Hydroxyectoine Metabolism in Halomonas elongata
Hydroxyectoine Metabolism in Halomonas elongata
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DissertationPrüfungsdatum
15.04.2013Datum der Veröffentlichung
16.04.2013Erstgutachter
Galinski, Erwin A.Zweitgutachter
Kunte, Hans-JörgMetadaten
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Inhalt
The marine Gram-negative bacterium Halomonas elongata is known to produce hydroxyectoine (8), a molecule that is valuable because of its broad spectrum of potential industrial applications. However, the widespread use of hydroxyectoine has been partially limited because of the high cost of production. This is in part due to the purification steps required to remove its precursor ectoine (6), which even under the best conditions for hydroxyectoine production, is still present in a relatively high proportion (>20 %). Since conversion of hydroxyectoine to ectoine has been observed, a better understanding of the mechanisms used by H. elongata to regulate the intracellular concentration of hydroxyectoine might provide a means to optimize its production. With this goal in mind, enzymes potentially involved in the catabolism of hydroxyectoine were identified and integrated with enzymes catalyzing the known steps in the metabolism of ectoines, creating a model that helps to understand the currently undefined pathways for hydroxyectoine degradation and its conversion to ectoine.
The genes doeA, eutB and eutC from H. elongata were studied in vivo as recombinant proteins expressed in Escherichia coli, but only DoeA was proven to act on the hydroxyectoine structure, leading to its cleavage. The precise cleavage product could not be defined because at least three metabolites were produced as a result of the DoeA activity. In similar conditions, it could be proved that the E. coli strain expressing DoeA cleaved ectoine accumulating Nγ-acetyl-2,4-diaminobutyric acid (5), Nα-acetyl-2,4-diaminobutyric acid (7) and 2,4-diaminobutyric acid (4). Therefore, it is likely that the hydroxyectoine cleavage products include Nγ-acetyl-2,4-diamino-3-hydroxybutyric acid (9), Nα-acetyl-2,4-diamino-3-hydroxybutyric acid (10) and 2,4-diamino-3-hydroxybutyric acid (11). Overall, the ectoines’ cleavage products due to DoeA activity must include at least one molecule that is further modified by enzymes coded by the genome of E. coli.
The genome of H. elongata was modified so as to create seven deletion mutants that were defective in genes potentially involved in the metabolism of both ectoine and hydroxyectoine. The mutants (ΔeutC, ΔdoeAΔectC, ΔdoeAΔeutC, ΔeutBΔectC, ΔeutBΔectCΔdoeA, ΔeutBΔdoeA and ΔectAΔdoeA) became part of a group of 16 strains (including also H. elongata WT, ΔdoeA, ΔdoeB, ΔeutB, ΔectC, ΔeutBΔeutCectB::ΩΔdoeD, ΔectA, ΔectB, ΔectR) that was used to study the metabolism of ectoines in H. elongata. The analysis of their ability to use hydroxyectoine as a carbon source suggests that, in addition to genes already known to be involved in the catabolism of ectoine, the genes eutB and eutC play also a role in the degradation of hydroxyectoine. Although the precise role of these two genes remains unclear, their function in the utilization of hydroxyectoine is strongly supported by the fact that some mutants accumulate a novel metabolite (2,4-diamino-3-hydroxybutyric acid, 11), which reaches very high levels in the strain with inactivated ectB, eutB, eutC and doeD when challenged to grow on hydroxyectoine as carbon source.
The genes doeA, eutB and eutC from H. elongata were studied in vivo as recombinant proteins expressed in Escherichia coli, but only DoeA was proven to act on the hydroxyectoine structure, leading to its cleavage. The precise cleavage product could not be defined because at least three metabolites were produced as a result of the DoeA activity. In similar conditions, it could be proved that the E. coli strain expressing DoeA cleaved ectoine accumulating Nγ-acetyl-2,4-diaminobutyric acid (5), Nα-acetyl-2,4-diaminobutyric acid (7) and 2,4-diaminobutyric acid (4). Therefore, it is likely that the hydroxyectoine cleavage products include Nγ-acetyl-2,4-diamino-3-hydroxybutyric acid (9), Nα-acetyl-2,4-diamino-3-hydroxybutyric acid (10) and 2,4-diamino-3-hydroxybutyric acid (11). Overall, the ectoines’ cleavage products due to DoeA activity must include at least one molecule that is further modified by enzymes coded by the genome of E. coli.
