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.