<jats:sec><jats:label /><jats:p>Molecular cloning of the biosynthetic gene cluster involved in the production of free 4‐chlorothreonine in <jats:italic><jats:styled-content style="fixed-case">S</jats:styled-content>treptomyces</jats:italic> sp. <jats:styled-content style="fixed-case">OH</jats:styled-content>‐5093 showed the presence of six <jats:styled-content style="fixed-case">ORF</jats:styled-content>s: <jats:styled-content style="fixed-case"><jats:italic>thr1</jats:italic></jats:styled-content>,<jats:styled-content style="fixed-case"> <jats:italic>thr2</jats:italic></jats:styled-content>,<jats:styled-content style="fixed-case"> <jats:italic>thr3</jats:italic></jats:styled-content>,<jats:styled-content style="fixed-case"> <jats:italic>orf1</jats:italic></jats:styled-content>,<jats:styled-content style="fixed-case"> <jats:italic>orf2</jats:italic></jats:styled-content> and <jats:styled-content style="fixed-case"><jats:italic>thr4</jats:italic></jats:styled-content>. According to bioinformatic analysis, <jats:styled-content style="fixed-case"><jats:italic>thr1</jats:italic></jats:styled-content>,<jats:styled-content style="fixed-case"> <jats:italic>thr2</jats:italic></jats:styled-content>,<jats:styled-content style="fixed-case"> <jats:italic>thr3</jats:italic></jats:styled-content> and <jats:styled-content style="fixed-case"><jats:italic>thr4</jats:italic></jats:styled-content> encode a free‐standing adenylation domain, a carrier protein, an <jats:styled-content style="fixed-case">F</jats:styled-content>e(II) nonheme α‐ketoglutarate‐dependent halogenase and a thioesterase, respectively, indicating the role of these genes in the activation and halogenation of threonine and the release of 4‐chlorothreonine in a pathway closely reflecting the formation of this amino acid in the biosynthesis of the lipodepsipeptide syringomycin from <jats:italic><jats:styled-content style="fixed-case">P</jats:styled-content>seudomonas syringae</jats:italic> pv. <jats:italic>syringae </jats:italic><jats:styled-content style="fixed-case">B</jats:styled-content>301<jats:styled-content style="fixed-case">DR</jats:styled-content>. <jats:styled-content style="fixed-case"><jats:italic>Orf1</jats:italic></jats:styled-content> and <jats:styled-content style="fixed-case"><jats:italic>orf2</jats:italic></jats:styled-content> show sequence similarity with alanyl/threonyl‐t<jats:styled-content style="fixed-case">RNA</jats:styled-content> synthetases editing domains and drug metabolite transporters, respectively. We show that <jats:styled-content style="fixed-case"><jats:italic>thr3</jats:italic></jats:styled-content> can replace the halogenase gene <jats:styled-content style="fixed-case"><jats:italic>syrB2</jats:italic></jats:styled-content> in the biosynthesis of syringomycin, by functional complementation of the mutant <jats:italic><jats:styled-content style="fixed-case">P</jats:styled-content>. s</jats:italic>. pv. <jats:italic>syringae</jats:italic> strain <jats:styled-content style="fixed-case">BR</jats:styled-content>135<jats:styled-content style="fixed-case">A</jats:styled-content>1 inactivated in <jats:styled-content style="fixed-case"><jats:italic>syrB2</jats:italic></jats:styled-content>. We also provide an insight into the structure–function relationship of halogenases <jats:styled-content style="fixed-case">T</jats:styled-content>hr3 and <jats:styled-content style="fixed-case">S</jats:styled-content>yr<jats:styled-content style="fixed-case">B</jats:styled-content>2 using homology modelling and site‐directed mutagenesis.</jats:sec><jats:sec><jats:title>Database</jats:title><jats:p>Nucleotide sequence data have been deposited in the ebi.ac.uk/<jats:styled-content style="fixed-case">EMBL</jats:styled-content>/GenBank databases under accession numbers: <jats:styled-content style="fixed-case"><jats:italic>thr1</jats:italic></jats:styled-content> (gi:378781338|<jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/CCF23454">CCF23454</jats:ext-link>); <jats:styled-content style="fixed-case"><jats:italic>thr2</jats:italic></jats:styled-content> (gi:378781340|<jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/CCF23456">CCF23456</jats:ext-link>); <jats:styled-content style="fixed-case"><jats:italic>thr3</jats:italic></jats:styled-content> (gi:378781341|<jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/CCF23457">CCF23457</jats:ext-link>); <jats:styled-content style="fixed-case"><jats:italic>orf1</jats:italic></jats:styled-content> (gi:378781342|<jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/CCF23458">CCF23458</jats:ext-link>); <jats:styled-content style="fixed-case"><jats:italic>orf2</jats:italic></jats:styled-content> (gi:378781343|<jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/CCF23459">CCF23459</jats:ext-link>); <jats:styled-content style="fixed-case"><jats:italic>thr4</jats:italic></jats:styled-content> (gi:378781344|<jats:ext-link xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/CCF23460">CCF23460</jats:ext-link>)</jats:sec>