Massively parallel fitness profiling reveals multiple novel enzymes in Pseudomonas putida 1 lysine metabolism 2

30 Despite intensive study for 50 years, the biochemical and genetic links between lysine 31 metabolism and central metabolism in Pseudomonas putida remain unresolved. To establish 32 these biochemical links, we leveraged Random Barcode Transposon Sequencing (RB-TnSeq), a 33 genome-wide assay measuring the fitness of thousands of genes in parallel, to identify multiple 34 novel enzymes in both Land D-lysine metabolism. We first describe three pathway enzymes 35 that catabolize L-2-aminoadipate (L-2AA) to 2-ketoglutarate (2KG), connecting D-lysine to the 36 TCA cycle. One of these enzymes, PP_5260, contains a DUF1338 domain, a family with no 37 previously described biological function. Our work also identified the recently described CoA 38 independent route of L-lysine degradation that metabolizes to succinate. We expanded on 39 previous findings by demonstrating that glutarate hydroxylase CsiD is promiscuous in its 240 oxoacid selectivity. Proteomics of select pathway enzymes revealed that expression of catabolic 41 genes is highly sensitive to particular pathway metabolites, implying intensive local and global 42 regulation. This work demonstrates the utility of RB-TnSeq for discovering novel metabolic 43 pathways in even well-studied bacteria, as well as a powerful tool for validating previous 44