Biosynthetic Investigationsof Ulbactins Unveil TwoNovel Thiazolinyl Imine Reductases Crucial for the Generation of SiderophoreActivity

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 Pseudoalteromonas flavipulchra 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 P. flavipulchra 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.