Identification of the SGR6065 gene product as a sesquiterpene cyclase involved in (+)-epicubenol biosynthesis in Streptomyces griseus

Abstract

Recent bacterial genome sequencing projects have shown the presence of many putative sesquiterpene cyclase (SC) genes, especially in the Gram-positive, filamentous bacterial genus Streptomyces. We describe here the characterization of a SC gene (SGR6065, named gecA) from Streptomyces griseus. Overexpression of gecA in Streptomyces lividans produced a sesquiterpene, which was isolated and determined to be (+)-epicubenol using spectroscopic analyses. The N-terminal histidine-tagged GecA protein was produced in Escherichia coli. Incubation of the recombinant GecA protein with farnesyl diphosphate (FPP) yielded (+)-epicubenol as the major product. The K(m) value for FPP and the k(cat) value for (+)-epicubenol formation were calculated to be 254 ± 7.1 nM and 0.026 ± 0.001 s(-1), respectively. The k(cat)/K(m) value (0.10 s(-1) μM(-1)) was broadly comparable to those reported for known bacterial SCs. (+)-Epicubenol was detected in the crude cell lysate of wild-type S. griseus, but not in a gecA-knockout mutant, indicating that GecA is a genuine (+)-epicubenol synthase. Although (+)-epicubenol synthases have been previously purified and characterized from the liverwort Heteroscyphus planus and Streptomyces sp. LL-B7, no (+)-epicubenol synthase gene has been cloned to date. The gecA gene is thus the first example of an (+)-epicubenol synthase-encoding gene. (+)-Epicubenol production was not controlled by the microbial hormone A-factor that induces morphological differentiation and production of several secondary metabolites in S. griseus.