Implementation of the β-hydroxyaspartate cycle increases growth performance of Pseudomonas putida on the PET monomer ethylene glycol
bioRxiv (Cold Spring Harbor Laboratory)
Abstract
Abstract Ethylene glycol (EG) is a promising next generation feedstock for bioprocesses. It is a key component of the ubiquitous plastic polyethylene terephthalate (PET) and other polyester fibers and plastics, used in antifreeze formulations, and can also be generated by electrochemical conversion of syngas, which makes EG a key compound in a circular bioeconomy. The majority of biotechnologically relevant bacteria assimilate EG via the glycerate pathway, a wasteful metabolic route that releases CO 2 and requires reducing equivalents as well as ATP. In contrast, the recently characterized β-hydroxyaspartate cycle (BHAC) provides a more efficient, carbon-conserving route for C2 assimilation. Here we aimed at overcoming the natural limitations of EG metabolism in the industrially relevant strain Pseudomonas putida KT2440 by replacing the native glycerate pathway with the BHAC. We first prototyped the core reaction sequence of the BHAC in Escherichia coli before establishing the complete four-enzyme BHAC in Pseudomonas putida . Directed evolution on EG resulted in an improved strain that exhibits 35% faster growth and 20% increased biomass yield compared to a recently reported P. putida strain that was evolved to grow on EG via the glycerate pathway. Genome sequencing and proteomics highlight plastic adaptations of the genetic and metabolic networks in response to the introduction of the BHAC into P. putida and identify key mutations for its further integration during evolution. Taken together, our study shows that the BHAC can be utilized as ‘plug-and-play’ module for the metabolic engineering of two important microbial platform organisms, paving the way for multiple applications for a more efficient and carbon-conserving upcycling of EG in the future.
Authors 9
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Leiden University · Max Planck Institute for Terrestrial Microbiology
Affiliation as printed
Department of Biochemistry & Synthetic Metabolism, Max Planck Institute for Terrestrial Microbiology
Institute of Biology Leiden, Leiden University
Department of Biochemistry & Synthetic Metabolism, Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch-Str. 0, 35043 Marburg, Germany
Institute of Biology Leiden, Leiden University, Sylviusweg 7, 333 BE Leiden, The Netherlands
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Max Planck Institute of Molecular Plant Physiology · Max Planck Institute for Terrestrial Microbiology
Affiliation as printed
Department of Biochemistry & Synthetic Metabolism, Max Planck Institute for Terrestrial Microbiology
Max Planck Institute of Molecular Plant Physiology
Department of Biochemistry & Synthetic Metabolism, Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch-Str. 0, 35043 Marburg, Germany
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Max Planck Institute for Terrestrial Microbiology
Affiliation as printed
Department of Biochemistry & Synthetic Metabolism, Max Planck Institute for Terrestrial Microbiology
Department of Biochemistry & Synthetic Metabolism, Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch-Str. 0, 35043 Marburg, Germany
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Max Planck Institute for Terrestrial Microbiology
Affiliation as printed
Department of Biochemistry & Synthetic Metabolism, Max Planck Institute for Terrestrial Microbiology
Department of Biochemistry & Synthetic Metabolism, Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch-Str. 0, 35043 Marburg, Germany
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Max Planck Institute of Molecular Plant Physiology · Max Planck Institute for Terrestrial Microbiology
Affiliation as printed
Department of Biochemistry & Synthetic Metabolism, Max Planck Institute for Terrestrial Microbiology
Max Planck Institute of Molecular Plant Physiology
Department of Biochemistry & Synthetic Metabolism, Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch-Str. 0, 35043 Marburg, Germany
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Max Planck Institute for Terrestrial Microbiology
Affiliation as printed
Facility for Mass Spectrometry and Proteomics, Max Planck Institute for Terrestrial Microbiology
Facility for Mass Spectrometry and Proteomics, Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch-Str. 10, 3503 Marburg, Germany
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Max Planck Institute of Molecular Plant Physiology
Affiliation as printed
Max Planck Institute of Molecular Plant Physiology
Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany
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Steffen N. Lindner corresponding
Max Planck Institute of Molecular Plant Physiology · Charité - Universitätsmedizin Berlin
Affiliation as printed
Department of Biochemistry, Charité Universitätsmedizin
Max Planck Institute of Molecular Plant Physiology
Department of Biochemistry, Charité Universitätsmedizin, Charitéplatz 1, 10117 Berlin, Germany
Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany
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Tobias J. Erb corresponding
Philipps University of Marburg · Loewe Center for Synthetic Microbiology · Max Planck Institute for Terrestrial Microbiology
Affiliation as printed
Department of Biochemistry & Synthetic Metabolism, Max Planck Institute for Terrestrial Microbiology
LOEWE-Center for Synthetic Microbiology, Philipps-University Marburg
Department of Biochemistry & Synthetic Metabolism, Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch-Str. 0, 35043 Marburg, Germany
LOEWE-Center for Synthetic Microbiology, Philipps-University Marburg, Karl-von-Frisch-Str. 8, 35043 Marburg, Germany
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