RT Journal Article SR Electronic T1 Massively parallel fitness profiling reveals multiple novel enzymes in Pseudomonas putida lysine metabolism JF bioRxiv FD Cold Spring Harbor Laboratory SP 450254 DO 10.1101/450254 A1 Mitchell G. Thompson A1 Jacquelyn M. Blake-Hedges A1 Cruz-Morales Pablo A1 Jesus F. Barajas A1 Samuel C. Curran A1 Nicholas C. Harris A1 Veronica T. Benites A1 Jennifer W. Gin A1 Christopher B. Eiben A1 William A. Sharpless A1 Rohith N. Krishna A1 Edward E. K. Baidoo A1 Christopher J. Petzold A1 Adam P. Arkin A1 Adam M. Deutschbauer A1 Jay D. Keasling YR 2018 UL http://biorxiv.org/content/early/2018/10/22/450254.abstract AB 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 remain 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 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 a 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 both 2OA and 2KG as a co-substrate in the hydroxylation of glutarate. Finally we demonstrate that the cellular abundance of D- and L-lysine pathway proteins are highly sensitive to pathway specific substrates. This work demonstrates the utility of RB-TnSeq for discovering novel metabolic pathways in even well-studied bacteria.ImportanceP. putida is an attractive host for metabolic engineering as its lysine metabolism can be utilized for the production of multiple important commodity chemicals.We demonstrate the first biochemical evidence of a bacterial 2OA catabolic pathway to central metabolites.DUF1338 proteins are widely dispersed across many kingdoms of life. Here we demonstrate the first biochemical evidence of function for a member of this protein family.