<jats:title>ABSTRACT</jats:title> <jats:p> The emergence of antibiotic-resistant pathogenic bacteria within the last decades is one reason for the urgent need for new antibacterial agents. A strategy to discover new anti-infective compounds is the evaluation of the genetic capacity of secondary metabolite producers and the activation of cryptic gene clusters (genome mining). One genus known for its potential to synthesize medically important products is <jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">Amycolatopsis</jats:named-content> . However, <jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">Amycolatopsis japonicum</jats:named-content> does not produce an antibiotic under standard laboratory conditions. In contrast to most <jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">Amycolatopsis</jats:named-content> strains, <jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">A. japonicum</jats:named-content> is genetically tractable with different methods. In order to activate a possible silent glycopeptide cluster, we introduced a gene encoding the transcriptional activator of balhimycin biosynthesis, the <jats:italic>bbr</jats:italic> gene from <jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">Amycolatopsis balhimycina</jats:named-content> ( <jats:italic>bbr</jats:italic> <jats:sub>Aba</jats:sub> ), into <jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">A. japonicum</jats:named-content> . This resulted in the production of an antibiotically active compound. Following whole-genome sequencing of <jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">A. japonicum</jats:named-content> , 29 cryptic gene clusters were identified by genome mining. One of these gene clusters is a putative glycopeptide biosynthesis gene cluster. Using bioinformatic tools, ristomycin (syn. ristocetin), a type III glycopeptide, which has antibacterial activity and which is used for the diagnosis of von Willebrand disease and Bernard-Soulier syndrome, was deduced as a possible product of the gene cluster. Chemical analyses by high-performance liquid chromatography and mass spectrometry (HPLC-MS), tandem mass spectrometry (MS/MS), and nuclear magnetic resonance (NMR) spectroscopy confirmed the <jats:italic>in silico</jats:italic> prediction that the recombinant <jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">A. japonicum</jats:named-content> /pRM4- <jats:italic>bbr</jats:italic> <jats:sub>Aba</jats:sub> synthesizes ristomycin A.