Using a Reverse Genetics approach the 66-kb gene cluster encoding the biosynthesis of balhimycin was identified and sequenced. By a combination of genetics, biochemistry and analytical organic chemistry the biosynthetic pathway was elucidated and functions were assigned to almost all genes of the cluster.

The biosynthesis commences with the pathway-specific provision of the non-proteinogenic amino acids ß-hydroxytyrosine (ß-Ht), hydroxyphenylglycine (Hpg) and dihydroxyphenylglycine (Dpg) which form together with (N-methyl)-leucine and asparagine the heptapeptide backbone of balhimycin. The amino acids are linked by non-ribosomal peptide synthetases (NRPS), and the aromatic side chains are interconnected by P450 monooxygenases in a stepwise fashion. The NADH/FAD-dependent halogenase BhaA catalyzes the chlorination of the two ß-hydroxytyrosines in the peptide backbone. The resulting cross-linked heptapeptide is then finally modified by methylation and glycosylation.

The substrates of the oxygenases as well as of the halogenase are not free biosynthetic precursors, but rather intermediates which are bound to the NRPS. For the efficient excretion of balhimycin an ABC-transporter is required. A StrR-type regulator is involved in the transcriptional control of the biosynthetic genes.

Non-producing mutants were used as recipients in cloning experiments as well as in approaches of precursor-directed biosynthesis by feeding chemically synthesized alternative precursors. Thus novel balhimycin derivatives were generated.