Functional characterization of the dbu locus for D-branched-chain amino acid catabolism in Pseudomonas putida

Abstract

ABSTRACT Pseudomonas putida is a metabolically robust soil bacterium that employs a diverse set of pathways to utilize a wide range of nutrients. The versatility of this microorganism contributes to both its environmental ubiquity and its rising popularity as a bioengineering chassis. In P. putida , the newly named dbu locus encodes a transcriptional regulator (DbuR), D-amino acid oxidase (DbuA), Rid2 protein (DbuB), and a putative transporter (DbuC). Current annotation implicates this locus in the utilization of D-arginine. However, data obtained in this study showed that genes in the dbu locus are not required for D-arginine utilization, but, rather, this locus is involved in the catabolism of multiple D-branched-chain amino acids (D-BCAA). The oxidase DbuA was required for catabolism of each D-BCAA and D-phenylalanine, while the requirements for DbuC and DbuB were less stringent. The functional characterization of the dbu locus contributes to our understanding of the metabolic network of P. putida and proposes divergence in function between proteins annotated as D-arginine oxidases across the Pseudomonas genus. IMPORTANCE Pseudomonas putida is a non-pathogenic bacterium that is broadly utilized as a host for bioengineering and bioremediation efforts. The popularity of P. putida as a chassis for such efforts is attributable to its physiological versatility and ability to metabolize a wide variety of compounds. Pathways for L-amino acid metabolism in this microbe have been rather well studied, primarily because of their relevance to efforts in foundational physiology research, as well as the commercial production of economically pertinent compounds. However, comparatively little is known about the metabolism of D-amino acids despite evidence showing the ability of P. putida to metabolize these enantiomers. In this work, we characterize the D-BCAA catabolic pathway of P. putida and its integration with the essential L-BCAA biosynthetic pathway. This work expands our understanding of the metabolic network of Pseudomonas putida , which has potential applications in efforts to model and engineer the metabolic network of this organism.