The objectives of this proposal are to understand key aspects of the multicomponent assembly line of enzymes that produce the important antibiotic vancomycin and its congeners. A cluster of almost 30 genes encodes enzymes that provide dedicated monomers to be incorporated, enzymes that constitute a 27 step assembly line for producing the D-D-L-D-D-L-L- acyclic heptapeptide scaffold and tailoring enzymes that crosslink and glycosylate the peptide backbone to create the active antibiotic. Specific aim 1 proposes continued study of the biogenesis of 3,,5-dihydroxyphenyiglycine (Dpg), a key residue for subsequent crosslinking of the scaffold, and also the 3-OH-meta-chlorotyrosines incorporated at residues 2 and 6 of the peptide. Specific aim 2 examines the third subunit CepC of the chloroeremomycin assembly line and congeneric assembly lines (vancomycin, teicoplanin) that contains the seventh (last) module. This chain termination modules activates and incorporates Dpg as the terminal residue of the heptapeptidyl chain and then releases this full length peptide from its covalent attachment to the last carrier protein domain, PCP7, by action of the thioesterase domain in CepC. Aim 3 of the proposal studies two types of post-assembly line tailoring enzymes. The first group are hemeprotein oxidases involved in making the three crosslinks in vancomycin (2-4, 4-6, 5-7) and the four crosslinks in teicoplanin (2-4, 4-6, 5-7, 1-3), creating the rigidified peptide skeleton required for recognition of bacterial cell wall peptidoglycan. The second group is the acyl transferases that act on the teicoplanin subgroup to convert the glycopeptides to lipoglycopeptides, modifications that provide regain of potency against some phenotypic forms of vancomycin resistant enterococci. The knowledge base gained in these three aims will provide insight into molecular logic to enable subsequent combinatorial biosynthetic antibiotic variants.