The echinulin family alkaloids can be grouped into three series depending on the number of the exo double bonds adjacent to the diketopiperazine core structure. Heterologous expression of the putative echinulin biosynthetic gene cluster from Aspergillus ruber in Aspergillus nidulans led to accumulation of echinulin without a double bond and neoechinulin A with one double bond (Δ¹⁰) as major products. Their analogues with a different number of prenyl moieties were detected as minor products. Neoechinulin B and analogues with two double bonds (Δ^10,14) were not observed. Feeding experiments confirmed that the cytochrome P450 enzyme EchP450 only catalyzes the formation of the double bond between C10 and C11. Coincubation and substrate concentration dependent assays with the prenyltransferase EchPT2 revealed that the reversely C2-prenylated preechinulin without a double bond is a much better substrate than neoechinulin A. These results prove that preechinulin serves as a common substrate for the formation of echinulin by two regiospecific prenylation steps with EchPT2 or for EchP450 to introduce one double bond and subsequent prenylations with low regioselectivity.

中文翻译：

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曲霉橡胶中Echinulin家族生物碱的生物合成中作为分支点的C 2-反向烯丙基化二酮哌嗪的烯丙基化和脱氢。

根据与二酮哌嗪核心结构相邻的外切双键的数目，棘球蛋白家族生物碱可分为三个系列。从推定的echinulin生物合成基因簇的异源表达曲霉属曲霉在构巢曲霉导致echinulin的积累没有双键，neoechinulin A和一个双键的（Δ ¹⁰）作为主要产物。他们的异戊二烯基数量不同的类似物被检测为次要产物。具有两个双键的新棘球蛋白B和类似物（^Δ10,14）没有被观察到。补料实验证实，细胞色素P450酶EchP450仅催化C10和C11之间的双键形成。异戊二烯基转移酶EchPT2的共孵育和底物浓度依赖性分析表明，没有双键的反向C 2-戊烯基化的preechinulin比新echinulin A更好。这些结果证明preechinulin是两种区域特异性形成echinulin的常见底物用EchPT2或EchP450进行烯丙基化步骤，以引入一个双键和随后的烯丙基化反应，降低区域选择性。