Production of the Ramoplanin Activity Analogue by Double Gene Inactivation

Abstract

Glycopeptides such as vancomycin and telavancin are essential for treating infections caused by Gram-positive bacteria. But the dwindling availability of new antibiotics and the emergence of resistant bacteria are making effective antibiotic treatment increasingly difficult. Ramoplanin, an inhibitor of bacterial cell wall biosynthesis, is a highly effective antibiotic against a wide range of Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus, vancomycin-intermediate resistant Clostridium difficile and vancomycin-resistant Enterococcus sp. Here, two tailoring enzyme genes in the biosynthesis of ramoplanin were deleted by double in-frame gene knockouts to produce new ramoplanin derivatives. The deschlororamoplanin A2 aglycone was purified and its structure was identified with LC-MS/MS. Deschlororamoplanin A2 aglycone and ramoplanin aglycone showed similar activity to ramoplanin A2. The results showed that α-1,2-dimannosyl disaccharide at Hpg11 and chlorination at Chp17 in the ramoplanin structure are not essential for its antimicrobial activity. This work provides new precursor compounds for the semisynthetic modification of ramoplanin.

Enzymatic Halogenation and Dehalogenation Reactions: Pervasive and Mechanistically Diverse

“…164–166 However, unlike 3 , chlorination is not essential for the bioactivity of 47 . 167 In a recent study using degenerate primers to screen for FDHs in mangrove actinomycetes, gene clusters for the production of 48 and ansamacrolactams were detected and novel producers of these antibiotics were identified. 168 Using a similar approach but for Arctic marine actinomycetes, a homolog of the FDH Asm12 was identified, among several other FDHs that likely participate in the biosynthesis of as yet unidentified halogenated natural products.…”

Development of Halogenase Enzymes for Use in Synthesis

“…[212][213][214][215] Despite exhibiting high sequence similarity, End30 is responsible for dichlorination of Hpg13 in enduracidin, whilst Ram20 monohalogenates the Hpg17 residue of ramoplanin. Interestingly, deletion of ram20 from the ramoplanin biosynthetic gene cluster and complementation with end30 resulted in mono-halogenation of Hpg17, whilst the same complementation experiment in a strain lacking the ramoplanin mannosyltransferase resulted in halogenation of Hpg13.…”

Discovery of Six Ramoplanin Family Gene Clusters and the Lipoglycodepsipeptide Chersinamycin**

“…Structure-activity relationship (SAR) studies have illuminated several shared structural features of these antibiotics that are critical for lipid II binding and antimicrobial activity: 1) Conserved amino acid type and stereochemistry within the 17-residue depsipeptide, which influences the overall peptide receptor-like conformation, [20][21][22][23] promotes antibiotic dimerization, [21,24,25] and facilitates binding to its lipid II target; [5,7,26,27] 2) Conformational constraint imparted by the 49-atom macrocycle; [26][27][28][29][30][31][32] and 3) N-terminal acylation, which promotes bacterial membrane association and influences its amphipathic C2 symmetrical dimeric conformation that is adopted upon membrane binding (Figure 1). [21,22] Conversely, features such as mannosylation, [33,34] halogenation, [35][36][37] and desaturation of the N-acyl lipid [12,38] have been shown dispensable for activity. We correlated the most functionally important architectural features with corresponding BGC-encoded enzymes that are responsible for their assembly to identify a minimum suite of probes for genome mining for new ramoplanin congeners.…”