Fig 1.
Chemical structures of polyenes cited in the text.
1: Amphotericin B; 2a: CE-108; 2b: CE-108B; 3a: CE-108D; 3b: CE-108E; 4a: Rimocidin; 4b: Rimocidin B. Note that the depicted stereochemistry of 2a, 2b, 3a, 3b and 4b was deduced from the known stereoschemistry of 4a and has not been experimentally established.
Table 1.
Bacterial strains and plasmids used in this study.
Table 2.
1H NMR and 13C NMR Data of 3a and 3b in DMSO-d6.
Fig 2.
(A) HPLC analysis of the fermentation broth of S. diastaticus var. 108/PM1-709B (rimJ disruptant). (B) HPLC analysis of the fermentation broth of the rimJ disruptant carrying pcsA under the control of the constitutive promoter ermEP* (S. diastaticus var. 108::PM1-709B/860); the numbers on the peaks in A and B correspond to the polyenes shown in Fig 1. Peaks marked with 753 and 752 are cited in the text. (C) Percentage of CE-108 and rimocidin production. Polyenes production was measured from liquid cultures by HPLC as described in Material and Methods. The data shown are the mean of three independent experiments. The standard deviation of the mean is indicated by error bars. 1, WT control S. diastaticus var. 108/780 carrying the empty vector; 2, WT derivative carrying rimJ under the control of the ermEP* promoter.
Fig 3.
Biological activities for the polyenes tested.
(A) In vitro susceptibilities of Issatchenkia orientalis, Filobasidiella neoformans, Aspergillus niger and Penicillium chrysogenum to seven antifungal agents. Minimal and maximum values of the range of concentrations for the MICs (μM) are detailed. Minimal values correspond to the lowest concentration of polyene in which growth appeared and maxima values were the lowest concentration that completely inhibited growth. Data are shown at 24 hours for I. orientalis and A. niger and at 48 hours for F. neoformans and P. chrysogenum. (B) Hemolytic activity of 1, 4b, 2b and 3b. Concentration in μM of the polyenes for 50% and 100% hemolysis is expressed. The standard deviation is shown in parentheses.
Fig 4.
Relative antifungal activity and toxicity.
(A) Number of times that 1 is more active against all the fungi tested and toxic in terms of hemolytic activity than the polyenes 4b, 2b and 3b. (B) Ratio between relative toxicity and relative antifungal activities. The values indicate the times that the polyene is less toxic than 1 for the same level of inhibition against the fungi tested.
Fig 5.
Proposed model for 3a and 3b biosynthesis.