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Title: Systems Analysis of NADH Dehydrogenase Mutants Reveals Flexibility and Limits of Pseudomonas taiwanensis VLB120’s Metabolism

Abstract

Obligate aerobic organisms rely on a functional electron transport chain for energy conservation and NADH oxidation. Because of this essential requirement, the genes of this pathway are likely constitutively and highly expressed to avoid a cofactor imbalance and energy shortage under fluctuating environmental conditions. In this work, we investigated the essentiality of the three NADH dehydrogenases of the respiratory chain of the obligate aerobe Pseudomonas taiwanensis VLB120 and the impact of the knockouts of corresponding genes on its physiology and metabolism. While a mutant lacking all three NADH dehydrogenases seemed to be nonviable, the single or double knockout mutant strains displayed no, or only a weak, phenotype. Only the mutant deficient in both type 2 dehydrogenases showed a clear phenotype with biphasic growth behavior and a strongly reduced growth rate in the second phase. In-depth analyses of the metabolism of the generated mutants, including quantitative physiological experiments, transcript analysis, proteomics, and enzyme activity assays revealed distinct responses to type 2 and type 1 dehydrogenase deletions. An overall high metabolic flexibility enables P. taiwanensis to cope with the introduced genetic perturbations and maintain stable phenotypes, likely by rerouting of metabolic fluxes. This metabolic adaptability has implications for biotechnological applications. While themore » phenotypic robustness is favorable in large-scale applications with inhomogeneous conditions, the possible versatile redirecting of carbon fluxes upon genetic interventions can thwart metabolic engineering efforts. While Pseudomonas has the capability for high metabolic activity and the provision of reduced redox cofactors important for biocatalytic applications, exploitation of this characteristic might be hindered by high, constitutive activity of and, consequently, competition with the NADH dehydrogenases of the respiratory chain. The in-depth analysis of NADH dehydrogenase mutants of Pseudomonas taiwanensis VLB120 presented here provides insight into the phenotypic and metabolic response of this strain to these redox metabolism perturbations. This high degree of metabolic flexibility needs to be taken into account for rational engineering of this promising biotechnological workhorse toward a host with a controlled and efficient supply of redox cofactors for product synthesis.« less

Authors:
 [1];  [2];  [3];  [4];  [4];  [4];  [2];  [5];  [6];  [1]; ORCiD logo [7]
  1. RWTH Aachen Univ. (Germany). Inst. of Applied Microbiology
  2. RWTH Aachen Univ. (Germany)
  3. Joint BioEnergy Institute (JBEI), Emeryville, CA (United States)
  4. Technical Univ. of Denmark, Lyngby (Denmark). Novo Nordisk Foundation Center for Biosustainability
  5. Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
  6. Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Technical Univ. of Denmark, Lyngby (Denmark). Novo Nordisk Foundation Center for Biosustainability; Univ. of California, Berkeley, CA (United States); Shenzhen Inst. for Advanced Technologies (China). Synthetic Biochemistry Center, Inst. for Synthetic Biology
  7. RWTH Aachen Univ. (Germany). Inst. of Applied Microbiology; Univ. of Queensland, Brisbane, QLD (Australia). Australian Inst. for Bioengineering and Nanotechnology (AIBN); Commonwealth Scientific and Industrial Research Organization (CSIRO), Canberra, ACT (Australia). CSIRO Future Science Platform in Synthetic Biology
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES)
OSTI Identifier:
1775385
Grant/Contract Number:  
AC02-05CH11231
Resource Type:
Accepted Manuscript
Journal Name:
Applied and Environmental Microbiology
Additional Journal Information:
Journal Volume: 86; Journal Issue: 11; Journal ID: ISSN 0099-2240
Publisher:
American Society for Microbiology
Country of Publication:
United States
Language:
English
Subject:
36 MATERIALS SCIENCE; Pseudomonas; NADH dehydrogenase; respiratory activity; oxidative stress; electron transport chain; pseudomonads; redox metabolism

