Attempting to Create a Pathway to 15-Deacetylcalonectrin with Limited Accumulation in Cultures of Fusarium Tri3 Mutants: Insight into Trichothecene Biosynthesis Machinery.

The compound 15-deacetylcalonectrin (15-deCAL) is a common pathway intermediate in the biosynthesis of Fusarium trichothecenes. This tricyclic intermediate is metabolized to calonectrin (CAL) by trichothecene 15-O-acetyltransferase encoded by Tri3. Unlike other trichothecene pathway Tri gene mutants, the Δtri3 mutant produces lower amounts of the knocked-out enzyme's substrate 15-deCAL, and instead, accumulates higher quantities of earlier bicyclic intermediate and shunt metabolites. Furthermore, evolutionary studies suggest that Tri3 may play a role in shaping the chemotypes of trichothecene-producing Fusarium strains. To better understand the functional role of Tri3p in biosynthesis and evolution, we aimed to develop a method to produce 15-deCAL by using transgenic Fusarium graminearum strains derived from a trichothecene overproducer. Unfortunately, introducing mutant Tri3, encoding a catalytically impaired but structurally intact acetylase, did not improve the low 15-deCAL production level of the ΔFgtri3 deletion strain, and the bicyclic products continued to accumulate as the major metabolites of the active-site mutant. These findings are discussed in light of the enzyme responsible for 15-deCAL production in trichothecene biosynthesis machinery. To efficiently produce 15-deCAL, we tested an alternative strategy of using a CAL-overproducing transformant. By feeding a crude CAL extract to a Fusarium commune strain that was isolated in this study and capable of specifically deacetylating C-15 acetyl, 15-deCAL was efficiently recovered. The substrate produced in this manner can be used for kinetic investigations of this enzyme and its possible role in chemotype diversification.

A Role in 15-Deacetylcalonectrin Acetylation in the Non-Enzymatic Cyclization of an Earlier Bicyclic Intermediate in Fusarium Trichothecene Biosynthesis.

The trichothecene biosynthesis in Fusarium begins with the cyclization of farnesyl pyrophosphate to trichodiene, followed by subsequent oxygenation to isotrichotriol. This initial bicyclic intermediate is further cyclized to isotrichodermol (ITDmol), a tricyclic precursor with a toxic trichothecene skeleton. Although the first cyclization and subsequent oxygenation are catalyzed by enzymes encoded by Tri5 and Tri4, the second cyclization occurs non-enzymatically. Following ITDmol formation, the enzymes encoded by Tri101, Tri11, Tri3, and Tri1 catalyze 3-O-acetylation, 15-hydroxylation, 15-O-acetylation, and A-ring oxygenation, respectively. In this study, we extensively analyzed the metabolites of the corresponding pathway-blocked mutants of Fusarium graminearum. The disruption of these Tri genes, except Tri3, led to the accumulation of tricyclic trichothecenes as the main products: ITDmol due to Tri101 disruption; a mixture of isotrichodermin (ITD), 7-hydroxyisotrichodermin (7-HIT), and 8-hydroxyisotrichodermin (8-HIT) due to Tri11 disruption; and a mixture of calonectrin and 3-deacetylcalonectrin due to Tri1 disruption. However, the ΔFgtri3 mutant accumulated substantial amounts of bicyclic metabolites, isotrichotriol and trichotriol, in addition to tricyclic 15-deacetylcalonectrin (15-deCAL). The ΔFgtri5ΔFgtri3 double gene disruptant transformed ITD into 7-HIT, 8-HIT, and 15-deCAL. The deletion of FgTri3 and overexpression of Tri6 and Tri10 trichothecene regulatory genes did not result in the accumulation of 15-deCAL in the transgenic strain. Thus, the absence of Tri3p and/or the presence of a small amount of 15-deCAL adversely affected the non-enzymatic second cyclization and C-15 hydroxylation steps.

Interlayer Modification of a Layered Silicate RUB-18 with 4-Phosphonophenylsilane and Its Surface Acidic Functions.

