Discovery and Characterization of a New Family of Diterpene Cyclases in Bacteria and Fungi

Abstract

Diterpene cyclases from bacteria and basidiomycete fungi are seldom studied. Here,wepresented the identification and verification of EriG,amember of the UbiA superfamily,as the enzyme responsible for the cyclization of the cyathane skeleton in the mushroom Hericium erinaceum. Genome mining using the EriG protein sequence as ap robe led to the discovery of anew family of ubiquitous UbiA-related diterpene cyclases in bacteria and fungi. We successfully characterized seven new diterpene cyclases from bacteria or basidiomycete fungi with the help of an engineered Escherichia coli strain and determined the structures of their corresponding products.A new diterpene with an unusual skeleton was generated during this process.T he discovery of this new family of diterpene cyclases provides new insight into the UbiA superfamily.Supportinginformation and the ORCID identification number(s) for the author(s) of this article can be found under: http://dx.

Bacterial Diterpene Biosynthesis

“…Chemie mechanism proceeds via the same intermediates as for lydicene up to C56 + ;h owever,w hereas the full stereochemistry of the intermediates for lydicene cannot be inferred from the structure of the final product, this is possible for the mechanism towards 57 and 58,a st he stereochemical information is retained in these products.T he intermediate C56 + can undergo another cyclisation to A57 + ,the precursor of 57 on attack of water.Arearrangement of the cyclopropylcarbinyl cation to A58 + ,similar to the rearrangements described for cyclooctat-9-en-7-ol (Scheme 2) and tsukubadiene (Scheme 3), leads to 58. [7]…”

“…Thes uggested cyclisation mechanism operates via GLPP to explain the formation of the Z-configured double bond in the cyclisation to A56 + .Asubsequent ring expansion and cyclisation to B56 + is followed by ar ing expansion to C56 + .This secondary cation is stabilised by a1,2hydride shift, and as ubsequent deprotonation affords 56. [7]…”

“…[6] Amore recently identified third family of DTSs are UbiA-related enzymes. [7] TheUbiA archetype is aprenyltransferase involved in ubiquinone biosynthesis that also acts by abstraction of diphosphate from the oligoprenyl diphosphate precursor, [8] but the amino acid sequence is not related to the sequence of type IT Ss,w hich suggests that these enzymes have evolved independently.F or all three enzyme classes,the substrate is folded into areactive conformation in the hydrophobic active site,f rom which water is excluded to avoid quenching of the highly reactive cationic intermediates. Thec omplex cyclisation cascades then make use of the intrinsic reactivity of the substrate.…”

Ancestral diterpene cyclases show increased thermostability and substrate acceptance

“…Bacterial diterpene cyclases have recently emerged as potent biocatalysts both by contributing to enhanced understanding of fundamental secondary metabolism and in the context of synthetic biology programs . At present, the availability of bacterial diterpene cyclase sequences is significantly limited , and our fundamental understanding of associated cyclization mechanisms remains incomplete . We reasoned that ASR of class I enzymes would constitute an interesting opportunity to expand terpene cyclase sequence space, and to investigate the potential, as well as limitations, of ASR when applied to an essentially unexplored family of important biosynthetic enzymes.…”

Genomic-wide analysis and prediction of genes involved in biosynthesis of polysaccharide and bioactive secondary metabolites in high-temperature-tolerance of wild Flammulina filiformis

“…For instances, genome-wide investigation of 66 cosmopolitan strains of Aspergillus fumigatus revealed 5 general types of variation in secondary genes cluster [37]. Identification of the tricyclic diterpene antibiotic pleuromuyilin gene clusters in genome-scale increased antibiotic production in Clitopilus passeckerianus [38]; Prediction of gene clusters involved in biosynthesis of terperoid/PKS in medicinal Hericium erinaceus by genome and transcriptome sequencing discovered a new family of diterpene cyclases in fungi [39,40] and identification of the candidate cytochromes P450s genes cluster possible related to triterpenoid biosynthesis in medicinal mushroom Ganoderma lucidum by genome sequencing improved the production of the medicinal effective compounds [41,42].…”