A Hybrid Modelling Approach to Identify Inhibiting Factors in Cell-Free Protein Synthesis

Cell-free protein synthesis provides lower production rates compared to conventional protein synthesis in prokaryotic organisms and is limited in reaction time. Efforts were made to investigate limiting steps in the in vitro synthesis to decrease the gap between in vivo and in vitro. The latter shows significantly lower productivities due to an unfavorable protein composition in the translation machinery as well as limited synthesis time caused by accumulating by-products that inhibit the translation machinery. Recent results have shown that different intermediates of the glycolysis as substrates for ATP regeneration strongly influence the overall duration of the cell-free reaction. It is supposed that the by-products of the mixed acid fermentation lead to a pH drop that stalls protein synthesis.

Another phenomenon that might limit synthesis time is the release of organic phosphate by some of the intermediates like PEP or G6P. Released phosphate molecules are indicated to bind free magnesium ions, which then are no longer available to bind ATP. Therefore phosphate release restricts the amount of energy available for protein synthesis.

Many efforts were performed on an experimental basis, however without the support of mathematical modelling. Influences of organic acids and phosphate were just published as indications.

Our goal is to identify the mechanisms that lead to shortened reaction duration based on dynamic flux analysis of a coupled energy regeneration and sequence based transcription-translation system. Focus will be the incorporation of a system to calculate free magnesium ions and its influence on the available ATP pool. This hybrid system allows incorporation of boundaries in terms of critical concentrations for expected by-products.