The interlayer silylation of a layered silicate H-RUB-18 (Si4O7(OH)2) using a new aromatic silylating reagent containing a phosphonic acid group (4-phosphonophenylsilane: PPS) was demonstrated (H-PPS-RUB-18). The phosphonic acid groups were attached to the silicate layers through the reaction of H-RUB-18 with (4-diethoxyphosphorylphenyl)-triethoxysilane (p-PPS-E), and the ester moieties were subsequently hydrolyzed with hydrochloric acid. H-PPS-RUB-18 is a solid acid, as indicated by the intercalation of various alkylamines and the catalytic acetalization of ketones. A systematic increase in interlayer spacing leading to surface acidic properties was obtained through intercalation with a series of alkylamines. In addition, H-PPS-RUB-18 was exfoliated, resulting in single-layer nanosheets with ca. 2.0 nm thickness. The catalytic acetalization of ketones was related to the interlayer spacing of the modified RUB-18.

Accumulation of 4-Deoxy-7-hydroxytrichothecenes, but Not 4,7-Dihydroxytrichothecenes, in Axenic Culture of a Transgenic Nivalenol Chemotype Expressing the NX-Type FgTri1 Gene.

Fusarium graminearum species complex produces type B trichothecenes oxygenated at C-7. In axenic liquid culture, F. graminearum mainly accumulates one of the three types of trichothecenes, namely 3-acetyldeoxyinvalenol, 15-acetyldeoxyinvalenol, or mixtures of 4,15-diacetylnivalenol/4-acetylnivalenol, depending on each strain's genetic background. The acetyl groups of these trichothecenes are slowly deacetylated to give deoxynivalenol (DON) or nivalenol (NIV) on solid medium culture. Due to the evolution of F. graminearum FgTri1, encoding a cytochrome P450 monooxygenase responsible for hydroxylation at both C-7 and C-8, new derivatives of DON, designated as NX-type trichothecenes, have recently emerged. To assess the risks of emergence of new NX-type trichothecenes, we examined the effects of replacing FgTri1 in the three chemotypes with FgTri1_NX chemotype, which encodes a cytochrome P450 monooxygenase that can only hydroxylate C-7 of trichothecenes. Similar to the transgenic DON chemotypes, the transgenic NIV chemotype strain accumulated NX-type 4-deoxytrichothecenes in axenic liquid culture. C-4 oxygenated trichothecenes were marginal, despite the presence of a functional FgTri13 encoding a C-4 hydroxylase. At present, outcrossing of the currently occurring NX chemotype with NIV chemotype strains of F. graminearum in the natural environment likely will not yield a new strain that produces a C-4 oxygenated NX-type trichothecene.

Impact of nitrogen metabolism-associated culture pH changes on regulation of Fusarium trichothecene biosynthesis: revision of roles of polyamine agmatine and transcription factor AreA.

Fusarium graminearum produces trichothecene mycotoxins in infected grains and axenic liquid culture. A proposed regulatory model of trichothecene biosynthesis was examined in relation to nitrogen utilization. First, we showed that an important factor for the stimulation of trichothecene biosynthesis was not the occurrence of agmatine as a specific inducer molecule, but rather continuous acidification of the liquid culture medium arising from agmatine catabolism. When the pH of the L-Gln synthetic medium was frequently adjusted to the pH of the agmatine culture, trichothecene productivity of the L-Gln culture was equal to that of the agmatine culture. For efficient trichothecene biosynthesis, the culture pH should be lowered at an appropriate time point during the early growth stage. Second, we re-evaluated the role of the nitrogen regulatory GATA transcription factor AreA in trichothecene biosynthesis. Since Tri6 encodes a transcription factor indispensable for trichothecene biosynthesis, all fifteen AreA-binding consensus sequences in the Tri6 promoter were mutated. The mutant could catabolize L-Phe as the sole nitrogen source; furthermore, the pH profile of the synthetic L-Phe medium (initial pH 4.2) was the same as that of the wild-type (WT) strain. Under such conditions, the promoter mutant exhibited approximately 72% of the trichothecene productivity compared to the WT strain. Thus, F. graminearum AreA (FgAreAp) is dispensable for the functioning of the Tri6 promoter, but it contributes to the increased production of mycotoxin under mildly acidic conditions to some extent. Further investigations on the culture pH revealed that extremely low pH bypasses the function of FgAreAp.

Identification of amino acids negatively affecting Fusarium trichothecene biosynthesis.

