The lysine metabolism of Pseudomonas putida can produce multiple important commodity chemicals and is implicated in rhizosphere colonization. However, despite intensive study, the biochemical and genetic links between lysine metabolism and central metabolism remains unresolved in P. putida. Here, we leverage Random Barcode Transposon Sequencing (RB-TnSeq), a genome-wide assay measuring the fitness of thousands of genes in parallel, to identify multiple novel enzymes and regulators in both L- and D-lysine metabolism. We first describe three pathway enzymes that catabolize 2-aminoadipate (2AA) to 2-ketoglutarate (2KG) connecting D-lysine to the TCA cycle. One of these enzymes, PP_5260, contains a DUF1338 domain, a family without previously described biological function. We demonstrate PP_5260 converts 2-oxoadipate (2OA) to 2-hydroxyglutarate (2HG), a novel biochemical reaction. We expand on recent work showing that the glutarate hydroxylase CsiD can co-utilize 2OA and 2KG. We further show glutarate metabolism is locally regulated by two independent glutarate responsive transcription factors, and that cellular abundance of D- and L-lysine pathway proteins are highly sensitive to pathway specific substrates. This work demonstrates the utility of RB-TnSeq of discovering novel metabolic pathways in even well-studied bacterial species.

Targeted proteomics - more information is available in the paper reference.