Search

Polyene macrolides are important antibiotics used to treat fungal infections in humans. In this work, acyltransferase (AT) domain swaps, mutagenesis, and cross-complementation with heterologous polyketide synthase domain (PKS) loading modules were performed in order to facilitate production of new analogues of the polyene macrolide nystatin. Replacement of AT 0 in the nystatin PKS loading module NysA with the propionate-specific AT 1 from the nystatin PKS NysB, construction of hybrids between NysA and the loading module of rimocidin PKS RimA, and stepwise exchange of specific amino acids in the AT 0 domain by site-directed mutagenesis were accomplished. However, none of the NysA mutants constructed was able to initiate production of new nystatin analogues. Nevertheless, many NysA mutants and hybrids were functional, providing for different levels of nystatin biosynthesis. An interplay between certain residues in AT 0 and an active site residue in the ketosynthase (KS)-like domain of NysA in initiation of nystatin biosynthesis was revealed. Some hybrids between the NysA and RimA loading modules carrying the NysA AT 0 domain were able to prime rimocidin PKS with both acetate and butyrate units upon complementation of a rimA -deficient mutant of the rimocidin/CE-108 producer Streptomyces diastaticus . Expression of the PimS0 loading module from the pimaricin producer in the same host, however, resulted in production of CE-108 only. Taken together, these data indicate relaxed substrate specificity of NysA AT 0 domain, which is counteracted by a strict specificity of the first extender module KS domain in the nystatin PKS of Streptomyces noursei .

The growth inhibitory effects on Trypanosoma cruzi of several natural tetraene macrolides and their derivatives were studied and compared with that of amphotericin B. All tetraenes strongly inhibited in vitro multiplication. Proliferation of epimastigotes was arrested by all these drugs at < or =3.6 microM, which were also active on amastigotes proliferating in fibroblasts. Compared with amphotericin B, the compounds were less effective but also less toxic, showing no effect on the proliferation of J774 and NCTC 929 mammalian cells at concentrations active against the parasites. CE-108B (a polyene amide) appeared to be an especially potent trypanocidal compound, with strong in vivo trypanocidal activity and very low or no toxic side effects, and thus should be considered for further studies.

Streptomyces diastaticus var. 108, a newly isolated strain [1], produces two closely related tetraene macrolides (rimocidin and CE-108) as well as oxytetracycline. A region of 19,065 base pairs of DNA from the S. diastaticus var. 108 genome was isolated, sequenced, and characterized. Ten complete genes and one truncated ORF were located. Disruption of these genes proved that this genomic region is part of the biosynthetic cluster for the two tetraenes. The choice of starter units by the loading module and the in vivo availability of the starter metabolites are crucial for the final ratio of the two macrolides. A second type I PKS, unrelated to tetraene biosynthesis, was also identified; disruption of these genes suggests that they would code for enzymes involved in the biosynthesis of a polyketide that might compete metabolically with rimocidin production.

Here we report the systematic study of the anti-trypanocidal activity of some new products derived from S. diastatus on 14 different T. cruzi strains spanning the six genetic lineages of T. cruzi. As the traditional growth inhibition curves giving similar IC 50 showed great differences on antibiotic and lineage tested, we decided to preserve the wealth of information derived from each inhibition curve and used an algorithm related to potency of the drugs, combined in a matrix data set used to generate a cluster tree. The cluster thus generated based just on drug susceptibility data closely resembles the phylogenies of the lineages derived from genetic data and provides a novel approach to correlate genetic data with phenotypes related to pathogenesis of Chagas disease. Furthermore we provide clues on the drugs mechanism of action. © 2012 Aquilino et al., Fondo de Investigaciones Sanitarias-Ministerio de Sanidad (FIS-PI08/0960), (FIS-PI08/0101); ChagasEpiNet (European VII framework Program); Network RICET (Red Investigacion Cooperativa en Enfermedades Tropicales); FIS (Fondo de Investigaciones Sanitarias); Ministerio de Sanidad; Fundacion Ramon Areces