Nitrogen sources in media have a significant impact on the onset of secondary metabolism in fungi. For transcriptional activation of many nitrogen catabolic genes, an AreA transcription factor is indispensable. This also holds true for Fusarium graminearum that produces trichothecenes, an important group of mycotoxin, in axenic culture. Despite the presence of numerous consensus AreA-binding sites in the promoters of Tri genes in the trichothecene cluster core region, the effect of medium amino acids on trichothecene biosynthesis is poorly understood. In this study, we examined the effect of certain amino acids, which were predicted to activate AreA function and increase Tri gene transcription, on trichothecene production in liquid culture. By frequent monitoring and adjustments in the pH of the culture medium, including replacement of the spent medium with fresh medium, we demonstrate the suppressive effects of the amino acids, used as the sole nitrogen source, on trichothecene biosynthesis. When the medium pH was maintained at 4.0, Gly, L-Ser, and L-Thr suppressed trichothecene production by F. graminearum. Enhanced trichothecene-inducing effects were observed when the medium pH was 3.5, with only L-Thr suppressing trichothecene synthesis.

Exploring an Artificial Metabolic Route in Fusarium sporotrichioides: Production and Characterization of 7-Hydroxy T-2 Toxin.

An artificial metabolic route to an unnatural trichothecene was designed by taking advantage of the broad substrate specificities of the T-2 toxin biosynthetic enzymes of Fusarium sporotrichioides. By feeding 7-hydroxyisotrichodermin, a shunt pathway metabolite of F. graminearum, to a trichodiene synthase-deficient mutant of F. sporotrichioides, 7-hydroxy T-2 toxin (1) was obtained as the final metabolite. Such an approach may have future applications in the metabolic engineering of a variety of fungal secondary metabolites. The toxicity of 7-hydroxy T-2 toxin was 10 times lower than that of T-2 toxin in HL-60 cells.

L-Threonine and its analogue added to autoclaved solid medium suppress trichothecene production by Fusarium graminearum.

Fusarium graminearum produces trichothecene mycotoxins under certain nutritional conditions. When L-Thr and its analogue L-allo-threonine were added to brown rice flour solid medium before inoculation, trichothecene production after 4 days of incubation was suppressed. A time-course analysis of gene expression demonstrated that L-Thr suppressed transcription of Tri6, a trichothecene master regulator gene, and a terpene cyclase Tri5 gene. Regulation of trichothecene biosynthesis by altering major primary metabolic processes may open up the possibility to develop safe chemicals for the reduction of mycotoxin contamination might be developed.

Identification of a trichothecene production inhibitor by chemical array and library screening using trichodiene synthase as a target protein.

Trichothecene mycotoxins often accumulate in apparently normal grains of cereal crops. In an effort to develop an agricultural chemical to reduce trichothecene contamination, we screened trichothecene production inhibitors from the compounds on the chemical arrays. By using the trichodiene (TDN) synthase tagged with hexahistidine (rTRI5) as a target protein, 32 hit compounds were obtained from chemical library of the RIKEN Natural Product Depository (NPDepo) by chemical array screening. At 10µgmL-1, none of the 32 chemicals inhibited trichothecene production by Fusarium graminearum in liquid culture. Against the purified rTRI5 enzyme, however, NPD10133 [progesterone 3-(O-carboxymethyl)oxime amide-bonded to phenylalanine] showed weak inhibitory activity at 10µgmL-1 (18.7µM). For the screening of chemicals inhibiting trichothecene accumulation in liquid culture, 20 analogs of NPD10133 selected from the NPDepo chemical library were assayed. At 10µM, only NPD352 [testosterone 3-(O-carboxymethyl)oxime amide-bonded to phenylalanine methyl ester] inhibited rTRI5 activity and trichothecene production. Kinetic analysis suggested that the enzyme inhibition was of a mixed-type. The identification of NPD352 as a TDN synthase inhibitor lays the foundation for the development of a more potent inhibitor via systematic introduction of wide structural diversity on the gonane skeleton and amino acid residues.

Hydroxylations of trichothecene rings in the biosynthesis of Fusarium trichothecenes: evolution of alternative pathways in the nivalenol chemotype.

