Fusarium species are traditionally grouped into type A and type B trichothecene producers based on structural differences in the mycotoxin they synthesize. The type B trichothecene-producing Fusarium graminearum strains are further divided into 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), and nivalenol (NIV) chemotypes. The former two chemotypes, collectively termed a deoxynivalenol (DON) chemotype, evolved from a NIV chemotype by inactivation of FgTri13, which encodes trichothecene C-4 hydroxylase, during the evolutionary process. Despite stable overexpression of FgTri13, however, both 3-acetylnivalenol (3-ANIV) and 3-ADON accumulate equally in shake flask culture of a transgenic 3-ADON chemotype. In this study, we investigated why the "3-ANIV chemotype" could not be obtained using this strategy. When analysis was extended to the transgenic NIV chemotype, in which FgTri7 C-4 acetylase gene was disrupted and FgTri8 deacetylase gene was replaced with the 3-ADON chemotype's orthologue, C-4 unoxygenated 3-ADON, as well as C-4 oxygenated 3-ANIV, accumulated as the end product. A feeding experiment with an ΔFgtri5ΔFgtri3 double gene disruptant, a trichothecene non-producing mutant unable to acetylate C-15 of the trichothecene ring, revealed the importance of the 15-O-acetyl group for efficient C-4 hydroxylation of DON-type trichothecenes. This implies that traditional DON and NIV chemotype diversification is not solely explained by FgTri13, but is also explained by the function of the FgTri8 trichothecene deacetylase gene. None of the crude cell extracts from existing chemotypes showed highly specific C-15 deacetylation activity against 3,15-diacetylnivalenol (3,15-diANIV) without deacetylating C-15 of the C-4 unoxygenated earlier intermediate, 3,15-diacetyldeoxynivalenol. Thus, an unnatural Fusarium trichothecene, 3-ANIV, could only be synthesized as part of a mixture with 3-ADON, unless the esterase encoded by FgTri8 evolves to act on the 15-O-acetyl of 3,15-diANIV with high specificity. We also explain why the transgenic "15-ANIV chemotype", which can be generated through functional inactivation of FgTri7, uses an engineered pathway via 3,15-diANIV, but not 15-ADON, to generate 15-ANIV. Tri genes appear to evolve continuously, and altered functions of trichothecene pathway enzymes result in the generation of new trichothecenes, such as NX-2 and NX-3, which have been recently discovered in field isolates of F. graminearum. As recombination of FgTri8 between existing F. graminearum isolates could give rise to a strain that produces mixtures of DON and NIV-type trichothecenes, it may also be noteworthy to monitor the emergence of a field isolate that invalidates traditional chemotype classification.

中文翻译：

---

镰刀菌生物合成酶的底物特异性解释了产生脱氧雪茄烯醇和雪茄烯醇型三态鞘氨醇的混合化学型的遗传基础。

根据镰刀菌属合成的霉菌毒素的结构差异，镰刀菌通常被分为A型和B型单端孢菌素生产者。产生B型单端孢菌素的禾谷镰刀菌菌株进一步分为3-乙酰基脱氧雪茄烯醇（3-ADON），15-乙酰基脱氧雪茄烯醇（15-ADON）和nivalenol（NIV）化学型。前两种化学型统称为脱氧雪腐烯（DON）化学型，是在进化过程中通过失活FgTri13编码而从NIV化学型演变而来的，该FgTri13编码天花粉C-4羟化酶。尽管FgTri13的稳定过表达，但是，3-乙酰基萘烯醇（3-ANIV）和3-ADON在转基因3-ADON化学型的摇瓶培养物中均等积累。在这项研究中，我们调查了为什么无法使用此策略获得“ 3-ANIV化学型”。当分析扩展到转基因NIV化学型时，其中FgTri7 C-4乙酰化酶基因被破坏，FgTri8脱乙酰基酶基因被3-ADON化学型直系同源物取代，C-4未氧化3-ADON，以及C-4氧化3 -ANIV，作为最终产品积累。饲喂ΔFgtri5ΔFgtri3双基因破坏剂（一种不能使三茂金属环的C-15乙酰化的三茂金属非生产突变体）的饲喂实验揭示了15-O-乙酰基对于DON型三茂金属有效C-4羟基化的重要性。这意味着传统的DON和NIV化学型多样化不仅是由FgTri13解释的，而且还由FgTri8上四茂铁烯脱乙酰酶基因的功能来解释。现有化学型的粗细胞提取物中，没有一种具有针对3的高特异性C-15脱乙酰活性，15-二乙酰基新戊烯醇（3,15-diANIV）不会使C-4未加氧的较早中间体3,15-二乙酰基脱氧新萘烯醇的C-15脱乙酰基。因此，除非由FgTri8编码的酯酶进化为以高特异性作用于3,15-diANIV的15-O-乙酰基，否则只能合成与3-ADON混合物的一部分的非天然镰孢镰刀菌3-ANIV。 。我们还解释了为什么可以通过功能失活FgTri7生成的转基因“ 15-ANIV化学型”使用经过3,15-diANIV而非15-ADON的工程途径来生成15-ANIV。Tri基因似乎不断进化，并且单端孢菌途径酶的功能改变导致新的单端孢菌的产生，例如NX-2和NX-3，它们最近已在禾谷镰孢的田间分离物中发现。作为现有F之间的FgTri8的重组。