Biosynthetic Investigations of Ulbactins Unveil Two Novel Thiazolinyl Imine Reductases Crucial for the Generation of Siderophore Activity

Siderophores are ferric ion chelating natural products secreted by microorganisms to survive in an iron-deficient environment. During screening for siderophores from marine bacteria, four interconvertible ulbactins C₁-C₄ and oxidized derivatives were identified in the bacterium <i>Pseudoalteromonas flavipulchra</i> S16. Iron-ion chelating activity assays revealed that the reduction of thiazoline rings is critical for the activity. The ulbactins biosynthetic gene cluster (<i>ubt</i>) was identified in the genome of <i>P. flavipulchra</i> S16 and validated <i>via</i> gene knockout and heterologous expression. A unique feature of ulbactin biosynthetic machinery involves UbtA, an atypical didomain enzyme (A_Sal-SalS) converting chorismate to salicyl-AMP. Two novel thiazolinyl imine reductases, UbtL and UbtM, were characterized as being essential for thiazolidine formation. Substitution of the native promoter of the <i>ubt</i> gene cluster with a constitutive promoter yielded new ulbactin variants and intermediate compounds, including unprecedented hydroxylated and dehydroalanine-containing ulbactins. <i>In vitro</i> reconstitution of ulbactins uncovered a distinctive synergistic catalytic mechanism between UbtL and UbtM for heterocycle reduction, which drives structural diversification during ulbactin biosynthesis.