With its ability to catabolize a wide variety of carbon sources and a growing engineering toolkit, Pseudomonas putida KT2440 is emerging as an important chassis organism for metabolic engineering. Despite advances in our understanding of this organism, many gaps remain in our knowledge of the genetic basis of its metabolic capabilities. These gaps are particularly noticeable in our understanding of both fatty acid and alcohol catabolism, where many paralogs putatively coding for similar enzymes co-exist making biochemical assignment via sequence homology difficult. To rapidly assign function to the enzymes responsible for these metabolisms, we leveraged Random Barcode Transposon Sequencing (RB-TnSeq). Global fitness analyses of transposon libraries grown on 13 fatty acids and 10 alcohols produced strong phenotypes for hundreds of genes. Fitness data from mutant pools grown on varying chain length fatty acids indicated specific enzyme substrate preferences, and enabled us to hypothesize that DUF1302/DUF1329 family proteins potentially function as esterases. From the data we also postulate catabolic routes for the two biogasoline molecules isoprenol and isopentanol, which are catabolized via leucine metabolism after initial oxidation and activation with CoA. Because fatty acids and alcohols may serve as both feedstocks or final products of metabolic engineering efforts, the fitness data presented here will help guide future genomic modifications towards higher titers, rates, and yields. IMPORTANCE To engineer novel metabolic pathways into P. putida, a comprehensive understanding of the genetic basis of its versatile metabolism is essential. Here we provide functional evidence for the putative roles of hundreds of genes involved in the fatty acid and alcohol metabolism of this bacterium. These data provide a framework facilitating precise genetic changes to prevent product degradation and channel the flux of specific pathway intermediates as desired.

30Despite 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 L-and 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 2-40 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 research. 45 Importance 46 P. putida lysine metabolism can produce multiple commodity chemicals, conferring great 47 biotechnological value. Despite much research, connecting lysine catabolism to central 48 metabolism in P. putida remained undefined. Herein we use Random Barcode Transposon 49Sequencing to fill in the gaps of lysine metabolism in P. putida. We describe a route of 2-50 oxoadipate (2OA) catabolism in bacteria, which utilizes DUF1338 containing protein PP_5260. 51Despite its prevalence in many domains of life, DUF1338 containing proteins had no known 52 biochemical function. We demonstrate PP_5260 is a metalloenzyme which catalyzes an unusual 53 2OA to D-2HG decarboxylation. Our screen also identified a recently described novel glutarate 54 metabolic pathway. We validate previous results, and expand the understanding of glutarate 55hydroxylase CsiD by showing can it use either 2OA or 2KG as a cosubstrate. Our work 56 demonstrates biological novelty can be rapidly identified using unbiased experimental genetics, 57and that RB-TnSeq can be used to rapidly validate previous results. 58 Introduction 59Pseudomonas putida is an ubiquitous saprophytic soil bacterium and is a model organism 60for bioremediation (1). Interest in utilizing P. putida KT2440 as a chassis organism for metabolic 61 engineering has recently surged due to the existence of well-established genetic tools and its 62 robust metabolism of aromatic compounds that resemble lignin hydrolysis products (2-4). As 63 lignin valorization remains essential for the economic feasibility of cellulosic bioproducts, a 64 nuanced and predictable understanding of P. putida metabolism is highly desirable (5). 65Although its aromatic metabolism has garnered mu...