title: 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.
authors.affiliation: Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.
authors.forename: Chiaki
authors.initials: C
authors.lastname: Nakano
authors.forename: Takeaki
authors.initials: T
authors.lastname: Tezuka
authors.forename: Sueharu
authors.initials: S
authors.lastname: Horinouchi
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Ohnishi
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D019755:Carbon-Carbon Lyases / Q000235:genetics* / Q000378:metabolism*; D004269:DNA, Bacterial / Q000235:genetics; D008401:Gas Chromatography-Mass Spectrometry; D055786:Gene Knockout Techniques; D005798:Genes, Bacterial; D007535:Isomerases / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D009154:Mutation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012717:Sesquiterpenes / Q000737:chemistry / Q000378:metabolism*; D013237:Stereoisomerism; D013305:Streptomyces griseus / Q000201:enzymology* / Q000235:genetics*; D059467:Transcriptome
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D012717:Sesquiterpenes; C506317:epicubenol; D019755:Carbon-Carbon Lyases; C051130:trichodiene synthetase; D007535:Isomerases; C404719:epicubenol synthase