The nysL gene in the S. noursei nystatin biosynthetic gene cluster is located downstream of nysK, the gene encoding the last module of the nystatin PKS and a terminal thioesterase (). Amino acid sequence analysis of the deduced nysL product revealed a high degree of similarity of this polypeptide to bacterial P450 monooxygenases, including those involved in antibiotic biosynthesis (data not shown). Further phylogenetic analysis (Fig. ) revealed clustering of the NysL amino acid sequence with those of PimD and AmphL, proven to act as an epoxidase and a hydroxylase in the biosynthesis of the polyene macrolides pimaricin and amphotericin, respectively. Although we have suggested earlier that NysL is most probably a C-10 hydroxylase, the experimental evidence for this assumption was lacking. Repeated attempts to inactivate nysL by in-frame deletion via double homologous recombination in an S. noursei wild-type strain failed. We then used a previously constructed mutant that does not produce nystatin, NDA59, with an inactivated nysA gene encoding the nystatin PKS loading module, which can be complemented with the wild-type copy of nysA to restore nystatin production (). The use of the latter mutant, along with the extension of flanking regions for the gene replacement construct up to 2 kb, allowed isolation of the ΔnysL mutant designated NLD101. The scheme for NDL101 mutant construction is presented in Fig. . The deletion within the nysL gene in NLD101 was confirmed by Southern blot analysis (data not shown).
Based on our data and previous findings, a refined treatment for amphotericin B-resistant infection can be considered. If the resistance mechanism is found to involve mutation or downregulation of the ERG3 gene, flucytosine in combination with a nonazole sterol biosynthesis inhibitor would be advised. Otherwise, an azole, such as fluconazole or itraconazole, would be recommended due to azole sensitivity observed with some clinical isolates that are resistant to amphotericin B. Further studies evaluating the role of the ERG3 gene and other mechanisms in antifungal resistance are warranted to better help direct clinical therapy for patients with amphotericin B-resistant C. lusitaniae infection, but our preliminary evaluations reveal promising directions in the tailoring of therapy according to specific fungal genotypes.
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P450 monooxygenases play an important role in the biosynthesis of many secondary metabolites produced by Streptomyces bacteria. Recent advances in engineered antibiotic biosynthesis in streptomycetes make P450 monooxygenases attractive targets for genetic manipulation that can lead to the production of novel antibiotic analogues (, , , , ). The biosynthesis of all polyene macrolide antibiotics known to possess potent antifungal activity involves oxidative steps apparently catalyzed by the P450 monooxygenases (). Inactivation of the monooxygenase genes in the producers of the polyene macrolides pimaricin and amphotericin yielded new analogues of these antibiotics with properties different from those of the final products of the respective biosynthetic pathways (, , ).
The late stages of amphotericin biosynthesis ..
Based on the current model for the polyene macrolide mode of action, which involves the formation of ion-permeable channels in fungal membranes (), it could be assumed that removal of the C-10 hydroxyl group from the nystatin polyol region will decrease channel permeability. Indeed, according to the model, hydroxyl groups in the polyol region of the molecule are located on the inner part of the channel, creating a hydrophilic environment allowing ions and other small molecules to leak out of the cell. However, we have demonstrated that the antifungal activity of 10-deoxynystatin is equal to that of nystatin, at least for C. albicans. This observation was in contrast to the data for 4,5-deepoxypimaricin and 8-deoxyamphotericin B, which were obtained upon inactivation of the genes encoding P450 monooxygenases performing epoxidation and hydroxylation of pimaricin and amphotericin precursors, respectively, in polyol regions of the molecules. In both cases, a notable decrease in antifungal activity was observed (, ). One possible explanation for this phenomenon might be that the conformation of the polyol region of the nystatin molecule is different from those of pimaricin and amphotericin. This difference might result in limited participation of the nystatin C-10 hydroxyl group in channel formation and, thus, its low significance for antifungal activity.
10/02/1999 · Genetic Analysis of Azole ..
In the present study, we have characterized the P450 monooxygenase NysL encoded by the nystatin biosynthetic gene cluster in S. noursei ATCC 11455. An alignment of the NysL amino acid sequence with those for P450 enzymes known to be involved in antibiotic biosynthesis, followed by phylogenetic analysis, clearly revealed that NysL is related to PimD and AmphL, an epoxidase and a hydroxylase in the pimaricin and amphotericin biosynthetic pathways, respectively. PimD, AmphL, and NysL represent a separate clade on the phylogenetic tree which is distant from that containing P450 monooxygenases from the polyene macrolide biosynthetic pathways known or assumed to catalyze the oxidation of an exocyclic methyl group (, , ).