads from valerian root. From the assembled transcripts, two sesquiterpene synthases were identified (VoTPS1 and VoTPS2), both of which showed predominant expression patterns in root. Transgenic yeast expressing VoTPS1 and VoTPS2 produced germacrene C/germacrene D and valerena‐4,7(11)‐diene, respectively, as major terpene products. Purified VoTPS1 and VoTPS2 recombinant enzymes confirmed these activities in vitro, with competent kinetic properties (Km of ∼ 10 μm and kcat of 0.01 s−1 for both enzymes). The structure of the valerena‐4,7(11)‐diene produced from the yeast expressing VoTPS2 was further substantiated by 13C‐NMR and GC‐MS in comparison with the synthetic standard. This study demonstrates an integrative approach involving next‐generation sequencing and metabolically engineered microbes to expand our knowledge of terpenoid diversity in medicinal plants.
Database
The sequences of cDNAs described in this work are available in the GenBank database under the following accession numbers: VoTPS1, JQ437839; VoTPS2, JQ437840
Valerian (Valeriana officinalis) root has been traditionally used as a mild sedative. Valerenic acid (C15 sesquiterpenoid) has been suggested as an active ingredient in valerian. Next generation sequencing is used to identify a novel sesquiterpene synthase that catalyzes the cyclization of farnesyl diphosphate (FPP) to a unique valerena‐4,7(11)‐diene sesquiterpene. A possible mechanism of valerena‐4,7(11)‐diene synthesis from FPP is proposed
Iterative type I polyketide synthases (PKSs) from fungi are multifunctional enzymes that use their active sites repeatedly in a highly ordered sequence to assemble complex natural products. A ...phytotoxic macrolide with anticancer properties, 10,11‐dehydrocurvularin (DHC), is produced by cooperation of a highly reducing (HR) iterative PKS and a non‐reducing (NR) iterative PKS. We have identified the DHC gene cluster in Alternaria cinerariae, heterologously expressed the active HR PKS (Dhc3) and NR PKS (Dhc5) in yeast, and compared them to corresponding proteins that make DHC in Aspergillus terreus. Phylogenetic analysis and homology modeling of these enzymes identified variable surfaces and conserved motifs that are implicated in product formation.
DHC keys: We have identified and heterologously expressed two iterative type I polyketide synthases necessary to produce the phytotoxic anticancer agent 10,11‐dehydrocurvularin (DHC) in A. cinerariae. This allowed bioinformatic comparison to the analogous metabolite gene cluster in A. terreus, which highlighted key structural features necessary for metabolite production.
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