Antimicrobial activity of Actinobacteria isolated from dry land soil in Yazd, Iran.

Historically, many important secondary metabolites including antibiotics used in clinic are purified from the cultural broths of Actinobacteria, which were inhabited in soil. Yazd is located in the center of Iran, the south of the Dasht-e Kavir and the west of the Dasht-e Lut; accordingly it has a hot, dry climate with long summers. In the present study, 18 strains of Actinobacteria isolated from 60 soil samples from Yazd-Iran. Pure isolates were screened for antibacterial activity against the ATCC strains by using two methods: single line streak method and spot inoculation method. ATCC strains include four antibiotic resistant ATCC strains (Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae and, Acinetobacter baumannii) and three antibiotic sensitive strains (Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli) and, Bacillus subtilis. Seven isolates exhibited antimicrobial activity against the ATCC strains (38.8%). Identification of type I and type II polyketide synthases (pksI, pksII) and nonribosomal peptide synthetase (NRPS) genes were done for these 7 isolates and all of 7 strains, possessed at least one of these genes. The results of this study confirm that soil Actinobacteria bear a great ability to produce antibacterial compounds against resistant and sensitive test organisms.

Identification and Characterization of a New Type III Polyketide Synthase from a Marine Yeast, Naganishia uzbekistanensis.

A putative Type III Polyketide synthase (PKSIII) encoding gene was identified from a marine yeast, Naganishia uzbekistanensis strain Mo29 (UBOCC-A-208024) (formerly named as Cryptococcus sp.) isolated from deep-sea hydrothermal vents. This gene is part of a distinct phylogenetic branch compared to all known terrestrial fungal sequences. This new gene encodes a C-terminus extension of 74 amino acids compared to other known PKSIII proteins like Neurospora crassa. Full-length and reduced versions of this PKSIII were successfully cloned and overexpressed in a bacterial host, Escherichia coli BL21 (DE3). Both proteins showed the same activity, suggesting that additional amino acid residues at the C-terminus are probably not required for biochemical functions. We demonstrated by LC-ESI-MS/MS that these two recombinant PKSIII proteins could only produce tri- and tetraketide pyrones and alkylresorcinols using only long fatty acid chain from C8 to C16 acyl-CoAs as starter units, in presence of malonyl-CoA. In addition, we showed that some of these molecules exhibit cytotoxic activities against several cancer cell lines.

Investigation of bacteria with polyketide synthase genes and antimicrobial activity isolated from South China Sea sponges.

AIMS: To obtain bacteria with PKS (polyketide synthase) genes and antimicrobial activity from sponges. METHODS AND RESULTS: Eighteen bacteria with KS (ketosynthase) genes were identified by polymerase chain reaction (PCR) screening of 98 isolates from South China Sea sponges, Stelletta tenuis, Halichondria rugosa, Dysidea avara and Craniella australiensis. 16S rRNA gene-based Blast analysis indicated that 15 isolates belonged to the phylum Firmicutes, among which 14 isolates were closely related to genus Bacillus, and 1 to Staphylococcus lentus. Two isolates were identified as actinomycetes, and one as Alcaligenes sp. in the phylum Proteobacteria. The 18 KS domains belong to trans-AT type I PKS and match PKS of marine bacterial symbionts. The 18 bacteria exhibited broad-spectrum antimicrobial activities against fungi, gram-positive and gram-negative bacteria. A 21.8-kb PKS gene cluster fragment containing five modules was isolated from the Staphylococcus lentus isolate A75 by screening of a fosmid library. CONCLUSIONS: The PKS gene diversity and different antimicrobial spectra indicate the potential of bacteria associated with South China Sea sponges for diverse polyketide production. SIGNIFICANCE AND IMPACT OF THE STUDY: Combined with bioactivity assay the PKS gene-based approach can be applied to efficient screening of strains of pharmaceutical value and the prediction of related compounds.

Combinatorial biosynthesis--potential and problems.

Because of their ecological functions, natural products have been optimized in evolution for interaction with biological systems and receptors. However, they have not necessarily been optimized for other desirable drug properties and thus can often be improved by structural modification. Using examples from the literature, this paper reviews the opportunities for increasing structural diversity among natural products by combinatorial biosynthesis, i.e., the genetic manipulation of biosynthetic pathways. It distinguishes between combinatorial biosynthesis in a narrower sense to generate libraries of modified structures, and metabolic engineering for the targeted formation of specific structural analogs. Some of the problems and limitations encountered with these approaches are also discussed. Work from the author's laboratory on ansamycin antibiotics is presented which illustrates some of the opportunities and limitations.

Modular polyketide synthases: Investigating intermodular communication using 6 deoxyerythronolide B synthase module 2.

A novel variant of 6-deoxyerythronolide B synthase (DEBS) module 2 was constructed to explore the balance between protein-protein-mediated intermodular channeling and intrinsic substrate specificity within DEBS. This construct, termed (N3)Mod2+TE, was co-incubated with a complementary, donor form of the same module, (N5)Mod2(C2), as well as with a mutant of (N5)Mod2(C2) with an inactive ketosynthase domain, in order to determine the extent of intermediate channeling versus substrate diffusion into the downstream module.