RNA-seq for gene expression analysis.

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.