Elucidation of the substrate specificity of the ABC transporter Tba uncovers an unsuspected role in the biosynthesis of glycopeptide antibiotics

Glycopeptide antibiotics (GPA) such as vancomycin and teicoplanin are essential last-resort antibiotics produced by actinomycetes. Their biosynthesis is encoded within biosynthetic gene clusters, also harboring genes for regulation, and transport. Diverse types of GPAs have been characterized that differ in peptide backbone composition and modification patterns. However, little is known about the ATP-binding cassette (ABC) transporters facilitating GPA export. Employing cutting-edge techniques, we investigated the substrate specificity of GPA ABC-transporters towards the type-I GPA, balhimycin. Phylogenetic analysis and trans-complementation experiments revealed that balhimycin can only be exported by the related type I GPA transporters Tba and Tva (transporter of vancomycin). Molecular dynamics simulations and mutagenesis experiments showed that Tba exhibits specificity towards the peptide backbone rather than the attached modifications. Unexpectedly, deletion or functional inactivation of Tba halted balhimycin biosynthesis. Combined with proximity dependent biotinylation experiments this suggested that the interaction of the active transporter with the biosynthetic machinery is required for compound production. Overall design: To investigate the differential expression of genes from the type I gylcopeptide biosynthetic gene cluster of balhimycin in a mutant lacking the correspoding ABC transporter (Tba) we analyzed RNA-seq samples from this mutant and compared the expression levels to the wildtype. As a control, we used a mutant lacking an StrR-regulator, Bbr, which regulates the overexpression of the biosynthetic genes.