Language：English   Publishing type：Research paper (scientific journal)   Publisher：{MDPI} {AG}  

<jats:p>In trichothecene-producing fusaria, isotrichodermol (ITDol) is the first intermediate with a trichothecene skeleton. In the biosynthetic pathway of trichothecene, a 3-O-acetyltransferase, encoded by Tri101, acetylates ITDol to a less-toxic intermediate, isotrichodermin (ITD). Although trichothecene resistance has been conferred to microbes and plants transformed with Tri101, there are no reports of resistance in cultured mammalian cells. In this study, we found that a 3-O-acetyl group of trichothecenes is liable to hydrolysis by esterases in fetal bovine serum and FM3A cells. We transfected the cells with Tri101 under the control of the MMTV-LTR promoter and obtained a cell line G3 with the highest level of C-3 acetylase activity. While the wild-type FM3A cells hardly grew in the medium containing 0.40 μM ITDol, many G3 cells survived at this concentration. The IC50 values of ITDol and ITD in G3 cells were 1.0 and 9.6 μM, respectively, which were higher than the values of 0.23 and 3.0 μM in the wild-type FM3A cells. A similar, but more modest, tendency was observed in deoxynivalenol and 3-acetyldeoxynivalenol. Our findings indicate that the expression of Tri101 conferred trichothecene resistance in cultured mammalian cells.</jats:p>

DOI： 10.3390/toxins11110654

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media {LLC}  

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.

DOI： 10.1007/s10482-018-1172-z

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Association of Mycotoxicology  

DOI： 10.2520/myco.69-1-2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier {BV}  

While Fusarium graminearum readily produces trichothecenes in complex media containing sucrose as the carbon source (YS_60), the amount of the mycotoxin is quite limited when other sugars, such as glucose and fructose, are used. We found that autoclaving of media containing fructose and yeast extract (YF_60) results in the formation of inhibitors of trichothecene biosynthesis by F. graminearum JCM 9873, a strain that produces 15-acetyldeoxynivalenol (15-ADON) in liquid culture. Removal of the solvent fraction from the autoclaved media after ethyl acetate extraction attenuated the inhibitory activity against trichothecene production. In addition, extraction of the non-autoclaved complex media with ethyl acetate, followed by removal of the solvent fraction, similarly resulted in increased accumulation of the mycotoxin. Although the increase in trichothecene production differed considerably among fungal strains and yeast extract products, F. graminearum species complex generally responded to the medium treatments in the same way. These results suggest that some hydrophobic substances that arise during the drying and heating of yeast extract negatively affected trichothecene production in liquid culture. Modes of actions of inhibitory substances were partially characterized using strain JCM 9873, with focus on the transcriptional and functional analyses of Tri6, a key regulator gene in trichothecene biosynthesis. The presence of the ethyl acetate-extractable substances in autoclaved YF_60 media decreased the relative transcription level of Tri6, as well as that of a trichodiene synthase gene Tri5. Thus, the substances exerted their inhibitory action through suppression of Tri6 expression. By using a yeast extract lot that completely prevented trichothecene production by the wild-type strain in autoclaved YS_60 medium, we prepared YF_60 media and cultured a constitutive Tri6 overexpressor strain described by Maeda et al. (2018). Despite the high transcription level of Tri6, the presence of the ethyl acetate extractable-substances suppressed 15-ADON production. These results suggested that both Tri6p-independent initial activation of Tri6 expression and subsequent Tri6p-dependent activation of Tri expression were affected by the hydrophobic substances in the yeast extract products.

DOI： 10.1016/j.ijfoodmicro.2018.08.026

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Association of Mycotoxicology  

DOI： 10.2520/myco.68-2-1

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Authorship：Lead author   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society ({ACS})  

DOI： 10.1021/acschembio.8b00044

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Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society ({ACS})  

© 2018 American Chemical Society and American Society of Pharmacognosy. 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.