Two structurally related polyene macrolides are produced by Streptomyces diastaticus var. 108: rimocidin (3a) and CE-108 (2a). Both bioactive metabolites are biosynthesized from the same pathway through type I polyketide synthases by choosing a starter unit either acetate or butyrate, resulting in 2a or 3a formation, respectively. Two additional polyene amides, CE-108B (2b) and rimocidin B (3b), are also produced “in vivo” when this strain was genetically modified by transformation with engineered SCP2*-derived vectors carrying the ermE gene. The two polyene amides, 2b and 3b, showed improved pharmacological properties, and are generated by a tailoring activity involved in the conversion of the exocyclic carboxylic group of 2a and 3a into their amide derivatives. The improvement on some biological properties of the resulting polyenes, compared with that of the parental compounds, encourages our interest for isolating the tailoring gene responsible for the polyene carboxamide biosynthesis, aimed to use it as tool for generating new bioactive compounds. In this work, we describe the isolation from S. diastaticus var. 108 the corresponding gene, pcsA, encoding a polyene carboxamide synthase, belonging to the Class II glutamine amidotransferases and responsible for “in vivo” and “in vitro” formation of CE-108B (2b) and rimocidin B (3b). The fermentation broth from S. diastaticus var. 108 engineered with the appropriate pcsA gene construction, showed the polyene amides to be the major bioactive compounds.

The rimJ gene, which codes for a crotonyl-CoA carboxylase/reductase, lies within the biosynthetic gene cluster for two polyketides belonging to the polyene macrolide group (CE-108 and rimocidin) produced by Streptomyces diastaticus var. 108. Disruption of rimJ by insertional inactivation gave rise to a recombinant strain overproducing new polyene derivatives besides the parental CE-108 (2a) and rimocidin (4a). The structure elucidation of one of them, CE-108D (3a), confirmed the incorporation of an alternative extender unit for elongation step 13. Other compounds were also overproduced in the fermentation broth of rimJ disruptant. The new compounds are in vivo substrates for the previously described polyene carboxamide synthase PcsA. The rimJ disruptant strain, constitutively expressing the pcsA gene, allowed the overproduction of CE-108E (3b), the corresponding carboxamide derivative of CE-108D (3a), with improved pharmacological properties. peerReviewed

Summary We recently characterized rimocidin B ( 3b ) and CE-108B ( 4b ) as two polyene amides with improved pharmacological properties, produced by genetically modified Streptomyces diastaticus var. 108. In this work, genetic and biochemical analysis of the producer strain show that the two amides are derived from the parental polyenes rimocidin ( 3a ) and CE-108 ( 4a ) by a post-PKS modification of the free side chain carboxylic acid. This modification is mediated by an amidotransferase activity operating after the biosynthesis of rimocidin ( 3a ) and CE-108 ( 4a ) are completed. Two polyenes, intermediates of the biosynthetic pathway of rimocidin ( 3a ) and CE-108 ( 4a ), were also isolated and shown to have some improved pharmacological properties compared with the final products.

SummaryStreptomyces diastaticus var. 108, a newly isolated strain, was recently characterized as a producer of two polyene macrolide antibiotics (rimocidin and CE-108), and the biosynthetic gene cluster was partially characterized. When the producer strain was genetically modified by transformation with some engineered SCP2*-derived vectors carrying the ermE gene, two previously uncharacterized macrolides were detected in the fermentation broth of the recombinant strain and chemically characterized as the amides of the parental polyene carboxylic acids. The biological activity and some in vitro toxicity assays showed that this chemical modification resulted in pharmaceuticals with improved biological properties compared with the parental products.

In the search for strains producing antifungal compounds, a new tetraene macrolide CE-108 (3) has been isolated from culture broth of Streptomyces diastaticus 108. In addition, the strain also produces the previously described tetraene rimocidin (1) and also the aromatic polyketide oxytetracycline. Both tetraene compounds, structurally related, are produced in a ratio between 25 to 35% (CE-108 compared to rimocidin), although it can be inverted toward CE-108 production by changing the composition of the fermentation medium. This paper deals with the characterization of the producer strain, fermentation, purification, structure determination and biological properties of the new macrolide tetraene CE-108.