Heterologous Expression of Pseudouridimycin and Description of the Corresponding Minimal Biosynthetic Gene Cluster

• Published in: Molecules (Basel, Switzerland) Vol. 26; no. 2; p. 510
• Main Authors: Böhringer, Nils, Patras, Maria A, Schäberle, Till F
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 19.01.2021; MDPI
• Subjects: Animals; Anti-Bacterial Agents - biosynthesis; Anti-Bacterial Agents - pharmacology; antibiotic; Antibiotics; Antiinfectives and antibacterials; Bacteria; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Biosynthesis; Chromatography; Cloning; DNA-directed RNA polymerase; Enzymes; Fermentation; Gene Expression Regulation, Bacterial; Genes; heterologous expression; Kinases; Mice; Mode of action; Multigene Family; natural product; Nucleoside analogs; Nucleosides - analogs & derivatives; Peritonitis; Protein-serine/threonine kinase; Proteins; pseudouridimycin; PUM; RNA polymerase; Streptococcus infections; Streptomyces - genetics; Streptomyces - metabolism; Streptomyces coelicolor - genetics; Streptomyces coelicolor - metabolism; Transcription; antibiotic; PUM; heterologous expression; pseudouridimycin; natural product
• ISSN: 1420-3049; 1420-3049
• DOI: 10.3390/molecules26020510

Pseudouridimycin (PUM) was recently discovered from sp. DSM26212 as a novel bacterial nucleoside analog that competes with UTP for access to the RNA polymerase (RNAP) active site, thereby inhibiting bacterial RNAP by blocking transcription. This represents a novel antibacterial mode of action and it is known that PUM inhibits bacterial RNAP in vitro, inhibits bacterial growth in vitro, and was active in vivo in a mouse infection model of peritonitis. The biosynthetic gene cluster (BGC) was previously identified and characterized by knockout experiments. However, the minimal set of genes necessary for PUM production was not proposed. To identify the minimal BGC and to create a plug-and-play production platform for PUM and its biosynthetic precursors, several versions of a redesigned PUM BGC were generated and expressed in the heterologous host M1146 under control of strong promotors. Heterologous expression allowed identification of the putative serine/threonine kinase PumF as an enzyme essential for heterologous PUM production and thus corroboration of the PUM minimal BGC.