Fusarium sporotrichioides genes FsTri11, FsTri13, and FsTri1 encode cytochrome P450 monooxygenases (CYPs) responsible for hydroxylations at C-15, C-4, and C-8 of the trichothecene skeleton, respectively. However, the corresponding genes of nivalenol (NIV)-chemotype Fusarium graminearum remain to be functionally elucidated. In this study, we characterized the roles of these CYPs in NIV biosynthesis. Analyses of the metabolites of the F. graminearum Fgtri11- mutant, a disruptant of FgTri11 encoding isotrichodermin (ITD) C-15 hydroxylase, revealed a small amount of NIV-type trichothecenes suggesting that an alternative C-15 hydroxylase partially complemented FgTRI11p. In contrast, the C-7/C-8 hydroxylations depended solely on FgTRI1p, as suggested by the metabolite profiles of the Fgtri11- Fgtri1- double gene disruptant. Disruption of FgTri1 in both the wild-type and Fgtri13- mutant backgrounds revealed that FgTRI13p exhibits marginal activity toward calonectrin (CAL) and that it was the only C-4 hydroxylase. In addition, feeding experiments demonstrated that the C-4 hydroxylation of a 7-hydroxytrichothecene lacking C-8 ketone was extremely limited. The marginal activity of FgTRI13p toward CAL was advantageous for the C-7/C-8 hydroxylation steps in NIV biosynthesis, as transformation of a C-4 oxygenated trichothecene lacking C-7/C-8 modifications into NIV-type trichothecenes was quite inefficient. The significance of hydroxylation steps in the evolution of Fusarium trichothecenes is discussed.

Re-examination of genetic and nutritional factors related to trichothecene biosynthesis in Fusarium graminearum.

Disruption of two Fusarium genes that negatively regulate trichothecene biosynthesis was reported to cause a drastic increase in trichothecene production. However, careful inspection of these genes revealed that neither was significantly related to trichothecene production. Agmatine medium maintained the expression of trichothecene genes at significant levels, resulting in a 2-3-fold increase in the final yield, as compared to glutamine medium.

The secreted antifungal protein thionin 2.4 in Arabidopsis thaliana suppresses the toxicity of a fungal fruit body lectin from Fusarium graminearum.

Plants possess active defense systems and can protect themselves from pathogenic invasion by secretion of a variety of small antimicrobial or antifungal proteins such as thionins. The antibacterial and antifungal properties of thionins are derived from their ability to induce open pore formation on cell membranes of phytopathogens, resulting in release of potassium and calcium ions from the cell. Wheat thionin also accumulates in the cell walls of Fusarium-inoculated plants, suggesting that it may have a role in blocking pathogen infection at the plant cell walls. Here we developed an anti-thionin 2.4 (Thi2.4) antibody and used it to show that Thi2.4 is localized in the cell walls of Arabidopsis and cell membranes of F. graminearum, when flowers are inoculated with F. graminearum. The Thi2.4 protein had an antifungal effect on F. graminearum. Next, we purified the Thi2.4 protein, conjugated it with glutathione-S-transferase (GST) and coupled the proteins to an NHS-activated column. Total protein from F. graminearum was applied to GST-Thi2.4 or Thi2.4-binding columns, and the fungal fruit body lectin (FFBL) of F. graminearum was identified as a Thi2.4-interacting protein. This interaction was confirmed by a yeast two-hybrid analysis. To investigate the biological function of FFBL, we infiltrated the lectin into Arabidopsis leaves and observed that it induced cell death in the leaves. Application of FFBL at the same time as inoculation with F. graminearum significantly enhanced the virulence of the pathogen. By contrast, FFBL-induced host cell death was effectively suppressed in transgenic plants that overexpressed Thi2.4. We found that a 15 kD Thi2.4 protein was specifically expressed in flowers and flower buds and suggest that it acts not only as an antifungal peptide, but also as a suppressor of the FFBL toxicity. Secreted thionin proteins are involved in this dual defense mechanism against pathogen invasion at the plant-pathogen interface.

Ordered microporous layered lanthanide 1,3,5-benzenetriphosphonates pillared with cationic organic molecules.