DOI： 10.1021/acs.jnatprod.7b00398

Scopus

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1111/lam.12797

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media {LLC}  

DOI： 10.1007/s00203-017-1364-3

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Other Link： http://link.springer.com/content/pdf/10.1007/s00203-017-1364-3.pdf

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier {BV}  

DOI： 10.1016/j.pestbp.2017.03.006

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Association of Mycotoxicology  

<p>  Identification of small-molecule binding proteins is an important requirement for studies investigating the mode-of-action of specific inhibitors. We here report the characterization of <i>Fusarium graminearum</i> genes that were identified to encode potential target proteins of a trichothecene production inhibitor. Care should be taken when assessing the inhibitory role of candidate ligand-binding proteins identified by the assays.</p>

DOI： 10.2520/myco.67-1-3

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Other Link： http://search.jamas.or.jp/link/ui/2017203113

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Association of Mycotoxicology  

<p>  <i>Fusarium graminearum</i> produces type B trichothecenes in liquid culture as the sole trichothecene products. When methionine was used as a nitrogen source, a considerable amount of 3-deacetylcalonectrin (3-deCAL), devoid of a C-8 ketone, accumulated in the synthetic medium as one of the major end products. This indicates that a wild-type <i>F</i>. <i>graminearum</i> type B trichothecene producer could accumulate a substantial amount of a type A trichothecene, depending on culture conditions.</p>

DOI： 10.2520/myco.67-1-9

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.ijfoodmicro.2016.09.011

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Authorship：Lead author   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1111/1462-2920.13338

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Authorship：Lead author   Publishing type：Research paper (scientific journal)  

PubMed

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Association of Mycotoxicology  

We examined the effect of using xylose as a carbon source in the medium on trichothecene production by <i>Fusarium graminearum</i>. By frequently adjusting the pH of the submerged culture in response to the pH changes of the known trichothecene- inducing sucrose-agmatine culture, trichothecene became detectable from the xylose liquid culture initiated at neutral pH. In contrast to the low pH requirement of the xylose medium for trichothecene production, the fungus produced the mycotoxin in sucrose liquid culture at neutral pH.

DOI： 10.2520/myco.66.17

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Other Link： https://jlc.jst.go.jp/DN/JLC/20020547163?from=CiNii

Publishing type：Research paper (scientific journal)   Publisher：Informa UK Limited  

Abstract

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.

DOI： 10.1080/09168451.2015.1088374

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Association of Mycotoxicology  

We examined the localization of the full-length protein and zinc finger domain of the trichothecene regulator, TRI6. When fused to an enhanced green fluorescent protein (<i>EGFP</i>) gene, nuclear accumulation of the zinc finger domain, but not the full-length protein, was observed.

DOI： 10.2520/myco.66.13

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Association of Mycotoxicology  

Leptomycin B (LMB)-sensitive strains of <i>Fusarium graminearum</i> were generated. An attempt to detect TRI6 in the nucleus of LMB-treated cells was unsuccessful.

DOI： 10.2520/myco.66.9

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Other Link： https://jlc.jst.go.jp/DN/JLC/20020548184?from=CiNii

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Microbiology Research Foundation  

DOI： 10.2323/jgam.2016.05.002

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.ijfoodmicro.2015.07.014

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Association of Mycotoxicology  

DOI： 10.2520/myco.65.7

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Publishing type：Research paper (scientific journal)   Publisher：Microbiology Research Foundation  

DOI： 10.2323/jgam.61.93

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Authorship：Lead author   Publishing type：Research paper (scientific journal)   Publisher：Wiley-Blackwell  

DOI： 10.1111/lam.12289

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Authorship：Lead author,　Corresponding author   Language：Japanese   Publishing type：Research paper (conference, symposium, etc.)  

