Konstruktion synthetischer Stoffwechselwege zur Produktion von(R)-Benzylsuccinat und weiteren aromatischen Metaboliten
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Philipps-Universität Marburg
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Abstract
The aim of this work was the generation of a synthetic bacterial production strain, which produces
new fermentation products via modified anaerobic pathways. In a first step a synthetic module for
the production of benzoyl-CoA as biosynthetic precursor was established in the host organism
Escherichia coli. Therefore a benzoatetransporter (encoded in the benK gene from the benzoate
degrading bacterium Aromatoleum aromaticum) and a bezoate-CoA ligase (encoded in the bclAgene
from A. aromaticum) were linked. In this work this biosynthetic module for benzoyl-CoA formation
was used for the developement of bacterial pathwaysfor (R)-benzylsuccinate synthesis or for the
synthesis of biphenyls, which occour in nature as secondary plant metabolites. The usage of this
benzoyl-CoA biosynthesis module can be more expanded by using it as a building block for further
synthetic pathways, which are benzoyl-CoA depending. Hence, it can be used for the synthesis of
many useful natural products, which are produced via a benzoyl-CoA intermediate.
i) Benzylsuccinate, an aromatic dicarboxylic acid, formed as first intermediate during
anaerobic toluene degradation, is of potential biotechnological interest e. g. for the
formation of bio-polymers. For the biological production of benzylsuccinate a second
biosynthetic module was generated. This module contains genes coding for seven
enzymes from Geobacter metallireducens, which occur in anaerobic toluene degradation.
Normally these enzymes catalyse the degradation of the first intermediate
benzylsuccinate to benzoyl-CoA and succinate via a modified β-oxidation pathway.
During co-expression of both biosynthetic modules in E. coliand feeding with benzoate
indeed benzylsuccinate was formed by the reversed β-oxidation pathway in significant
amounts. The yield was increased 1000-fold by switching from aerobic respiration to
anaerobic mixed acid fermentation and fumarate respiration (from 3 nM up to 5 µM).
This result verifies the idea of the project, to form synthetic anaerobic production strains.
The production could be further increased by a factor of 3.5 by the addition of a gene
coding for an unspecific mechanosensitive channel, which exports the formed metabolite
into the medium. This system can now be further optimized by genetical modifications
supported by mathematical modelling of the pathway and by optimization of the
production conditions to increase the production upto a possible industrial application.
ii) For the synthesis of the secondary plant metabolite3,5-dihydroxybiphenyl the
biosynthetic module for benzoyl-CoA formation was combined with genes coding for a
biphenyl synthase from the rowan berry (lat. Sorbus aucuparia) and a malonyl-CoA
synthetase from Paracoccus denitrificans. These genes were coexpressed in the host organisms E. coliand Shimwellia blattae. The malonyl-CoA synthetase was added to
ensure the supply of the second precursor malonyl-CoA. Although all enzymes necessary
for this pathway were produced with sufficient activities, no biphenyl formation was
detected so far. Instead the generated synthetic bacteria produced large amounts of
indole, which is known as a stress metabolite in enterobacteria. In addition to indole
some further yet unknown metabolites were detected.One of these metabolites is
formed from malonate. Further investigations are necessary to identify this metabolite
and to figure out how it is linked to the syntheticbiphenyl pathway.
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Dates
Created: 2013Issued: 2013-06-12Updated: 2013-06-12
Faculty
Fachbereich Biologie
Publisher
Philipps-Universität Marburg
Language
ger
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DoctoralThesis
Keywords
benzylsuccinatebeta-OxidationBenzylsuccinatbiotechnologyBiphenylBiotechnologieEnzymologiesynthetic metabolic pathwayanaerobbeta-oxidationbiphenylBiokatalyseSynthetischer Stoffwechselweganaerobe,enzymemology
DFG-subjects
BenzylsuccinatBiotechnologieEnzymologieSynthetischer StoffwechselwegBiokatalysebeta-OxidationanaerobBiphenyl
DDC-Numbers
570
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Mock, Johanna (1035626314): Konstruktion synthetischer Stoffwechselwege zur Produktion von(R)-Benzylsuccinat und weiteren aromatischen Metaboliten. : Philipps-Universität Marburg 2013-06-12. DOI: https://doi.org/10.17192/z2013.0339.
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This item has been published with the following license: In Copyright