Design and Application of non native enzyme cascades in living cell factories

The general aim of this pilot project is to apply a retrosynthetic approach for the synthesis of chiral building blocks via a multi-enzymatic synthesis in living cells. Thereby, a de novo designed pathway of "non" related enzymes will be introduced into the well established model organism Escherichia coli. The enzymatic cascade is designed by coupling enzymes according to their functional group transformations of a particular class of substrates. In this study carboxylic esters will be hydrolyzed to the corresponding alcohol and subsequently oxidized to the aldehyde moiety. Hence, the de novo pathway will be linked to the central carbon metabolism of E.coli. Thereby the glycolytic substrate dihydroxy acetone and the corresponding product of the de novo pathway will undergo the final step of the enzymatic cascade. By merging both pathways the maintaining metabolism of the host and the new pathway gets highly inter connected. In order to study as many parameters as possible in the model systems it is the our general vision to combine methods and tools of metabolic engineering, synthetic biology, systems biology, and biocatalysis, in particular a) metabolic flux analysis (MFA), b) metabolomics (LC-NMR-MS; LC-MS/MS), c) mathematic modeling (flux balance analysis (FBA)), and d) retrosynthetic analysis to conduct de novo design and application of non-natural biosynthetic platforms for multi-step-reactions in microorganisms.

This research project aimed at the development of artificial cell factories. Here, a production line, consisting of enzymes that are necessary for the production of chemical compounds, was built in the simple bacterium Escherichia coli. The construction of this assembly line was done by genetic manipulation of the cell. After demonstrating that it is possible to develop such cell factories in principle, we have gone one step further and have begun to optimize the production line so that these cell factories can continue to be used industrially in the future. In doing so, we pursued different strategies and also realized new ideas. For example, instead of a large number of experiments in the laboratory, we resorted to computer simulations in order to quickly achieve the desired production optimization. In addition, we have developed a new concept for the construction of such cell conveyor belts. In the process, intermediates that had fallen off the assembly line were returned to the production line and resulted in an optimized overall process. Specifically, we have tried to produce modified sugar molecules, which are then used as starting materials for drugs or as building blocks in the food industry. Our research project was very successful and we were able to publish our results in renowned journals and present them at symposia.