Biosynthesis of the polyene antifungal antibiotic …
Hybrids C and D were tested for restoration of the polyene macrolide production in the rimA-deficient mutant of S. diastaticus. Both hybrid proteins differ only in the AT domains, which originate from NysA and RimA in hybrids C and D, respectively (). The corresponding hybrid genes were placed under the control of the ermEP* promoter in several steps as described in Table S1 in the supplemental material. The resulting plasmids (pLEC104C and pLEC106D for the hybrids C and D, respectively) were used to transform protoplasts of a new rimA disruptant (S. diastaticus variant 108::PM1/pLEC101). Several candidates were selected and confirmed by direct plasmid extraction (for the presence of recombinant plasmid) and by Southern blotting for the correct integration of PM1/pLEC101 phage into the chromosome. The HPLC analysis of the fermentation broth of the recombinant strains showed restoration of CE-108D, CE-108, rimocidin D, and rimocidin production in both cases (C and D), as evident from the appearance of peaks eluting, respectively, at ca. 9.5 min, 10.5 min, 11.5 min, and 12.3 min. The identities of these metabolites have previously been confirmed by LC-mass spectrometry (LC-MS) analyses of the extract from the wild-type S. diastaticus variant 108 (E. M. Seco et al., unpublished data). Some additional peaks appeared on the chromatogram within the ca. 2- to 8-min elution time upon complementation with hybrid C. However, we could not correlate these peaks with polyene UV spectra and do not know at present what they represent. In any case, successful complementation of the S. diastaticus rimA mutant with hybrids C and D strongly suggests that the AT0 domain of NysA under heterologous conditions is able to recognize and recruit both malonyl-CoA and butyryl-CoA. Moreover, hybrids C and D could functionally interact with the RimB PKS, which was surprising considering the presence of an intact NysA C terminus on both hybrids and the lack of such interaction between NysA and RimB (see above).
Molecular Control of Polyene Macrolide Biosynthesis
among polyene biosynthetic gene clusters
Thesis title: "Hybrid peptide-polyketide natural product biosynthesis : resistance to the bleomycin family of antitumor antibiotics, beta-amino acid activating adenylation domains, and oxazolomycin polyketide synthases that require discrete aclytransferases for two distinct extender units"
Polyene antibiotic biosynthesis gene clusters.
FscP encoded by the FR-008/candicidin gene cluster is highly homologous with its counterparts AmphN, NysN, and PimG of the other polyene pathways (, , -, ). FscP and FscFE (carrying the electron transfer of ferredoxin in the P450 system) represent a P450 monooxygenase system that is postulated to be responsible for the oxidation of the methyl branch into a carboxyl group, presumably introduced in the 13th elongation step by the methylmalonate-specific AT13 of FscD (Fig. ). It also remains ambiguous whether an additional oxidoreductase is required to convert a methyl branch into a carboxyl group. Our objective was to inactivate the fscP gene as an initial step toward understanding its functional role(s).