Massively Parallel Fitness Profiling Reveals Multiple Novel Enzymes in <italic toggle="yes">Pseudomonas putida</italic> Lysine Metabolism

ABSTRACT Despite intensive study for 50 years, the biochemical and genetic links between lysine metabolism and central metabolism in Pseudomonas putida remain unresolved. To establish these biochemical links, we leveraged random barcode transposon sequencing (RB-TnSeq), a genome-wide assay measuring...

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Autores principales:	Mitchell G. Thompson, Jacquelyn M. Blake-Hedges, Pablo Cruz-Morales, Jesus F. Barajas, Samuel C. Curran, Christopher B. Eiben, Nicholas C. Harris, Veronica T. Benites, Jennifer W. Gin, William A. Sharpless, Frederick F. Twigg, Will Skyrud, Rohith N. Krishna, Jose Henrique Pereira, Edward E. K. Baidoo, Christopher J. Petzold, Paul D. Adams, Adam P. Arkin, Adam M. Deutschbauer, Jay D. Keasling
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2019
Materias:	biochemistry biotechnology genomics metabolism transposons Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/a71a6236dda04344a17ff1e8947b3078
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spelling	oai:doaj.org-article:a71a6236dda04344a17ff1e8947b30782021-11-15T15:55:24ZMassively Parallel Fitness Profiling Reveals Multiple Novel Enzymes in <italic toggle="yes">Pseudomonas putida</italic> Lysine Metabolism10.1128/mBio.02577-182150-7511https://doaj.org/article/a71a6236dda04344a17ff1e8947b30782019-06-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.02577-18https://doaj.org/toc/2150-7511ABSTRACT Despite intensive study for 50 years, the biochemical and genetic links between lysine metabolism and central metabolism in Pseudomonas putida remain unresolved. To establish these biochemical links, we leveraged 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 l-2-aminoadipate (l-2AA) to 2-ketoglutarate (2KG), connecting d-lysine to the TCA cycle. One of these enzymes, P. putida 5260 (PP_5260), contains a DUF1338 domain, representing a family with no previously described biological function. Our work also identified the recently described coenzyme A (CoA)-independent route of l-lysine degradation that results in metabolization to succinate. We expanded on previous findings by demonstrating that glutarate hydroxylase CsiD is promiscuous in its 2-oxoacid selectivity. Proteomics of selected pathway enzymes revealed that expression of catabolic genes is highly sensitive to the presence of particular pathway metabolites, implying intensive local and global regulation. This work demonstrated the utility of RB-TnSeq for discovering novel metabolic pathways in even well-studied bacteria, as well as its utility a powerful tool for validating previous research. IMPORTANCE P. putida lysine metabolism can produce multiple commodity chemicals, conferring great biotechnological value. Despite much research, the connection of lysine catabolism to central metabolism in P. putida remained undefined. Here, we used random barcode transposon sequencing to fill the gaps of lysine metabolism in P. putida. We describe a route of 2-oxoadipate (2OA) catabolism, which utilizes DUF1338-containing protein P. putida 5260 (PP_5260) in bacteria. Despite its prevalence in many domains of life, DUF1338-containing proteins have had no known biochemical function. We demonstrate that PP_5260 is a metalloenzyme which catalyzes an unusual route of decarboxylation of 2OA to d-2-hydroxyglutarate (d-2HG). Our screen also identified a recently described novel glutarate metabolic pathway. We validate previous results and expand the understanding of glutarate hydroxylase CsiD by showing that can it use either 2OA or 2KG as a cosubstrate. Our work demonstrated that biological novelty can be rapidly identified using unbiased experimental genetics and that RB-TnSeq can be used to rapidly validate previous results.Mitchell G. ThompsonJacquelyn M. Blake-HedgesPablo Cruz-MoralesJesus F. BarajasSamuel C. CurranChristopher B. EibenNicholas C. HarrisVeronica T. BenitesJennifer W. GinWilliam A. SharplessFrederick F. TwiggWill SkyrudRohith N. KrishnaJose Henrique PereiraEdward E. K. BaidooChristopher J. PetzoldPaul D. AdamsAdam P. ArkinAdam M. DeutschbauerJay D. KeaslingAmerican Society for MicrobiologyarticlebiochemistrybiotechnologygenomicsmetabolismtransposonsMicrobiologyQR1-502ENmBio, Vol 10, Iss 3 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	biochemistry biotechnology genomics metabolism transposons Microbiology QR1-502
