Characterisation of ascocorynin biosynthesis in the purple jellydisc fungus Ascocoryne sarcoides.
Characterisation of ascocorynin biosynthesis in the purple jellydisc fungus Ascocoryne sarcoides.
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DOI：
10.1186/s40694-022-00138-7
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发表时间：
2022-04-27
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非核糖体肽合成酶样（NRPS-like）酶在真菌基因组中高度富集，可分为还原酶和非还原酶。非还原性nrps类酶具有c端硫酯酶结构域，催化两种相同的芳香α-酮酸在形成酶特异性底物相互连接的核心结构下缩合，如terphenylquinones， furanones， butyroactones或dioxolanones。抗坏血碱类结节产生大量的抗坏血碱，其结构类似于对羟基苯基丙酮酸酯和苯基丙酮酸酯缩合产生的terphenylquinone。由于一种非还原性nrp样酶同时使用两种不同的底物是极不寻常的，我们研究了a . sarcoides中抗坏血凝素的生物合成。在这里，我们搜索了A. sarcoides的基因组，寻找编码非还原性nrps样酶的基因。一个单一的候选基因被确定为acyN。异源基因表达证实AcyN参与抗坏血氨酸的产生，但只产生非羟基化前体聚磷酸。虽然acyN嵌入在抗坏血酸生物合成基因簇中，但编码聚磷酸羟基化所需的单加氧酶的基因不存在。对所有单加氧酶编码基因的表达分析发现了一个与acyN表达模式相同的候选基因。因此，acyN和单加氧酶基因的异种共表达导致了抗坏血凝素的产生。单加氧酶的结构模型表明，疏水底物聚磷酸从面向膜的进入位点进入单加氧酶，并转化为更亲水的产物抗坏血酸，这阻止了它再次进入进行第二轮羟基化。本研究描述了第一个从子囊菌中自然产生的聚磷酸合成酶。它证实了这种非还原性nrp样酶的高底物和产物特异性，并强调了单加氧酶产生terphenylquinone ascocoryin的必要性。在线版本包含补充材料，可在10.1186/s40694-022-00138-7获得。
Non-ribosomal peptide synthetase-like (NRPS-like) enzymes are highly enriched in fungal genomes and can be discriminated into reducing and non-reducing enzymes. Non-reducing NRPS-like enzymes possess a C-terminal thioesterase domain that catalyses the condensation of two identical aromatic α-keto acids under the formation of enzyme-specific substrate-interconnecting core structures such as terphenylquinones, furanones, butyrolactones or dioxolanones. Ascocoryne sarcoides produces large quantities of ascocorynin, which structurally resembles a terphenylquinone produced from the condensation of p-hydroxyphenylpyruvate and phenylpyruvate. Since the parallel use of two different substrates by a non-reducing NRPS-like enzyme appeared as highly unusual, we investigated the biosynthesis of ascocorynin in A. sarcoides. Here, we searched the genome of A. sarcoides for genes coding for non-reducing NRPS-like enzymes. A single candidate gene was identified that was termed acyN. Heterologous gene expression confirmed that AcyN is involved in ascocorynin production but only produces the non-hydroxylated precursor polyporic acid. Although acyN is embedded in an ascocorynin biosynthesis gene cluster, a gene encoding a monooxygenase required for the hydroxylation of polyporic acid was not present. Expression analyses of all monooxygenase-encoding genes from A. sarcoides identified a single candidate that showed the same expression pattern as acyN. Accordingly, heterologous co-expression of acyN and the monooxygenase gene resulted in the production of ascocorynin. Structural modelling of the monooxygenase suggests that the hydrophobic substrate polyporic acid enters the monooxygenase from a membrane facing entry site and is converted into the more hydrophilic product ascocorynin, which prevents its re-entry for a second round of hydroxylation. This study characterises the first naturally occurring polyporic acid synthetase from an ascomycete. It confirms the high substrate and product specificity of this non-reducing NRPS-like enzyme and highlights the requirement of a monooxygenase to produce the terphenylquinone ascocorynin. The online version contains supplementary material available at 10.1186/s40694-022-00138-7.