Enhanced Oxytetracycline Production by Streptomyces rimosus in Submerged Co-Cultures with Streptomyces noursei

In the present study, Streptomyces rimosus was confronted with Streptomyces noursei, Penicillium rubens, Aspergillus niger, Chaetomium globosum, or Mucor racemosus in two-species submerged co-cultures in shake flasks with the goal of evaluating the oxytetracycline production and morphological development. The co-culture of S. rimosus with S. noursei exhibited stimulation in oxytetracycline biosynthesis compared with the S. rimosus monoculture, whereas the presence of M. racemosus resulted in a delay in antibiotic production. Different strategies of initiating the “S. rimosus + S. noursei” co-cultures were tested. The improvement in terms of oxytetracycline titers was recorded in the cases where S. noursei was co-inoculated with S. rimosus in the form of spores. As the observed morphological changes were not unique to the co-culture involving S. noursei, there was no evidence that the improvement of oxytetracycline levels could be attributed mainly to morphology-related characteristics.

Bioinformatic and Reactivity-Based Discovery of Linaridins

Linaridins are members of the ribosomally synthesized and post-translationally modified peptide (RiPP) family of natural products. Five linaridins have been reported, which are defined by the presence of dehydrobutyrine, a dehydrated threonine residue. This work describes the development of a linaridin specific scoring module for Rapid ORF Description and Evaluation Online (RODEO), a genome-mining tool tailored towards RiPP discovery. Upon mining publicly accessible genomes available in the NCBI database, RODEO identified 561 (382 non-redundant) linaridin biosynthetic gene clusters (BGCs). Linaridin BGCs with unique gene architectures and precursor sequences markedly different from previous predictions were uncovered during these efforts. To aid in dataset validation, two new linaridins, pegvadin A and B, were detected through reactivity-based screening (RBS) and isolated from Streptomyces noursei and Streptomyces auratus, respectively. RBS involves the use of a reactive chemical probe that chemoselectively modifies a functional group present in the natural product. The dehydrated amino acids present in linaridins as α/β-unsaturated carbonyls were appropriate electrophiles for nucleophilic 1,4 addition using a thiol-functionalized probe. The data presented within significantly expands the number of predicted linaridin BGCs and serves as a road map for future work in the area. The combination of bioinformatics and RBS is a powerful approach to accelerate natural product discovery.

Streptomyces staurosporininus sp. nov., a staurosporine-producing actinomycete

The taxonomic position of a staurosporine-producing actinomycete isolated from a hay meadow soil was determined using a polyphasic approach. The organism had chemical and morphological characteristics consistent with its classification in the genus Streptomyces and formed a distinct branch between the Streptomyces lydicus and Streptomyces noursei clades in the 16S rRNA Streptomyces gene tree. DNA–DNA relatedness values between the isolate and its nearest phylogenetic neighbours, namely Streptomyces lydicus NBRC 13058T and Streptomyces chattanoogensis NBRC 12754T, were 53 % and 40 %, respectively. The isolate was also readily distinguished from the type strains of these species using a combination of morphological and other phenotypic properties. On the basis of these results, it is proposed that isolate BK179T ( = KACC 20912T = NRRL B-24850T) be classified as the type strain of Streptomyces staurosporininus sp. nov.

Initiation of Polyene Macrolide Biosynthesis: Interplay between Polyketide Synthase Domains and Modules as Revealed via Domain Swapping, Mutagenesis, and Heterologous Complementation

ABSTRACTPolyene 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 AT0in the nystatin PKS loading module NysA with the propionate-specific AT1from 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 AT0domain 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 AT0and 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 AT0domain were able to prime rimocidin PKS with both acetate and butyrate units upon complementation of arimA-deficient mutant of the rimocidin/CE-108 producerStreptomyces 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 AT0domain, which is counteracted by a strict specificity of the first extender module KS domain in the nystatin PKS ofStreptomyces noursei.