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Title: Massively Parallel Fitness Profiling Reveals Multiple Novel Enzymes in Pseudomonas putida 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 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 greatmore » 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.« less

Authors:
 [1];  [1];  [2];  [3];  [1];  [1];  [4];  [5];  [5];  [1];  [4];  [4];  [1];  [5];  [5];  [5];  [1];  [6];  [6];  [7] more »;  [8] « less
+ Show Author Affiliations
  1. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Univ. of California, Berkeley, CA (United States)
  2. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Tecnológico de Monterrey (Mexico)
  3. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Dept. of Energy Agile BioFoundry, Emeryville, CA (United States)
  4. Univ. of California, Berkeley, CA (United States)
  5. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
  6. Univ. of California, Berkeley, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
  7. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Univ. of California, Berkeley, CA (United States); Technical Univ. of Denmark, Lyngby (Denmark)
  8. Korea Advanced Inst. of Science and Technology, Daejeon (South Korea)
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER)
OSTI Identifier:
1572007
Grant/Contract Number:  
AC02-05CH11231
Resource Type:
Accepted Manuscript
Journal Name:
mBio (Online)
Additional Journal Information:
Journal Name: mBio (Online); Journal Volume: 10; Journal Issue: 3; Journal ID: ISSN 2150-7511
Publisher:
American Society for Microbiology
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; biochemistry; biotechnology; genomics; metabolisml; transposons

Citation Formats

Thompson, Mitchell G., Blake-Hedges, Jacquelyn M., Cruz-Morales, Pablo, Barajas, Jesus F., Curran, Samuel C., Eiben, Christopher B., Harris, Nicholas C., Benites, Veronica T., Gin, Jennifer W., Sharpless, William A., Twigg, Frederick F., Skyrud, Will, Krishna, Rohith N., Pereira, Jose Henrique, Baidoo, Edward E. K., Petzold, Christopher J., Adams, Paul D., Arkin, Adam P., Deutschbauer, Adam M., Keasling, Jay D., and Lee, Sang Yup. Massively Parallel Fitness Profiling Reveals Multiple Novel Enzymes in Pseudomonas putida Lysine Metabolism. United States: N. p., 2019. Web. doi:10.1128/mBio.02577-18.
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Thompson, Mitchell G., Blake-Hedges, Jacquelyn M., Cruz-Morales, Pablo, Barajas, Jesus F., Curran, Samuel C., Eiben, Christopher B., Harris, Nicholas C., Benites, Veronica T., Gin, Jennifer W., Sharpless, William A., Twigg, Frederick F., Skyrud, Will, Krishna, Rohith N., Pereira, Jose Henrique, Baidoo, Edward E. K., Petzold, Christopher J., Adams, Paul D., Arkin, Adam P., Deutschbauer, Adam M., Keasling, Jay D., & Lee, Sang Yup. Massively Parallel Fitness Profiling Reveals Multiple Novel Enzymes in Pseudomonas putida Lysine Metabolism. United States. https://doi.org/10.1128/mBio.02577-18
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Thompson, Mitchell G., Blake-Hedges, Jacquelyn M., Cruz-Morales, Pablo, Barajas, Jesus F., Curran, Samuel C., Eiben, Christopher B., Harris, Nicholas C., Benites, Veronica T., Gin, Jennifer W., Sharpless, William A., Twigg, Frederick F., Skyrud, Will, Krishna, Rohith N., Pereira, Jose Henrique, Baidoo, Edward E. K., Petzold, Christopher J., Adams, Paul D., Arkin, Adam P., Deutschbauer, Adam M., Keasling, Jay D., and Lee, Sang Yup. Tue . "Massively Parallel Fitness Profiling Reveals Multiple Novel Enzymes in Pseudomonas putida Lysine Metabolism". United States. https://doi.org/10.1128/mBio.02577-18. https://www.osti.gov/servlets/purl/1572007.
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@article{osti_1572007,
title = {Massively Parallel Fitness Profiling Reveals Multiple Novel Enzymes in Pseudomonas putida Lysine Metabolism},
author = {Thompson, Mitchell G. and Blake-Hedges, Jacquelyn M. and Cruz-Morales, Pablo and Barajas, Jesus F. and Curran, Samuel C. and Eiben, Christopher B. and Harris, Nicholas C. and Benites, Veronica T. and Gin, Jennifer W. and Sharpless, William A. and Twigg, Frederick F. and Skyrud, Will and Krishna, Rohith N. and Pereira, Jose Henrique and Baidoo, Edward E. K. and Petzold, Christopher J. and Adams, Paul D. and Arkin, Adam P. and Deutschbauer, Adam M. and Keasling, Jay D. and Lee, Sang Yup},
abstractNote = {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.},
doi = {10.1128/mBio.02577-18},
journal = {mBio (Online)},
number = 3,
volume = 10,
place = {United States},
year = {Tue May 07 00:00:00 EDT 2019},
month = {Tue May 07 00:00:00 EDT 2019}
}
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https://doi.org/10.1128/mBio.02577-18
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    An iron (II) dependent oxygenase performs the last missing step of plant lysine catabolism
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    • Thompson, Mitchell G.; Blake-Hedges, Jacquelyn M.; Pereira, Jose Henrique
    • Nature Communications, Vol. 11, Issue 1
    • DOI: 10.1038/s41467-020-16815-3
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