Citation Formats

Nies, Salome C., Dinger, Robert, Chen, Yan, Wordofa, Gossa G., Kristensen, Mette, Schneider, Konstantin, Büchs, Jochen, Petzold, Christopher J., Keasling, Jay D., Blank, Lars M., and Ebert, Birgitta E.. Systems Analysis of NADH Dehydrogenase Mutants Reveals Flexibility and Limits of Pseudomonas taiwanensis VLB120’s Metabolism. United States: N. p., 2020. Web. https://doi.org/10.1128/aem.03038-19.
Nies, Salome C., Dinger, Robert, Chen, Yan, Wordofa, Gossa G., Kristensen, Mette, Schneider, Konstantin, Büchs, Jochen, Petzold, Christopher J., Keasling, Jay D., Blank, Lars M., & Ebert, Birgitta E.. Systems Analysis of NADH Dehydrogenase Mutants Reveals Flexibility and Limits of Pseudomonas taiwanensis VLB120’s Metabolism. United States. https://doi.org/10.1128/aem.03038-19
Nies, Salome C., Dinger, Robert, Chen, Yan, Wordofa, Gossa G., Kristensen, Mette, Schneider, Konstantin, Büchs, Jochen, Petzold, Christopher J., Keasling, Jay D., Blank, Lars M., and Ebert, Birgitta E.. Tue . "Systems Analysis of NADH Dehydrogenase Mutants Reveals Flexibility and Limits of Pseudomonas taiwanensis VLB120’s Metabolism". United States. https://doi.org/10.1128/aem.03038-19. https://www.osti.gov/servlets/purl/1775385.
@article{osti_1775385,
title = {Systems Analysis of NADH Dehydrogenase Mutants Reveals Flexibility and Limits of Pseudomonas taiwanensis VLB120’s Metabolism},
author = {Nies, Salome C. and Dinger, Robert and Chen, Yan and Wordofa, Gossa G. and Kristensen, Mette and Schneider, Konstantin and Büchs, Jochen and Petzold, Christopher J. and Keasling, Jay D. and Blank, Lars M. and Ebert, Birgitta E.},
abstractNote = {Obligate aerobic organisms rely on a functional electron transport chain for energy conservation and NADH oxidation. Because of this essential requirement, the genes of this pathway are likely constitutively and highly expressed to avoid a cofactor imbalance and energy shortage under fluctuating environmental conditions. In this work, we investigated the essentiality of the three NADH dehydrogenases of the respiratory chain of the obligate aerobe Pseudomonas taiwanensis VLB120 and the impact of the knockouts of corresponding genes on its physiology and metabolism. While a mutant lacking all three NADH dehydrogenases seemed to be nonviable, the single or double knockout mutant strains displayed no, or only a weak, phenotype. Only the mutant deficient in both type 2 dehydrogenases showed a clear phenotype with biphasic growth behavior and a strongly reduced growth rate in the second phase. In-depth analyses of the metabolism of the generated mutants, including quantitative physiological experiments, transcript analysis, proteomics, and enzyme activity assays revealed distinct responses to type 2 and type 1 dehydrogenase deletions. An overall high metabolic flexibility enables P. taiwanensis to cope with the introduced genetic perturbations and maintain stable phenotypes, likely by rerouting of metabolic fluxes. This metabolic adaptability has implications for biotechnological applications. While the phenotypic robustness is favorable in large-scale applications with inhomogeneous conditions, the possible versatile redirecting of carbon fluxes upon genetic interventions can thwart metabolic engineering efforts. While Pseudomonas has the capability for high metabolic activity and the provision of reduced redox cofactors important for biocatalytic applications, exploitation of this characteristic might be hindered by high, constitutive activity of and, consequently, competition with the NADH dehydrogenases of the respiratory chain. The in-depth analysis of NADH dehydrogenase mutants of Pseudomonas taiwanensis VLB120 presented here provides insight into the phenotypic and metabolic response of this strain to these redox metabolism perturbations. This high degree of metabolic flexibility needs to be taken into account for rational engineering of this promising biotechnological workhorse toward a host with a controlled and efficient supply of redox cofactors for product synthesis.},
doi = {10.1128/aem.03038-19},
journal = {Applied and Environmental Microbiology},
number = 11,
volume = 86,
place = {United States},
year = {2020},
month = {5}
}

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