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LC-mass spectrometry (LC-MS) analyses of the extract from the wild-type S. diastaticus variant 108 (E. M. Seco et al., unpublished data).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

Two polyene amides produced by genetically modified Streptomyces diastaticus var.

among polyene biosynthetic gene clusters

Ph.D. (2008)

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"

Publications:

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).

Streptomyces nodosus host strain optimized for polyene glycosylation engineering[J].
Engineered biosynthesis of disac-charide-modified polyene macrolides[J].

The polyene antibiotics difficidin and oxydifficidin ..

There are several genes in the cluster that might encode enzymes involved in tailoring reactions on the assembled polyketide chain. SimC6 is similar to several putative KRs (), and SimC7 is most similar to SnoAW, a putative hydroxylase or DH from Streptomyces nogalater (). SimC6 and SimC7 might catalyze the KR and DH reactions needed to complete the biosynthesis of the tetraene moiety. Similar enzymes acting on the assembled polyketide have been described for the biosynthesis of avermectin (). SimC4 is similar to a putative dioxygenase from S. coelicolor A3(2) () and a neoxanthine-cleaving enzyme from Arabidopsis thaliana (), and the deduced amino acid sequence of simC5 is similar to aldehyde dehydrogenases from different sources (). Both SimC4 and SimC5 might be involved in the formation of the second carboxyl group of the tetraene moiety, which seems to be generated after the formation of the side chain and the attachment of the side chain to -olivose (Fig. ). The deduced amino acid sequences of simC2 () and simC3 () are similar to putative TEs, providing yet further examples of TE genes located in an antibiotic biosynthetic gene cluster. The deduced amino acid sequence of simC8 is similar to that of NysF, a putative phosphopantetheinyl transferase from Streptomyces noursei (), and it is also similar to Svp, a phosphopantetheinyl transferase from Streptomyces verticillus (). SimC8 might posttranslationally modify the ACP domains of SimC1A, SimC1B, and SimC1C. As Svp was characterized as a flexible phosphopantetheinyl transferase which is able to modify different types of ACPs and peptidyl carrier proteins (PCPs), we propose that SimC8 might also be involved in modifying the PCP domain of SimD6 (). Further experiments have to be performed to study the specificities of SimA11 and SimC8 and their exact functions during simocyclinone biosynthesis.

coli and the purified DNA yielded phage plaques on Streptomyces lividans after polyethylene glycol (PEG)-mediated transfection of protoplasts.

Polyene antibiotic bosynthesis gene clusters.

1. Galm, U.; Wendt-Pienkowski, E.; Wang, L.; George, N. P.; Oh, T.-J.; Yi, F.; Tao, M.; Coughlin, J. M; Shen, B. (2009) The biosynthetic gene cluster of zorbamycin, a member of the bleomycin family of antitumor antibiotics, from Streptomyces flavoviridis ATCC21892. Mol. BioSyst. 4:77-90.

Improved antifungal polyene macrolides via engineering of the nystatin biosynthetic genes in Strep-tomyces noursei[J].

the antibiotic biosynthetic gene clusters is ..

Each of the culture extracts from these four strains showed a typical polyene-like high-pressure liquid chromatography (HPLC) chromatogram profile, and strong antifungal activity against Candida albicans .