spellingShingle	biochemistry biotechnology genomics metabolism transposons Microbiology QR1-502 Mitchell G. Thompson Jacquelyn M. Blake-Hedges Pablo Cruz-Morales Jesus F. Barajas Samuel C. Curran Christopher B. Eiben Nicholas C. Harris Veronica T. Benites Jennifer W. Gin William A. Sharpless Frederick F. Twigg Will Skyrud Rohith N. Krishna Jose Henrique Pereira Edward E. K. Baidoo Christopher J. Petzold Paul D. Adams Adam P. Arkin Adam M. Deutschbauer Jay D. Keasling Massively Parallel Fitness Profiling Reveals Multiple Novel Enzymes in <italic toggle="yes">Pseudomonas putida</italic> Lysine Metabolism
description	ABSTRACT Despite intensive study for 50 years, the biochemical and genetic links between lysine metabolism and central metabolism in Pseudomonas putida remain unresolved. To establish these biochemical links, we leveraged 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 l-2-aminoadipate (l-2AA) to 2-ketoglutarate (2KG), connecting d-lysine to the TCA cycle. One of these enzymes, P. putida 5260 (PP_5260), contains a DUF1338 domain, representing a family with no previously described biological function. Our work also identified the recently described coenzyme A (CoA)-independent route of l-lysine degradation that results in metabolization to succinate. We expanded on previous findings by demonstrating that glutarate hydroxylase CsiD is promiscuous in its 2-oxoacid selectivity. Proteomics of selected pathway enzymes revealed that expression of catabolic genes is highly sensitive to the presence of particular pathway metabolites, implying intensive local and global regulation. This work demonstrated the utility of RB-TnSeq for discovering novel metabolic pathways in even well-studied bacteria, as well as its utility a powerful tool for validating previous research. IMPORTANCE P. putida lysine metabolism can produce multiple commodity chemicals, conferring great biotechnological value. Despite much research, the connection of lysine catabolism to central metabolism in P. putida remained undefined. Here, we used random barcode transposon sequencing to fill the gaps of lysine metabolism in P. putida. We describe a route of 2-oxoadipate (2OA) catabolism, which utilizes DUF1338-containing protein P. putida 5260 (PP_5260) in bacteria. Despite its prevalence in many domains of life, DUF1338-containing proteins have had no known biochemical function. We demonstrate that PP_5260 is a metalloenzyme which catalyzes an unusual route of decarboxylation of 2OA to d-2-hydroxyglutarate (d-2HG). Our screen also identified a recently described novel glutarate metabolic pathway. We validate previous results and expand the understanding of glutarate hydroxylase CsiD by showing that can it use either 2OA or 2KG as a cosubstrate. Our work demonstrated that biological novelty can be rapidly identified using unbiased experimental genetics and that RB-TnSeq can be used to rapidly validate previous results.
format	article
author	Mitchell G. Thompson Jacquelyn M. Blake-Hedges Pablo Cruz-Morales Jesus F. Barajas Samuel C. Curran Christopher B. Eiben Nicholas C. Harris Veronica T. Benites Jennifer W. Gin William A. Sharpless Frederick F. Twigg Will Skyrud Rohith N. Krishna Jose Henrique Pereira Edward E. K. Baidoo Christopher J. Petzold Paul D. Adams Adam P. Arkin Adam M. Deutschbauer Jay D. Keasling
author_facet	Mitchell G. Thompson Jacquelyn M. Blake-Hedges Pablo Cruz-Morales Jesus F. Barajas Samuel C. Curran Christopher B. Eiben Nicholas C. Harris Veronica T. Benites Jennifer W. Gin William A. Sharpless Frederick F. Twigg Will Skyrud Rohith N. Krishna Jose Henrique Pereira Edward E. K. Baidoo Christopher J. Petzold Paul D. Adams Adam P. Arkin Adam M. Deutschbauer Jay D. Keasling
author_sort	Mitchell G. Thompson
title	Massively Parallel Fitness Profiling Reveals Multiple Novel Enzymes in <italic toggle="yes">Pseudomonas putida</italic> Lysine Metabolism
title_short	Massively Parallel Fitness Profiling Reveals Multiple Novel Enzymes in <italic toggle="yes">Pseudomonas putida</italic> Lysine Metabolism
title_full	Massively Parallel Fitness Profiling Reveals Multiple Novel Enzymes in <italic toggle="yes">Pseudomonas putida</italic> Lysine Metabolism
title_fullStr	Massively Parallel Fitness Profiling Reveals Multiple Novel Enzymes in <italic toggle="yes">Pseudomonas putida</italic> Lysine Metabolism
title_full_unstemmed	Massively Parallel Fitness Profiling Reveals Multiple Novel Enzymes in <italic toggle="yes">Pseudomonas putida</italic> Lysine Metabolism
title_sort	massively parallel fitness profiling reveals multiple novel enzymes in <italic toggle="yes">pseudomonas putida</italic> lysine metabolism
publisher	American Society for Microbiology
publishDate	2019
url	https://doaj.org/article/a71a6236dda04344a17ff1e8947b3078
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Massively Parallel Fitness Profiling Reveals Multiple Novel Enzymes in <italic toggle="yes">Pseudomonas putida</italic> Lysine Metabolism

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