Novel isomorphous pillared-layer-type crystalline lanthanide 1,3,5-benzenetriphosphonates were prepared with bpy and dbo as organic pillars (LnBP-bpy and LnBP-dbo; Ln: Ce, Pr, and Nd). Ab initio crystal structure solution using synchrotron X-ray powder diffraction data revealed that the organic pillars do not exist as neutral coordinating ligands but as cationic molecules. Especially the LnBP-dbo phases have ordered interlayer space filled with water molecules between the dbo pillars, and the interlayer water is successfully removed by heating under vacuum with slightly distorted but basically retained pillared layer structures. Microporosity of the materials is confirmed by adsorption of nitrogen, carbon dioxide, and hydrogen gases. Such microporous layered metal phosphonates pillared with cationic molecules should be unprecedented and should offer new strategies to design ordered microporous materials.

A New Synthetic Route to Microporous Silica with Well-Defined Pores by Replication of a Metal-Organic Framework.

Microporous amorphous hydrophobic silica materials with well-defined pores were synthesized by replication of the metal-organic framework (MOF) [Cu3 (1,3,5-benzenetricarboxylate)2 ] (HKUST-1). The silica replicas were obtained by using tetramethoxysilane or tetraethoxysilane as silica precursors and have a micro-meso binary pore system. The BET surface area, the micropore volume, and the mesopore volume of the silica replica, obtained by means of hydrothermal treatment at 423 K with tetraethoxysilane, are 620 m(2) g(-1) , 0.18 mL g(-1) , and 0.55 mL g(-1) , respectively. Interestingly, the silica has micropores with a pore size of 0.55 nm that corresponds to the pore-wall thickness of the template MOF. The silica replica is hydrophobic, as confirmed by adsorption analyses, although the replica has a certain amount of silanol groups. This hydrophobicity is due to the unique condensation environment of the silica precursors in the template MOF.

Mesoporous Zirconium Phenylenesiliconate-phosphonate Hybrids with Ordered Lamellar Nanostructures.

Novel ordered lamellar mesostructure pZrPS-2 was hydrothermally prepared by using zirconium propoxide and 4-(EtO)2OPC6H4Si(OEt)3 (pPPS-E), which was hydrolyzed to organic building units substituted with both siliconate and phosphonate groups, in the presence of Cn TAB and TMAOH. The pZrPS-2 materials were obtained at a Zr/PPS ratio of 2 or higher and the basal spacing was increased by using a longer-chain surfactant (n = 12-18). Removal of the occluded surfactants at 300 °C resulted in retention of the lamellar structure with negligible shrinkage of the interlayer distance. Nitrogen adsorption studies revealed the ordered mesoporous nature of pZrPS-2 with a pore diameter of approximately 2 to 3 nm. The lamellar structure is assumed to be composed of layers that include zirconia-based crystalline nanodomains and interlayer pillars mainly based on PPS units. Although lamellar structures with the same crystalline phase also formed when no surfactant was added or when the meta isomer of PPS was used, no mesoporous materials were obtained except pZrPS-2. A possible schematic model to elucidate these results is also proposed.

Feasibility and safety of intracoronary nicorandil infusion as a novel hyperemic agent for fractional flow reserve measurements.

Fractional flow reserve (FFR) is a useful modality to assess the functional significance of coronary stenoses. Although adenosine triphosphate (ATP) is generally used as the hyperemic stimulus, we sometimes encounter adverse events like hypotension during FFR measurement. Nicorandil, an ATP-sensitive potassium channel opener, recognized as an epicardial and resistance vessel dilator, has not been fully evaluated as a possible alternative hyperemic agent. The aim of this study was to evaluate the feasibility and safety of intracoronary nicorandil infusion compared to intravenous ATP for FFR measurement in patients with coronary artery disease. A total of 102 patients with 124 intermediate lesions (diameter stenosis >40 and <70% by visual assessment) were enrolled. All vessels underwent FFR measurements with both ATP (150 µg/kg/min) and nicorandil (2.0 mg) stimulus. FFR, hemodynamic values, and periprocedural adverse events between the two groups were evaluated. A strong correlation was observed between FFR with ATP and FFR with nicorandil (r = 0.954, p < 0.001). The agreement between the two sets of measurements was also high, with a mean difference of 0.01 ± 0.03. The mean aortic pressure drop during pharmacological stimulus was significantly larger with ATP compared to nicorandil (9.6 ± 9.6 vs. 5.5 ± 5.8 mmHg, p < 0.001). During FFR measurement, transient atrioventricular block was frequently observed with ATP compared to nicorandil (4.0 vs. 0%, p = 0.024). This study suggests that intracoronary nicorandil infusion is associated with clinical utility and safety compared to ATP as an alternative hyperemic agent for FFR measurement.

