Caerulomycin A—An Antifungal Compound Isolated from Marine Actinomycetes

Actinomycetes have been prolific sources of novel secondary metabolites with a range of biological activities that may ultimately find application as therapeutic compounds. Hence several drug discovery companies are engaged in isolation of novel bioactive metabolites from these microbial sources. Antibiotics form the major class of such bioactive metabolites and have been widely used for treating infectious diseases. One of the most critical problems in clinical practice is the increase of prevalence of drug resistant strains, especially azole resistance among fungi. Due to this, there is a constant need for development of new antifungal antibiotics having novel scaffolds and/or mechanism of action. In our in-house screening program in the quest of novel and superior antifungal compounds, an actinomycetes strain PM0525875 was isolated from a marine invertebrate. The extracts of this microbe showed potent in-vitro antifungal activity against drug resistant fungal strains. The antifungal active peak from the extract obtained by shake flask fermentation was identified by chromatographic and other analytical techniques during bioactivity guided isolation. Later the fermentation conditions were optimized in 30 L fermentor for the production of sufficient amount antifungal compound for complete structural characterization. Consequently the fermented broth extract was subjected to bioactivity-guided fractionation, to isolate the active principle using different preparative chromatographic techniques followed by its characterization. The active principle was characterized to be Caerulomycin A. Minimum inhibitory concentration (MIC) of the compound was found in the range of 0.39 - 1.56 μg/ml against pathogenic fungal test strains. The phylogenetic analysis of producer strain using 16S rRNA sequence showed closest match with Actinoalloateichus cyanogriseus. Herewith we report the isolation of Caerulomycin A from marine invertebrate-associated Actinoalloteichus sp. using optimized medium and fermentation conditions.

Characterization of the metallo-dependent amidohydrolases responsible for“auxiliary”leucinyl removal in the biosynthesis of 2,20-bipyridine antibiotics

2,20-Bipyridine(2,20-BiPy)is an attractive core structure present in a number of biologically active natural products,including the structurally related antibiotics caerulomycins(CAEs)and collismycins(COLs).Their biosynthetic pathways share a similar key 2,20-BiPy-L-leucine intermediate,which is desulfurated or sulfurated at C5,arises from a polyketide synthase/nonribosomal peptide synthetase hybrid assembly line.Focusing on the common off-line modification steps,we here report that the removal of the“auxiliary”L-leucine residue relies on the metallo-dependent amidohydrolase activity of CaeD or ColD.This activity leads to the production of similar 2,20-BiPy carboxylate products that then receive an oxime functionality that is characteristic for both CAEs and COLs.Unlike many metallo-dependent amidohydrolase superfamily proteins that have been previously reported,these proteins(particularly CaeD)exhibited a strong zinc ion-binding capacity that was proven by site-specific mutagenesis studies to be essential to proteolytic activity.The kinetics of the conversions that respectively involve CaeD and ColD were analyzed,showing the differences in the efficiency and substrate specificity of these two proteins.These findings would generate interest in the metallo-dependent amidohydrolase superfamily proteins that are involved in the biosynthesis of bioactive natural products.