The genome of H. elongata was modified so as to create seven deletion mutants that were defective in genes potentially involved in the metabolism of both ectoine and hydroxyectoine. The mutants (ΔeutC, ΔdoeAΔectC, ΔdoeAΔeutC, ΔeutBΔectC, ΔeutBΔectCΔdoeA, ΔeutBΔdoeA and ΔectAΔdoeA) became part of a group of 16 strains (including also H. elongata WT, ΔdoeA, ΔdoeB, ΔeutB, ΔectC, ΔeutBΔeutCectB::ΩΔdoeD, ΔectA, ΔectB, ΔectR) that was used to study the metabolism of ectoines in H. elongata. The analysis of their ability to use hydroxyectoine as a carbon source suggests that, in addition to genes already known to be involved in the catabolism of ectoine, the genes eutB and eutC play also a role in the degradation of hydroxyectoine. Although the precise role of these two genes remains unclear, their function in the utilization of hydroxyectoine is strongly supported by the fact that some mutants accumulate a novel metabolite (2,4-diamino-3-hydroxybutyric acid, 11), which reaches very high levels in the strain with inactivated ectB, eutB, eutC and doeD when challenged to grow on hydroxyectoine as carbon source.
Klassifikation (DDC)
570 Biowissenschaften, BiologieCorrea Acosta, Jhonny Edmith: Hydroxyectoine Metabolism in Halomonas elongata. - Bonn, 2013. - Dissertation, Rheinische Friedrich-Wilhelms-Universität Bonn.
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5n-31905
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5n-31905
@phdthesis{handle:20.500.11811/5671,
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5n-31905,
author = {{Jhonny Edmith Correa Acosta}},
title = {Hydroxyectoine Metabolism in Halomonas elongata},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2013,
month = apr,
note = {The marine Gram-negative bacterium Halomonas elongata is known to produce hydroxyectoine (8), a molecule that is valuable because of its broad spectrum of potential industrial applications. However, the widespread use of hydroxyectoine has been partially limited because of the high cost of production. This is in part due to the purification steps required to remove its precursor ectoine (6), which even under the best conditions for hydroxyectoine production, is still present in a relatively high proportion (>20 %). Since conversion of hydroxyectoine to ectoine has been observed, a better understanding of the mechanisms used by H. elongata to regulate the intracellular concentration of hydroxyectoine might provide a means to optimize its production. With this goal in mind, enzymes potentially involved in the catabolism of hydroxyectoine were identified and integrated with enzymes catalyzing the known steps in the metabolism of ectoines, creating a model that helps to understand the currently undefined pathways for hydroxyectoine degradation and its conversion to ectoine.
The genes doeA, eutB and eutC from H. elongata were studied in vivo as recombinant proteins expressed in Escherichia coli, but only DoeA was proven to act on the hydroxyectoine structure, leading to its cleavage. The precise cleavage product could not be defined because at least three metabolites were produced as a result of the DoeA activity. In similar conditions, it could be proved that the E. coli strain expressing DoeA cleaved ectoine accumulating Nγ-acetyl-2,4-diaminobutyric acid (5), Nα-acetyl-2,4-diaminobutyric acid (7) and 2,4-diaminobutyric acid (4). Therefore, it is likely that the hydroxyectoine cleavage products include Nγ-acetyl-2,4-diamino-3-hydroxybutyric acid (9), Nα-acetyl-2,4-diamino-3-hydroxybutyric acid (10) and 2,4-diamino-3-hydroxybutyric acid (11). Overall, the ectoines’ cleavage products due to DoeA activity must include at least one molecule that is further modified by enzymes coded by the genome of E. coli.
