Massively parallel fitness profiling reveals multiple novel enzymes inPseudomonas putidalysine metabolism

Abstract

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...

Identification, characterization, and application of a highly sensitive lactam biosensor fromPseudomonas putida

“…While OplR-based biosensors may be able to aid in the selection of mutant acyl-coA ligases with enhanced activity, there remains a sizeable thermodynamic barrier for the cyclization of the 7-membered ring 1 Since naturally-occuring genetically encoded biosensors for chemicals of interest have the potential to be much more sensitive than those repurposed or evolved in the laboratory, it is critical to pursue rapid and efficient means of identifying them. The recent development of other high-throughput methods to associate genotypes with phenotypes, such as RB-TnSeq and CRISPRi, has created a large reservoir of data that can be easily mined for transcription factors useful in synthetic biology 22,[29][30][31] . Bacteria often locally regulate catabolism, thus allowing inference of genetic control by adjacent transcription factors once a catabolic pathway has been discovered.…”

Catabolism of biogenic amines in Pseudomonas species

“…) involves the participation, in the first step, of a decarboxylase (LdcA) that generates cadaverine (Espinosa‐Urgel and Ramos, ; Revelles et al . , ; Idurthi et al ., ; Thompson et al ., ). In P. putida KT2440, cadaverine is then deaminated and cyclised to 1‐piperideine by a cadaverine aminotransferase (Revelles et al .…”

“…One of the multiple pathways reported in Pseudomonas species for the degradation of L-lysine (Fig. 6) involves the participation, in the first step, of a decarboxylase (LdcA) that generates cadaverine (Espinosa-Urgel and Ramos, 2001;Revelles et al 2004Revelles et al , 2005Idurthi et al, 2016;Thompson et al, 2019a). In P. putida KT2440, cadaverine is then deaminated and cyclised to 1-piperideine by a cadaverine aminotransferase (Revelles et al 2005).…”

Glutarate metabolism in Pseudomonas putida is regulated by two distinct glutarate sensing transcription factors

“…Recently this utility has inspired much work to uncover missing steps in the lysine catabolism of P. putida. These missing steps included the discovery of an additional route of glutarate catabolism through a coA-independent route to succinate, in addition to the known coA-dependent route to acetyl-coA 14,16 (Figure 1). Recent work has also demonstrated that both glutarate catabolic pathways are highly upregulated in the presence of glutarate 16 .…”

“…These missing steps included the discovery of an additional route of glutarate catabolism through a coA-independent route to succinate, in addition to the known coA-dependent route to acetyl-coA 14,16 (Figure 1). Recent work has also demonstrated that both glutarate catabolic pathways are highly upregulated in the presence of glutarate 16 . While work in Pseudomonas aeruginosa has characterized a homolog of the regulator of the ketogenic pathway (GcdR) 17 , and work in Escherichia coli has preliminarily characterized the glucogenic pathway regulator (CsiR), no work has directly investigated the regulation of the P. putida L-lysine catabolic pathways.…”