Project dates: 01/10/2010 - 30/09/2027
Analysis of the flux and accumulation of intermediates of metabolic pathways is currently possible using analytical chemistry. However, these methods are expensive, labour intensive and can only generate snapshots of a highly dynamic process. We aim to develop a microbial platform allowing the monitoring of initially the key steps, and eventually every single step, of the MEP pathway developing protein based biosensors expressed in living cells. This system will allow us to observe the metabolism in real time and follow the dynamics of the carbon flux and the change in intermediate pools that are generated in response to environmental or industrial perturbations. We aim to generate a microbial platform for real time monitoring and HTP evaluation of newly engineered isoprenoid producing strains using protein-based biosensors.
The first step in this program is generation of a protein biosensor for the MEP pathway intermediate MecPP. MecPP is a critical intermediate molecule that accumulates in the cell when MEP pathway flux increases. The two enzymes downstream of this key intermediate are oxygen – sensitive iron-sulphur dependent enzymes. Moreover, when MEcPP accumulates in plant chloroplasts (where the MEP pathway is hosted) it is exported to the nucleus as a retrograde signalling molecule, driving expression of stress response genes that produce proteins that help mitigate oxidative stress in the chloroplast. We are developing protein biosensors based on chemically induced dimerization (CID) of components with high affinity and specificity identified in mRNA display screenings. These dedicated biosensors will allow the rapid and accurate measurement of MecPP accumulation in in vitro devices and in living cells. This will help us better understand the biological roles of this key intermediate and develop engineering strategies to increase flux through the pathway.
Chief Investigators
Project team member
- Irfan Farabi (QUT)
Project funding
