Fig 1.
Structures of several important BIQ antibiotics and medermycin biosynthetic gene cluster.
(A) Structures of three BIQ antibiotics and their producing strains. The numbering given is based on the biosynthetic origin of these compounds. (B) Organization of the gene cluster for medermycin biosynthesis. The genes (med-ORF8, 14, 15, 16, 17, 18 and 20) indicated with numbers are involved in C-glycosylation and med-ORF7 for oxygenation in the tailoring steps in the biosynthesis of medemycin.
Table 1.
Bacteria used in this study.
Fig 2.
LC/MS analysis of the metabolites by the medermycin-producing Streptomyces sp. AM-7161 and heterologous expression strain.
(A) UV absorption at 254 nm of authentic medermycin (upper) and the metabolites by Streptomyces sp. AM-7161 (bottom) (medermycin has a characteristic absorption at 254 nm). (B) UV absorption at 254 nm of metabolites by the wild type strain AM-7161 and heterologous expression strain CH999/pIK340. (C and E) Full-wavelength absorption spectra for two peaks in Fig. 2A at 10.99 min and 19.97 min respectively. (D and F) MS spectra of two peaks in Fig. 2A at 10.99 min and 19.97 min respectively.
Fig 3.
LC/MS analysis of the metabolites by the medermycin-producing Streptomyces sp AM-7161 (A, B and C) and kalafungin-producing Streptomyces tanashiensis Kala DSM731 (D, E and F).
(A) and (D) UV absorption at 254 nm of metabolites by the wild type strain AM-7161 and DSM731. (B and E) Full-wavelength absorption spectra for two peaks in A at 18.002 min (compound X) and in D at 18.048 min (kalafungin) respectively. (C and F) MS spectra for compound X at 18.002 min in A and kalafungin at 18.048 min in D respectively.
Fig 4.
Purification and structural elucidation of compound X isolated from AM-7161.
Absorption profile at 254 nm (A, HPLC analysis), bioassay (B, DMSO as blank control), and high resolution mass spectrum (C) of compound X purified from AM-7161 are shown.
Fig 5.
Comparison of pigmentation and bioassay between the wild type AM-7161 and mutant strains.
(A) and (B) Pigmentation of AM-7161 and mutant strains on solid and liquid R4 medium. (C) Absorbance values at 254 nm of crude extracts from AM-7161 and mutant strains. (D) Plate assay of antimicrobial activity of AM-7161 and mutant strains against S. epidermidis ATCC 35984. EtoAc is used as blank control. (E) The ratio of the inhibition zone diameter by mutant strains (Dm) to that of the inhibition zone by AM-7161 (Dw).
Fig 6.
Metabolite analysis of AM-7161 and mutant strains by HPLC.
(A) UV absorption at 254 nm of AM-7161 and mutant strains. (B) The ratio of the peak area (Sm) by mutant strains to the peak area (Sw) by AM-7161 at 18.2 min, indicating the production of compound X.
Fig 7.
Proposed tailoring steps in the biosynthetic pathway of medermycin.
(S)-DNPA: 4-dihydro-9-hydroxy-1-methyl-10-oxo-3-H-naphtho[2,3-c]pyran-3-acetic acid, as a first chiral intermediate in the pathway of medermycin. NDP-angolosamine: the specific glycosyl moiety formed under the control of seven glycosylating genes indicated in the med gene cluster in Fig. 1B. The conversion from DDHK to DHK could be catalyzed by an oxygenase encoded by med-ORF7 in the med gene cluster [26]. The numbering given is based on the biosynthetic origin of these intermediates.