The teicoplanin acyltransferase (Atf) responsible for N-acylation of the glucosamine moiety to create the teicoplanin lipoglycopeptide scaffold has recently been identified. Here we use that enzyme (tAtf) and the cognate acyltransferase from the related A-40,926 biosynthetic cluster (aAtf) to evaluate specificity for glycopeptide scaffolds and for the acyl-CoA donor. In addition to acylation of 2-aminoglucosyl glycopeptide scaffolds with k(cat) values of 400-2000 min(-1), both Atfs transfer acyl groups to regioisomeric 6-aminoglucosyl scaffolds and to glucosyl scaffolds at rates of 0.2-0.5 min(-1) to create variant lipoglycopeptides. Using the teicoplanin glycosyltransferase tGtfA, tAtf, and GtfD, a glycosyltransferase from the vancomycin producer, it is possible to assemble a novel lipoglycopeptide with GlcNAc at beta-OH-Tyr(6) and an N(6)-acyl-glucosaminyl-vancosamine at Phegly(4). This study illustrates the utility of chemo- and regioselective acyltransferases and glycosyltransferases to create novel lipoglycopeptides.