Investigation of halogenation during the biosynthesis of ramoplanin in Actinoplanes sp. ATCC33076.

Ramoplanin and enduracidin are lipopeptide antibiotics effective against Gram-positive pathogens, which share close similarity in structure and biosynthetic pathway. Both compounds have chlorine atoms attached to 4-hydroxyphenylglycine (Hpg) but with different chlorinating sites and levels. Here, to probe the factor affecting the site and level of halogenation, gene inactivation and heterologous expression were carried out in Actinoplanes sp. ATCC33076 by homologous recombination. Metabolite analysis confirmed that ram20 encodes the only halogenase in ramoplanin biosynthetic pathway, and enduracidin halogenase End30 could heterologously complement the ram20-deficient mutant. Additionally, the mannosyltransferase-deficient mutant produces a dichlorinated ramoplanin aglycone with the halogenation site at Hpg(13). This study has refined our understanding of how halogenation occurs in ramoplanin biosynthetic pathway, and lays the foundation for further exploitation of ramoplanin and enduracidin halogenase in combinatorial biosynthesis.

Functional identification of the gene encoding the enzyme involved in mannosylation in ramoplanin biosynthesis in Actinoplanes sp.

Ramoplanin is a lipopeptide antibiotic active against multi-drug-resistant, Gram-positive pathogens. Structurally, it contains a di-mannose moiety attached to the peptide core at Hpg(11). The biosynthetic gene cluster of ramoplanin has already been reported and the assembly of the depsipeptide has been elucidated but the mechanism of transferring sugar moiety to the peptide core remains unclear. Sequence analysis of the biosynthetic gene cluster indicated ramo-orf29 was a mannosyltransferase candidate. To investigate the involvement of ramo-orf29 in ramoplanin biosynthesis, gene inactivation and complementation have been conducted in Actinoplanes sp. ATCC 33076 by homologous recombination. Metabolite analysis revealed that the ramo-orf29 inactivated mutant produced no ramoplanin but the ramoplanin aglycone. Thus, ramo-orf29 codes for the mannosyltransferase in the ramoplanin biosynthesis pathway. This lays the foundation for further exploitation of the ramoplanin mannosyltransferase and aglycone in combinatorial biosynthesis.