Implementation of thiamine pyrophosphate (TPP) riboswitches as synthetic biosensors and regulatory tools in cyanobacteria

Sammanfattning: The natural occurrence of the non-mevalonate (also called MEP after the compound methyl-erythriol phosphate) pathway in the model cyanobacterium Synechocystis sp. PCC 6803 allows for biosynthesis of various high-value terpenoid compounds. An important co-factor of this pathway is thiamine pyrophosphate (TPP), coenzyme to the 1- deoxy-D-xylulose-5-phosphate synthase (DXS) reaction in the initial step of the MEP pathway. Concurrently, TPP biosynthesis derives partially from 1-deoxy-D-xylulose phosphate, the product of DXS. This makes TPP a potentially significant measure of MEP pathway activity, and thus terpenoid productivity. The implementation of a molecular biosensor for TPP could be a promising approach towards on-line assessment and feedback regulation of MEP pathway activity and this application is therefore investigated in this work. Riboswitches have been suggested as versatile RNA-based tools for biotechnological applications in bacteria, including various cyanobacterial species. However, TPP-responsive riboswitches have not been addressed in cyanobacteria thus far. This project therefore aims at the evaluation and implementation of TPP-responsive riboswitches in Synechocystis, using a yellow fluorescent reporter protein as quantitative readout of translational regulation. Native putative OFF-switches from two cyanobacterial species are investigated along with one synthetic ON-switch, originally based on the native riboswitch from E. coli. The induction effects are assessed on both RNA and protein level for both TPP and its precursor thiamine. The synthetic riboswitch is found to be effective in Synechocystis and is further examined for its dynamic range. Several protocols for fluorescence and transcript level experiments are developed. Several continuation experiments are suggested, including further investigation of the cyanobacterial OFF-switches.