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Insights into an alternative pathway for glycerol metabolism in a glycerol kinase deficient Pseudomonas putida KT2440

Meg Walsh, William Casey, Shane T. Kenny, Tanja Narancic, Lars M. Blank, Nick Wierckx, Hendrik Ballerstedt, Kevin E O Connor
doi: https://doi.org/10.1101/567230
Meg Walsh
1School of biomolecular and biomedical science, University College Dublin, Belfield, Dublin 4, Ireland
2Bioplastech ltd Belfield Innovation park, University College Dublin, Dublin 4
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William Casey
2Bioplastech ltd Belfield Innovation park, University College Dublin, Dublin 4
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Shane T. Kenny
2Bioplastech ltd Belfield Innovation park, University College Dublin, Dublin 4
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Tanja Narancic
1School of biomolecular and biomedical science, University College Dublin, Belfield, Dublin 4, Ireland
3BEACON SFI bioeconomy research centre - iAMB, Aachen Biology and Biotechnology - ABBt, RWTH Aachen University, Worringerweg 1, Aachen 52074, Germany
4UCD Earth Institute - iAMB, Aachen Biology and Biotechnology - ABBt, RWTH Aachen University, Worringerweg 1, Aachen 52074, Germany
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Lars M. Blank
5Institute of Applied Microbiology - iAMB, Aachen Biology and Biotechnology - ABBt, RWTH Aachen University, Worringerweg 1, Aachen 52074, Germany
6Bioeconomy Science Center (BioSC), Forschungszentrum Jülich, Jülich, Germany
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Nick Wierckx
7Forschungszentrum Jülich, Institute of Bio- and Geosciences IBG-1: Biotechnology, 52425 Jülich, Germany
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Hendrik Ballerstedt
5Institute of Applied Microbiology - iAMB, Aachen Biology and Biotechnology - ABBt, RWTH Aachen University, Worringerweg 1, Aachen 52074, Germany
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Kevin E O Connor
1School of biomolecular and biomedical science, University College Dublin, Belfield, Dublin 4, Ireland
2Bioplastech ltd Belfield Innovation park, University College Dublin, Dublin 4
3BEACON SFI bioeconomy research centre - iAMB, Aachen Biology and Biotechnology - ABBt, RWTH Aachen University, Worringerweg 1, Aachen 52074, Germany
4UCD Earth Institute - iAMB, Aachen Biology and Biotechnology - ABBt, RWTH Aachen University, Worringerweg 1, Aachen 52074, Germany
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Abstract

Pseudomonas putida KT2440 is known to metabolise glycerol via glycerol-3-phosphate using glycerol kinase an enzyme previously described as critical for glycerol metabolism (1). However, when glycerol kinase was knocked out in P. putida KT2440 it retained the ability to use glycerol as the sole carbon source, albeit with a much-extended lag period and 2 fold lower final biomass compared to the wild type strain. A metabolomic study identified glycerate as a major and the most abundant intermediate in glycerol metabolism in this mutated strain with levels 21-fold higher than wild type. Erythrose-4-phosphate was detected in the mutant strain, but not in the wild type strain. Glyceraldehyde and glycraldehyde-3-phosphate were detected at similar levels in the mutant strain and the wild type. Transcriptomic studies identified 191 genes that were more than 2-fold upregulated in the mutant compared to the wild type and 175 that were down regulated. The genes involved in short chain length fatty acid metabolism were highly upregulated in the mutant strain. The genes encoding 3-hydroxybutyrate dehydrogenase were 5.8-fold upregulated and thus the gene was cloned, expressed and purified to reveal it can act on glyceraldehyde but not glycerol as a substrate.

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Posted March 12, 2019.
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Insights into an alternative pathway for glycerol metabolism in a glycerol kinase deficient Pseudomonas putida KT2440
Meg Walsh, William Casey, Shane T. Kenny, Tanja Narancic, Lars M. Blank, Nick Wierckx, Hendrik Ballerstedt, Kevin E O Connor
bioRxiv 567230; doi: https://doi.org/10.1101/567230
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Insights into an alternative pathway for glycerol metabolism in a glycerol kinase deficient Pseudomonas putida KT2440
Meg Walsh, William Casey, Shane T. Kenny, Tanja Narancic, Lars M. Blank, Nick Wierckx, Hendrik Ballerstedt, Kevin E O Connor
bioRxiv 567230; doi: https://doi.org/10.1101/567230

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