Stabilisation of molecular TiO4 species on the pore surface of mesoporous silica for photocatalytic H2 evolution.

Although molecular tetrahedral Ti-oxo species exhibit unique electronic and photochemical properties due to their discrete energy levels, which are different from those of anatase and rutile, such Ti-oxo species are generally unstable and readily transformed to amorphous/crystalline TiO2 (bulk phases, nanoparticles and clusters) via hydrolysis and condensation. Here, molecular Ti-oxo species were immobilised within mesoporous silica SBA-15 by grafting titanium(IV) oxyacetylacetonate using the surface silanol groups of SBA-15 as a scaffold, followed by chemical etching with dilute hydrochloric acid to form molecular TiO4 species. These Ti species mainly exist as isolated tetrahedrally coordinated structures, as was confirmed by diffuse reflectance UV-vis and Raman spectroscopy. The SBA-15-immobilised molecular TiO4 exhibited higher photocatalytic activity for H2 evolution from an aqueous methanol solution than conventional Ti-incorporated mesoporous silica (Ti-MCM-41) and reference TiO2 (P25).

Microporous brookite-phase titania made by replication of a metal-organic framework.

Metal-organic frameworks (MOFs) provide access to structures with nanoscale pores, the size and connectivity of which can be controlled by combining the appropriate metals and linkers. To date, there have been no reports of using MOFs as templates to make porous, crystalline metal oxides. Microporous titania replicas were made from the MOF template HKUST-1 by dehydration, infiltration with titanium isopropoxide, and subsequent hydrothermal treatment at 200 °C. Etching of the MOF with 1 M aqueous HCl followed by 5% H2O2 yielded a titania replica that retained the morphology of the parent HKUST-1 crystals and contained partially ordered micropores as well as disordered mesopores. Interestingly, the synthesis of porous titania from the HKUST-1 template stabilized the formation of brookite, a rare titania polymorph.

Identification and characterization of an inhibitor of trichothecene 3-O-acetyltransferase, TRI101, by the chemical array approach.

Trichothecene 3-O-acetyltransferase (TRI101) is an indispensable enzyme for the biosynthesis of trichothecenes, a group of mycotoxins produced by Fusarium graminearum. In this study, an inhibitor of TRI101 was identified by chemical array analysis using compounds from the RIKEN Natural Products Depository (NPDepo) library. Although the addition of the identified enzyme inhibitor to the fungal culture did not inhibit trichothecene production, it can serve as a candidate lead compound in the development of a mycotoxin inhibitor that inactivates fungal defense mechanisms.

Regulatory mechanism of trichothecene biosynthesis in Fusarium graminearum.

Among the genes involved in the biosynthesis of trichothecene (Tri genes), Tri6 and Tri10 encode a transcription factor with unique Cys2His2 zinc finger domains and a regulatory protein with no consensus DNA-binding sequences, respectively. Although various chemical factors, such as nitrogen nutrients, medium pH, and certain oligosaccharides, are known to influence trichothecene biosynthesis in Fusarium graminearum, the transcriptional regulatory mechanism of Tri6 and Tri10 genes is poorly understood. Particularly, culture medium pH is a major regulator in trichothecene biosynthesis in F. graminearum, but it is susceptible to metabolic changes posed by nutritional and genetic factors. Hence, appropriate precautions should be considered to minimize the indirect influence of pH on the secondary metabolism while studying the roles of nutritional and genetic factors on trichothecene biosynthesis regulation. Additionally, it is noteworthy that the structural changes of the trichothecene gene cluster core region exert considerable influence over the normal regulation of Tri gene expression. In this perspective paper, we consider a revision of our current understanding of the regulatory mechanism of trichothecene biosynthesis in F. graminearum and share our idea toward establishing a regulatory model of Tri6 and Tri10 transcription.