To examine whether there are other undiscovered geldanamycin (GDM) analogs in the gdmN disruption mutant of Streptomyces hygroscopicus 17997, a time-course monitoring of its fermentation culture was conducted. The fermentation supernatants were extracted with ethyl acetate, and a novel GDM analog was isolated via silica-gel TLC, HPLC, liquid chromatography-ESI(+)-MS and NMR analyses. The analog was identified as 7-O-descarbamoyl-7-hydroxygeldanamycin (designated CT-1-7x), which is the C-4,5 olefinic form of 4,5-dihydro-7-O-descarbamoyl-7-hydroxygeldanamycin (CT-1-7). Bioconversion experiments showed that CT-1-7x failed to be converted to GDM by GDM-pks- (a GDM PKS gene disruption mutant retaining post-PKS tailoring genes), whereas CT-1-7 could be converted to GDM. This indicates that the carbamoyltransferase in GDM biosynthesis exhibits substrate specificity for the C-4,5 single bond, and 7-O-carbamoylation must occur before C-4,5 oxidation in the post-polyketide synthase (PKS) tailoring process of GDM biosynthesis.