Table 1.
Deduced ORFs and their predicted functions in the xim gene cluster.
Figure 1.
Mutational analysis of the xiamenmycin gene cluster.
(A) Schematic representation of the construction of the gene inactivation mutant (ximA), where ximA was replaced by the apr cassette. The ximA mutant gives a 1041 bp PCR product, while the wild type strain is 1663 bp. Marker (1 kb DNA Ladder, Fermantas). (B) UPLC profiles of 1, 3 and products of wild type and xim mutants. Disruption of ximA to ximE individually abolished production of 1, but the intermediate 3 only accumulated in ΔximA.
Figure 2.
Heterologus expression of the xiamenmycin biosynthetic pathway in S. lividans.
UPLC- total ion chromatography MS profiles of the metabolites produced by (A) S. xiamenensis wild type; (B) S. lividans harboring the empty vector pSET152; (C) S. lividans containing pLMO09404.
Figure 3.
Proposed biosynthetic pathway for xiamenmycin.
Figure 4.
Confirmation of 4HB in the core structure of xiamenmycin.
HR-MS of 1 after [ring-13C6] 4HB was fed to S. xiamenensis wild type strain, (A) in the positive ionization mode; (B) in the negative ionization mode.
Figure 5.
GC-MS profiles from the in vitro assay of XimC. (A) 4HB; (B) heat-inactivated XimC incubated with chorismate; (C) XimC incubated with chorismate.
Figure 6.
UPLC-total ion chromatography MS in the negative ionization mode of the XimB in vitro assay. (A) HR-MS/MS of compound 2; the lines display the proposed permutations and combination pattern; (B) UPLC-total ion chromatography MS of the membrane fraction without XimB incubated with 4HB and GPP; (C) UPLC-total ion chromatography MS of XimB incubated with 4HB and GPP.
Figure 7.
Feeding experiments of the xim gene disruptants.
UPLC- total ion chromatography MS profiles of extracts of feeding experiments. (A) xiamenmycin (1); (B) ΔximC, supplemented with 0.1 mg/ml 4HB; (C) ΔximD mutant supplemented with 0.1 mg/ml xiamenmycin B (3); (D) ΔximE mutant supplemented with 0.1 mg/ml xiamenmycin B (3).
Figure 8.
UPLC-extracted ion chromatography MS (EIC-MS) in the negative ionization mode of XimA in vitro assay. (A) xiamenmycin (1); (B) XimA incubated with xiamenmycin B (3) and L-threonine; (C) boiled XimA incubated with xiamenmycin B (3) and L-threonine.