Hydroxynitrile lyases from cyanogenic millipedes: molecular cloning, heterologous expression, and whole-cell biocatalysis for the production of (R)-mandelonitrile

Abstract

Hydroxynitrile lyases (HNLs), which are key enzymes in cyanogenesis, catalyze the cleavage of cyanohydrins into carbonyl compounds and hydrogen cyanide. Since HNLs also catalyze the reverse reaction, they are used industrially for the asymmetric synthesis of cyanohydrins, which are valuable building blocks of pharmaceuticals and fine chemicals. HNLs have been isolated from cyanogenic plants and bacteria. Recently, an HNL from the cyanogenic millipede Chamberlinius hualienensis was shown to have the highest specific activity for (R)-mandelonitrile synthesis, along with high stability and enantioselectivity. However, no HNLs have been isolated from other cyanogenic millipedes. We identified and characterized HNLs from 10 cyanogenic millipedes in the Paradoxosomatidae and Xystodesmidae. Sequence analyses showed that HNLs are conserved among cyanogenic millipedes and likely evolved from one ancestral gene. The HNL from Parafontaria tonominea was expressed in Escherichia coli SHuffle T7 and showed high specific activity for (R)-mandelonitrile synthesis and stability at a range of pHs and temperatures. The stability of millipede HNLs is likely due to disulfide bond(s). The E. coli cells expressing HNL produced (R)-mandelonitrile with 97.6% enantiomeric excess without organic solvents. These results demonstrate that cyanogenic millipedes are a valuable source of HNLs with high specific activity and stability.

Molecular evolution of gland cell types and chemical interactions in animals

“…For example, certain millipedes and butterflies transform aliphatic and aromatic hydroxynitrileseither synthesized from amino acids or sequestered from the dietinto hydrogen cyanide (HCN) (Shear, 2015;Zagrobelny et al, 2008Zagrobelny et al, , 2018. HCN liberation is catalyzed by a highly specific enzyme, (S)-hydroxynitrile lyase, which functions solely for this purpose (Dadashipour et al, 2015;Sharma et al, 2005;Yamaguchi et al, 2018). The challenges of identifying and characterizing new classes of enzyme may lead to an underestimate of the prevalence of examples of de novo enzyme evolution.…”

R‐hydroxynitrile lyase from the cyanogenic millipede, Chamberlinius hualienensis—A new entry to the carrier protein family Lipocalines

“…Hydroxynitrile lyases (HNLs) have been known to occur mostly in plants [1,2], and recently, those from bacteria [3,4] and arthropod (cyanogenic millipedes) have been newly added to the group [5,6]. HNLs of plant origin have been studied well and their new distribution, characterization, and structure determination are reported.…”

High-throughput in-field bioprospecting for cyanogenic plants and hydroxynitrile lyases

“…Using the Feigl-Anger microfuge tube in this study, we have identified 25 plant species that -although not cyanogenicwere able to degrade racemic mandelonitrile which can be investigated further for the presence of mandelonitrile lyases. Although, HNLs may possibly still be present in specimens that do not have substrate available (Andexer et al 2007;Yamaguchi et al 2018), it may also be useful to test those plants for natural cyanogenic ability in other seasons, climates, as well as, tissue types and maturity to increase chances of extracting enough amounts of HNLs for downstream characterisation. It remains uncertain if species identified as non-cyanogenic (but able to degrade racemic mandelonitrile) in this study are, in fact, completely devoid of cyanogenic activity since this mechanism has been found to be influenced by seasonal changes, climate, tissue type and tissue maturity (Gleadow and Woodrow 2000).…”

“…Cyanogenesis, distinguished by the release of hydrogen cyanide, is believed to be triggered in response to herbivores, predators and infectious microorganisms (Jones 1998). This mechanism is predominantly found in plants, belonging to at least 90 plant families (Conn 1969;Jones 1998;Asano et al 2005;, however, it has also been reported alongside HNLs in bacteria (Hajnal et al 2013;Wiedner et al 2014) and several species of millipedes from the Paradoxosomatidae and Xystodesmidae families (Dadashipour et al 2015;Yamaguchi et al 2018). It has become an attractive option to screen for HNLs from natural sources closely related to those already known to produce HNLs, especially considering that plants do not necessarily have to be cyanogenic to contain HNLs and vice versa (Andexer et al 2007;Yamaguchi et al 2018).…”