University of Minnesota, Department of Biochemistry, Molecular Biology, and Biophysics, Saint Paul, MN, USA.
USDA ARS Cereal Disease Laboratory, Saint Paul, MN, USA.
Fungal Genet Biol. 2019 Mar;124:78-87. doi: 10.1016/j.fgb.2019.01.006. Epub 2019 Jan 18.
The sesquiterpenoid deoxynivalenol (DON) is an important trichothecene mycotoxin produced by the cereal pathogen Fusarium graminearum. DON is synthesized in specialized subcellular structures called toxisomes. The first step in DON synthesis is catalyzed by the sesquiterpene synthase (STS), Tri5 (trichodiene synthase), resulting in the cyclization of farnesyl diphosphate (FPP) to produce the sesquiterpene trichodiene. Tri5 is one of eight putative STSs in the F. graminearum genome. To better understand the F. graminearum terpenome, the volatile and soluble fractions of fungal cultures were sampled. Stringent regulation of sesquiterpene accumulation was observed. When grown in trichothecene induction medium, the fungus produces trichothecenes as well as several volatile non-trichothecene related sesquiterpenes, whereas no volatile terpenes were detected when grown in non-inducing medium. Surprisingly, a Δtri5 deletion strain grown in inducing conditions not only ceased accumulation of trichothecenes, but also failed to produce the non-trichothecene related sesquiterpenes. To test whether Tri5 from F. graminearum may be a promiscuous STS directly producing all observed sesquiterpenes, Tri5 was cloned and expressed in E. coli and shown to produce primarily trichodiene in addition to minor, related cyclization products. Therefore, while Tri5 expression in F. graminearum is necessary for non-trichothecene sesquiterpene biosynthesis, direct catalysis by Tri5 does not explain the sesquiterpene deficient phenotype observed in the Δtri5 strain. To test whether Tri5 protein, separate from its enzymatic activity, may be required for non-trichothecene synthesis, the Tri5 locus was replaced with an enzymatically inactive, but structurally unaffected tri5 allele. This allele restores non-trichothecene synthesis but not trichothecene synthesis. The tri5 allele also restores toxisome structure which is lacking in the Δtri5 deletion strain. Our results indicate that the Tri5 protein, but not its enzymatic activity, is also required for the synthesis of non-trichothecene related sesquiterpenes and the formation of toxisomes. Toxisomes thus not only may be important for DON synthesis, but also for the synthesis of other sesquiterpene mycotoxins such as culmorin by F. graminearum.
倍半萜脱氧雪腐镰刀菌烯醇（DON）是一种由谷物病原体禾谷镰刀菌产生的重要的单端孢霉烯族真菌毒素。DON 是在称为产毒体的专门亚细胞结构中合成的。DON 合成的第一步是由倍半萜合酶（STS）Tri5（毕赤酵母二烯合酶）催化的，导致法呢基二磷酸（FPP）环化产生倍半萜毕赤酵母二烯。Tri5 是禾谷镰刀菌基因组中八个假定 STS 之一。为了更好地了解禾谷镰刀菌的萜类化合物组，对真菌培养物的挥发性和可溶性部分进行了采样。观察到倍半萜积累受到严格调控。当在单端孢霉烯诱导培养基中生长时，真菌会产生单端孢霉烯以及几种挥发性非单端孢霉烯相关的倍半萜，而在非诱导培养基中则未检测到挥发性萜类化合物。令人惊讶的是，在诱导条件下生长的Δtri5 缺失菌株不仅停止了单端孢霉烯的积累，而且还未能产生非单端孢霉烯相关的倍半萜。为了测试禾谷镰刀菌的 Tri5 是否可能是一种直接产生所有观察到的倍半萜的混杂 STS，克隆并在大肠杆菌中表达了 Tri5，并发现其主要产生毕赤酵母二烯，此外还有少量相关的环化产物。因此，虽然 Tri5 在禾谷镰刀菌中的表达是非单端孢霉烯倍半萜生物合成所必需的，但 Tri5 的直接催化作用并不能解释在Δtri5 菌株中观察到的倍半萜缺乏表型。为了测试 Tri5 蛋白是否在没有其酶活性的情况下也可能需要用于非单端孢霉烯的合成，用具有酶失活但结构不受影响的 tri5 等位基因替换了 Tri5 基因座。该等位基因恢复了非单端孢霉烯的合成，但不能恢复单端孢霉烯的合成。tri5 等位基因还恢复了在Δtri5 缺失菌株中缺失的产毒体结构。我们的结果表明，Tri5 蛋白，而不是其酶活性，也是非单端孢霉烯相关倍半萜合成和产毒体形成所必需的。因此，产毒体不仅可能对 DON 合成很重要，而且对禾谷镰刀菌产生的其他倍半萜真菌毒素如 culmorin 的合成也很重要。
