a, Four fatty acid desaturases were identified in the genome of S. cattleya and targeted for deletion. Per cent identity (%ID) and per cent similarity (%S) compared to the closest sequenced homologue are shown with the host in brackets. Number of amino acids in protein also tabulated. SCAT_0184 is adjacent to type I polyketide synthase and may be part of a biosynthetic cluster. SCAT_p1525 may also have a metabolic role, although its genomic context is not highly informative. SCAT_2136 and SCAT_4823 are clustered with proteins thought to be involved in primary metabolism and rRNA transcription. We were unable to identify any deletion strains of SCAT_2136, suggesting that SCAT_2136 may be an essential fatty acid desaturase. Knockouts of SCAT_0184, SCAT_4823, and SCAT_p1525 were found to still produce βes by CuAAC derivatization with 3-azido-7-hydroxy-coumarin followed by liquid chromatography and quadrupole time of flight (LC–QTOF) monitoring of the βes-coumarin CuAAC product (βes-Cou). The reaction in which Cu(i) is omitted provides a negative control because Cu(i) is required for the cycloaddition to occur. b, Extracted ion chromatograms of the culture supernatant of S. catenulae with and without CuAAC derivatization, monitoring either the Pra-coumarin adduct (Pra-Cou; m/z = 317.0880) or Pra (m/z = 114.0550) compared to a synthetic Pra standard using LC–QTOF. Chromatograms shown are representative of at least three independent experimental replicates. c, We generated a list of approximately 650 gene clusters (>2 genes) that were conserved between both species. Clusters were generated using Cytoscape33. Nodes are individual genes and edges connect genes that are within two protein-coding regions of one another. Gene clusters comprise of connected nodes. Connected grey nodes are gene clusters also found in any of the other 26 Streptomyces spp. used in the analysis. Connected red nodes are gene clusters found to be unique between S. cattleya and S. catenulae. We then curated this list by removing gene clusters that are more closely related between S. cattleya and 26 other Streptomyces spp., narrowing down the number of conserved unique clusters to just 4. One of these unique clusters contains member nodes WP_014151494.1, WP_014151495.1, WP_014151496.1 and WP_014151497.1, which composes the putative bes gene cluster (indicated with a red arrow). Source data