Chemical diversity of labdane-type bicyclic diterpene biosynthesis in Actinomycetales microorganisms.

Five pairs of bacterial type-A and type-B diterpene synthases have been characterized: BAD86798/BAD86797, AHK61133/AHK61132, BAB39207/BAB39206, CldD/CldB and RmnD/RmnB, and are involved in the formation of pimara-9(11),15-diene, terpente-3,13,15-triene and labda-8(17),12(E),14-triene. Mining of bacterial genome data revealed an additional four pairs of type-A and type-B diterpene synthases: Sros_3191/Sros_3192 of Streptosporangium roseum DSM 43021, Sare_1287/Sare_1288 of Salinispora arenicola CNS-205, SCLAV_5671/SCLAV_5672 and SCLAV_p0491/SCLAV_p0490 of Streptomyces clavuligerus ATCC 27064. Since SCLAV_p0491/SCLAV_p0490 is similar to the labdane-type diterpene synthase pairs, CldD/CldB and RmnD/RmnB based on the alignment of the deduced amino acid sequences and phylogenetic analyses of the aligned sequences, these predicted diterpene synthases were characterized by an enzymatic reaction using a pair of recombinant type-A and type-B diterpene synthases prepared in Escherichia coli and the heterologous expression of two genes encoding type-A and type-B diterpene synthases in an engineered Streptomyces host. The generation of labda-8(17),12(E),14-triene (1) by CldB and CldD was reconfirmed by enzymatic synthesis. Furthermore, labda-8(17),13(16),14-triene (2) was generated by SCLAV_p0491 and CldB, and ladba-7,12(E),14-triene (3) by CldD and SCLAV_p0490. SCLAV_p0491 and SCLAV_p0490 catalyzed the generation of the novel diterpene hydrocarbon, labda-7,13(16),14-triene (4).