DOI： 10.2520/myco.64.69

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Association of Mycotoxicology  

DOI： 10.2520/myco.64.141

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Association of Mycotoxicology  

DOI： 10.2520/myco.64.147

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：Japanese Society of Mycotoxicology  

Marker genes are required for the genetic manipulation of living organisms. For selecting transformants after the process of transformation, two types of marker genes are employed: one for the positive selection of the transformants carrying the positive selection marker gene and another for the negative selection of the transformants lacking the negative selection marker gene. Here we have summarized the current advancements in genome engineering of <i>Fusarium</i> species. We have focused on introducing arbitrary mutations by applying both positive and negative selection approaches that result in the exclusion of the marker genes from the target genome. We have discussed the advantages and necessity of using this technique in studies on the regulation of biosynthetic gene expression in mycotoxin production.

DOI： 10.2520/myco.63.85

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Other Link： http://agriknowledge.affrc.go.jp/RN/2010851797

Publishing type：Research paper (scientific journal)  

DOI： 10.1371/journal.ppat.1003581

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japan Society for Bioscience, Biotechnology, and Agrochemistry  

Trichothecene 3-<i>O</i>-acetyltransferase (TRI101) is an indispensable enzyme for the biosynthesis of trichothecenes, a group of mycotoxins produced by <i>Fusarium graminearum</i>. 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.

DOI： 10.1271/bbb.130153

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Association of Mycotoxicology  

The central region of the trichothecene gene cluster contains two pathway and two regulatory genes (four <i>Tri</i> genes) that are necessary for forming the trichothecene skeleton. One of these genes, <i>Tri4</i>, encodes a multifunctional cytochrome P 450 monooxygenase, and the expression of this gene is highly induced under trichothecene-inducing conditions. Apart from this gene cluster, <i>Tri101</i> occurs as a single non-cluster gene, and the product of this gene contributes to the function of self-protection in <i>Fusarium graminearum</i>. The promoter activity of these <i>Tri</i> genes was compared with that of <i>TEF1α</i> (a highly expressed translation elongation factor 1-alpha) gene under toxin-inducing conditions by using the <i>β</i>-glucuronidase (GUS) gene as a reporter. The <i>Tri101</i> promoter- and <i>TEF1α</i> promoter-reporter fusions integrated at the end of the gene cluster showed activities that were similar to those of the original loci with respect to the timing and the level of GUS protein accumulation. However, as opposed to the normal pattern of <i>Tri4</i> transcription from the original locus, no GUS activity was detected when the <i>Tri4</i> promoter was placed at the end of the gene cluster. Thus, we can conclude that the position of the promoter in the gene cluster is important for native transcriptional regulation of <i>Tri4</i> in trichothecene biosynthesis.

DOI： 10.2520/myco.63.17

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Other Link： http://agriknowledge.affrc.go.jp/RN/2010851791

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Association of Mycotoxicology  

<i>Tri13</i> encodes a cytochrome P450 monooxygenase that catalyzes C-4 hydroxylation of some intermediates in the biosynthesis of type A and type B trichothecenes. This work demonstrated that <i>Tri13</i> (<i>FsTri13</i>) of <i>Fusarium sporotrichioides</i>, a typical type A trichothecene producer, can participate in the biosynthesis of type B trichothecenes. When <i>FsTri13</i> was expressed in <i>Fusarium graminearum</i> that produces 3-acetyldeoxynivalenol (3-ADON) as an end product, 3-acetylnivalenol (3-ANIV) was newly detected in the liquid medium. Although 3-ANIV can easily be prepared enzymatically from nivalenol (NIV) by using recombinant trichothecene 3-<i>O</i>-acetyltransferase (TRI101), there has been no conclusive report on the production of 3-ANIV in cultures of <i>Fusarium</i> strains. Possible biosynthetic pathway for production of 3-ANIV was suggested with regard to the function of <i>Tri8</i>, which is responsible for deacetylation of the side-chain <i>O</i>-acetyls in 3-ADON biosynthesis.

DOI： 10.2520/myco.62.83

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Other Link： https://jlc.jst.go.jp/DN/JALC/10009361699?from=CiNii

Authorship：Lead author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Springer Nature  

DOI： 10.1007/s10327-010-0250-5

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Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.bbrc.2007.09.055

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