The genome of H. elongata was modified so as to create seven deletion mutants that were defective in genes potentially involved in the metabolism of both ectoine and hydroxyectoine. The mutants (ΔeutC, ΔdoeAΔectC, ΔdoeAΔeutC, ΔeutBΔectC, ΔeutBΔectCΔdoeA, ΔeutBΔdoeA and ΔectAΔdoeA) became part of a group of 16 strains (including also H. elongata WT, ΔdoeA, ΔdoeB, ΔeutB, ΔectC, ΔeutBΔeutCectB::ΩΔdoeD, ΔectA, ΔectB, ΔectR) that was used to study the metabolism of ectoines in H. elongata. The analysis of their ability to use hydroxyectoine as a carbon source suggests that, in addition to genes already known to be involved in the catabolism of ectoine, the genes eutB and eutC play also a role in the degradation of hydroxyectoine. Although the precise role of these two genes remains unclear, their function in the utilization of hydroxyectoine is strongly supported by the fact that some mutants accumulate a novel metabolite (2,4-diamino-3-hydroxybutyric acid, 11), which reaches very high levels in the strain with inactivated ectB, eutB, eutC and doeD when challenged to grow on hydroxyectoine as carbon source.},
url = {https://hdl.handle.net/20.500.11811/5671}
}
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5n-31905,
author = {{Jhonny Edmith Correa Acosta}},
title = {Hydroxyectoine Metabolism in Halomonas elongata},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2013,
month = apr,
note = {The marine Gram-negative bacterium Halomonas elongata is known to produce hydroxyectoine (8), a molecule that is valuable because of its broad spectrum of potential industrial applications. However, the widespread use of hydroxyectoine has been partially limited because of the high cost of production. This is in part due to the purification steps required to remove its precursor ectoine (6), which even under the best conditions for hydroxyectoine production, is still present in a relatively high proportion (>20 %). Since conversion of hydroxyectoine to ectoine has been observed, a better understanding of the mechanisms used by H. elongata to regulate the intracellular concentration of hydroxyectoine might provide a means to optimize its production. With this goal in mind, enzymes potentially involved in the catabolism of hydroxyectoine were identified and integrated with enzymes catalyzing the known steps in the metabolism of ectoines, creating a model that helps to understand the currently undefined pathways for hydroxyectoine degradation and its conversion to ectoine.
The genes doeA, eutB and eutC from H. elongata were studied in vivo as recombinant proteins expressed in Escherichia coli, but only DoeA was proven to act on the hydroxyectoine structure, leading to its cleavage. The precise cleavage product could not be defined because at least three metabolites were produced as a result of the DoeA activity. In similar conditions, it could be proved that the E. coli strain expressing DoeA cleaved ectoine accumulating Nγ-acetyl-2,4-diaminobutyric acid (5), Nα-acetyl-2,4-diaminobutyric acid (7) and 2,4-diaminobutyric acid (4). Therefore, it is likely that the hydroxyectoine cleavage products include Nγ-acetyl-2,4-diamino-3-hydroxybutyric acid (9), Nα-acetyl-2,4-diamino-3-hydroxybutyric acid (10) and 2,4-diamino-3-hydroxybutyric acid (11). Overall, the ectoines’ cleavage products due to DoeA activity must include at least one molecule that is further modified by enzymes coded by the genome of E. coli.
The genome of H. elongata was modified so as to create seven deletion mutants that were defective in genes potentially involved in the metabolism of both ectoine and hydroxyectoine. The mutants (ΔeutC, ΔdoeAΔectC, ΔdoeAΔeutC, ΔeutBΔectC, ΔeutBΔectCΔdoeA, ΔeutBΔdoeA and ΔectAΔdoeA) became part of a group of 16 strains (including also H. elongata WT, ΔdoeA, ΔdoeB, ΔeutB, ΔectC, ΔeutBΔeutCectB::ΩΔdoeD, ΔectA, ΔectB, ΔectR) that was used to study the metabolism of ectoines in H. elongata. The analysis of their ability to use hydroxyectoine as a carbon source suggests that, in addition to genes already known to be involved in the catabolism of ectoine, the genes eutB and eutC play also a role in the degradation of hydroxyectoine. Although the precise role of these two genes remains unclear, their function in the utilization of hydroxyectoine is strongly supported by the fact that some mutants accumulate a novel metabolite (2,4-diamino-3-hydroxybutyric acid, 11), which reaches very high levels in the strain with inactivated ectB, eutB, eutC and doeD when challenged to grow on hydroxyectoine as carbon source.},
url = {https://hdl.handle.net/20.500.11811/5671}
}
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