Konstruktion synthetischer Stoffwechselwege zur Produktion von(R)-Benzylsuccinat und weiteren aromatischen Metaboliten

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.