Shotgun sequencing summary statistics (PacBio).

Hydrogen cyanide (HCN) is a highly toxic biogenic compound. Unlike most natural defensive chemicals, which are typically lineage-specific, the biosynthesis and liberation of HCN, called “cyanogenesis”, occur sporadically among arthropod and plant lineages. This suggests that cyanogenesis has evolved independently numerous times in the animal and plant kingdoms. Although cyanogenesis was identified in millipedes 140 years ago, the cyanogenesis-related enzymes in these arthropods have not yet been fully identified. Here, we report a complete set of cyanogenesis-related enzymes in the millipede Chamberlinius hualienensis based on an analysis combining genome sequencing and biological characterisation. The gene encoding hydroxynitrile lyase, which catalyses the liberation of HCN from (R)-mandelonitrile, and its paralogous genes were clustered, indicating sequential duplication of their coding genes, giving rise to hydroxynitrile lyase in millipedes. We discovered that (R)-mandelonitrile cyanohydrin biosynthesis in C. hualienensis utilises a flavin-dependent monooxygenase (ChuaMOxS) for the initial aldoxime synthesis step, similar to the process in ferns, instead of cytochrome P450 (CYP) as in higher plants and insects. Although a single CYP is responsible for subsequently converting aldoxime into cyanohydrin in plants and insects, the reaction involves two enzymes in millipedes. We found two millipede CYPs (CYP4GL4 and CYP30008A2) that catalyse aldoxime dehydration to produce nitrile, in addition to CYP3201B1, which then catalyses the formation of (R)-mandelonitrile from nitrile. The discovery of cyanogenesis-related enzymes in millipedes demonstrates that cyanogenic millipedes evolved these enzymes independently from plants and insects, providing a deeper understanding of the mechanisms underlying the evolution of metabolic pathways.氰化氢（Hydrogen cyanide, HCN）是一类剧毒生物源化合物。与多数通常具备谱系特异性的天然防御性化学物质不同，氰化氢的生物合成与释放过程（被称为"氰生成作用（cyanogenesis）"）在节肢动物与植物类群中呈零星分布。这一现象表明，氰生成作用在动物界与植物界中已独立演化多次。尽管早在140年前便在马陆中发现了氰生成作用，但这类节肢动物体内与氰生成相关的酶类尚未被完全鉴定。本研究结合基因组测序与生物学表征分析，报道了台湾腔马陆（Chamberlinius hualienensis）体内一套完整的氰生成相关酶系。催化(R)-扁桃腈释放氰化氢的羟腈裂合酶（hydroxynitrile lyase）编码基因及其旁系同源基因成簇分布，提示其编码基因经历了连续复制事件，最终演化出马陆体内的羟腈裂合酶。本研究发现，台湾腔马陆体内的(R)-扁桃腈氰醇生物合成途径，采用黄素依赖型单加氧酶（ChuaMOxS）完成初始醛肟合成步骤，该过程与蕨类植物相似，而非高等植物与昆虫所采用的细胞色素P450（cytochrome P450, CYP）途径。尽管在植物与昆虫体内，仅需单一细胞色素P450便可将醛肟转化为氰醇，但在马陆体内该反应需两种酶协同完成。本研究鉴定到两种马陆细胞色素P450——CYP4GL4与CYP30008A2——可催化醛肟脱水生成腈，另有CYP3201B1可进一步催化腈生成(R)-扁桃腈。马陆体内氰生成相关酶系的发现证实，产氰马陆与植物、昆虫独立演化出了这类酶系，这为解析代谢通路演化的底层机制提供了更为深入的认知。