Studies on the biosynthesis of antibiotic nystatin with emphasis on regulation

Nystatin is an antifungal polyene macrolide antibiotic produced by Streptomyces noursei, first described in 1950 (Hazen and Brown, 1950). Nystatin is currently being used for treatment of superficial fungal infections, and is considered a medically important drug. However, until recently, nothing was known about the biosynthesis of this antibiotic in S. noursei. This study is the part of the detailed investigation of the genetics and biochemistry of nystatin biosynthesis with emphasis on the regulation. First, the pleiotropic regulatory gene locus from S.noursei capable of enhancing actinorhodin (Act) production in S.lividans was cloned and sequenced. Two genes, designated ssmA and ssmB, have been suggested to be responsible for the phenomenon. Putative product of ssmA showed limited homology to the peptide encoded by afsR2, known as a pleiotropic regulator from S.lividans and S. coelicolor. Recombinant S.lividans strains carrying deletion derivatives of the locus were tested for Act production. The results of these experiments showed that ssmA is required for Act overproduction, while ssmB is possibly involved in the negative regulation of antibiotic production. Further experiments suggested that ssmA is involved in the carbon source-dependent regulation of nystatin production in S.noursei. Next, the entire nystatin biosynthetic gene cluster from S.noursei was cloned and sequenced, putative functions for the biosynthetic genes were implied, and a model for the nystatin biosynthesis was suggested. Six genes encoding PKS type I, genes for posttranslational modifications of nystatin aglycon, efflux of antibiotic out of the cell, and putative clusterspecific regulatory genes have been identified in the cluster. Inactivation of PKS-encoding genes in the cluster resulted in nystatin non-producing mutants, confirming their roles in the biosynthesis of this antibiotic. Genes presumably encoding nystatin efflux pump were studied via gene inactivation and analysis of resulting mutants. It was shown that the efflux is tightly linked to C-10 hydroxylation of the nystatin macrolactone ring. Several genes for post-PKS modifications have been found in the nystatin cluster, among them three genes for synthesis and attachment of mycosamine moiety to the nystatin aglycon. Effect of inactivation of these genes on nystatin biosynthesis was studied. Combined, these results have helped to refine the model of nystatin biosynthesis. The regulatory locus of 6 genes has been found on a right flank of nystatin biosynthetic cluster. Four of them were shown by gene inactivation to be directly involved in the regulation of nystatin biosynthesis. Promoter-probe studies revealed the main targets of regulation in the nystatin gene cluster, and cross-complementation experiments allowed establishing the hierarchy among the regulators. Finally, the model for regulatory cascade was suggested. The results of studies described above provided important information needed for rational engineering of novel polyene macrolides by manipulation of the nystatin biosynthetic genes. Seven analogs of nystatin with altered polyol region and carboxylic group have been obtained and subjected to in vitro antifungal and hemolitic activities tests. It was shown that combinations of several mutations could be beneficial for the activity-toxicity properties of the new compounds. The two most active and less toxic analogs were chosen for in vivo tests in a mouse model, where they proved to be considerably less toxic and at least as active as amphotericin B, the antifungal antibiotic used for treatment of systemic fungal infections. These results indicate that two obtained nystatin analogs can be used for further development into antifungal drugs for human therapy, and that genetic engineering is an effective tool for obtaining new compounds with improved therapeutic properties