Genetic Analysis of the Upper Phenylacetate Catabolic Pathway in the Production of Tropodithietic Acid by Phaeobacter gallaeciensis

Abstract

ABSTRACTProduction of the antibiotic tropodithietic acid (TDA) depends on the central phenylacetate catabolic pathway, specifically on the oxygenase PaaABCDE, which catalyzes epoxidation of phenylacetyl-coenzyme A (CoA). Our study was focused on genes of the upper part of this pathway leading to phenylacetyl-CoA as precursor for TDA.Phaeobacter gallaeciensisDSM 17395 encodes two genes with homology to phenylacetyl-CoA ligases (paaK1andpaaK2), which were shown to be essential for phenylacetate catabolism but not for TDA biosynthesis and phenylalanine degradation. Thus, inP. gallaeciensisanother enzyme must produce phenylacetyl-CoA from phenylalanine. Using random transposon insertion mutagenesis of apaaK1-paaK2double mutant we identified a gene (ior1) with similarity toiorAandiorBin archaea, encoding an indolepyruvate:ferredoxin oxidoreductase (IOR). Theior1mutant was unable to grow on phenylalanine, and production of TDA was significantly reduced compared to the wild-type level (60%). Nuclear magnetic resonance (NMR) spectroscopic investigations using13C-labeled phenylalanine isotopomers demonstrated that phenylalanine is transformed into phenylacetyl-CoA by Ior1. Using quantitative real-time PCR, we could show that expression ofior1depends on the adjacent regulator IorR. Growth on phenylalanine promotes production of TDA, induces expression ofior1(27-fold) andpaaK1(61-fold), and regulates the production of TDA. Phylogenetic analysis showed that the aerobic type of IOR as found in many roseobacters is common within a number of different phylogenetic groups of aerobic bacteria such asBurkholderia,Cupriavidis, andRhizobia, where it may also contribute to the degradation of phenylalanine.