Selected Derivatives of Erythromycin B-In Silico and Anti-Malarial Studies

Erythromycin A is an established anti-bacterial agent against Gram-positive bacteria, but it is unstable to acid. This led to an evaluation of erythromycin B and its derivatives because these have improved acid stability. These compounds were investigated for their anti-malarial activities, by their in silico molecular docking into segments of the exit tunnel of the apicoplast ribosome from Plasmodium falciparum. This is believed to be the target of the erythromycin A derivative, azithromycin, which has mild anti-malarial activity. The erythromycin B derivatives were evaluated on the multi-drug (chloroquine, pyrimethamine, and sulfadoxine)-resistant strain K1 of P. falciparum for asexual growth inhibition on asynchronous culture. The erythromycin B derivatives were identified as active in vitro inhibitors of asexual growth of P. falciparum with low micro-molar IC50 values after a 72 h cycle. 5-Desosaminyl erythronolide B ethyl succinate showed low IC50 of 68.6 µM, d-erythromycin B 86.8 µM, and erythromycin B 9-oxime 146.0 µM on the multi-drug-resistant K1 of P. falciparum. Based on the molecular docking, it seems that a small number of favourable interactions or the presence of unfavourable interactions of investigated derivatives of erythromycin B with in silico constructed segment from the exit tunnel from the apicoplast of P. falciparum is the reason for their weak in vitro anti-malarial activities.

Multiplexed Integrating Plasmids for Engineering of the Erythromycin Gene Cluster for Expression in Streptomyces spp. and Combinatorial Biosynthesis

ABSTRACTBacteria in the genusStreptomycesand its close relatives are prolific producers of secondary metabolites with antibiotic activity. Genome sequencing of these bacteria has revealed a rich source of potentially new antibiotic pathways, whose products have never been observed. Moreover, these new pathways can provide novel genes that could be used in combinatorial biosynthesis approaches to generate unnatural analogues of existing antibiotics. We explore here the use of multiple orthologous integrating plasmid systems, based on theint/attPloci from phages TG1, SV1, and ϕBT1, to express the polyketide synthase (PKS) for erythromycin in a heterologousStreptomyceshost.Streptomycesstrains containing the three polyketide synthase geneseryAI,eryAII, anderyAIIIexpressed from three different integrated plasmids produced the aglycone intermediate, 6-deoxyerythronolide B (6-dEB). A further pair of integrating plasmids, both derived from the ϕC31int/attPlocus, were constructed carrying a gene cassette for glycosylation of the aglycone intermediates, with or without the tailoring gene,eryF, required for the synthesis of erythronolide B (EB). Liquid chromatography-mass spectrometry of the metabolites indicated the production of angolosaminyl-6-dEB and angolosaminyl-EB. The advantages of using multiplexed integrating plasmids for engineering expression and for combinatorial biosynthesis were demonstrated.

Functional Analysis of OleY l-Oleandrosyl 3-O-Methyltransferase of the Oleandomycin Biosynthetic Pathway in Streptomyces antibioticus

ABSTRACT Oleandomycin, a macrolide antibiotic produced byStreptomyces antibioticus, contains two sugars attached to the aglycon: l-oleandrose and d-desosamine.oleY codes for a methyltransferase involved in the biosynthesis of l-oleandrose. This gene was overexpressed in Escherichia coli to form inclusion bodies and inStreptomyces lividans, producing a soluble protein.S. lividans overexpressing oleY was used as a biotransformation host, and it converted the precursorl-olivosyl-erythronolide B into its 3-O-methylated derivative,l-oleandrosyl-erythronolide B. Two other monoglycosylated derivatives were also substrates for the OleY methyltransferase:l-rhamnosyl- and l-mycarosyl-erythronolide B. OleY methyltransferase was purified yielding a 43-kDa single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native enzyme showed a molecular mass of 87 kDa by gel filtration chromatography, indicating that the enzyme acts as a dimer. It showed a narrow pH range for optimal activity, and its activity was clearly stimulated by the presence of several divalent cations, being maximal with Co2+. The S. antibioticus OleG2 glycosyltransferase is proposed to transfer l-olivose to the oleandolide aglycon, which is then converted intol-oleandrose by the OleY methyltransferase. This represents an alternative route for l-oleandrose biosynthesis from that in the avermectin producer Streptomyces avermitilis, in which l-oleandrose is transferred to the aglycon by a glycosyltransferase.