ME: Metabolic Engineering of PABA-Free Folic Acid Production

Carnegie Mellon University, Pittsburgh PA

Investigators

Abstract

The objective of this project is to metabolically engineer bacteria to produce high levels of folic acid directly from glucose as opposed to transforming a more expensive raw material such as para amino benzoic acid (PABA). Phosphoenolpyruvate (PEP), erythrose-4-phosphate (E4P), glutamate, and GTP are the glucose-derived precursors of folic acid. Thus, from the concentration and formation flux standpoints, these four precursors need to be engineered to be amply available. The metabolic engineering approach will initially focus on increasing further the formation of chorismate, a PEP and E4P-derived intermediate in folic acid biosynthesis. The proposed tasks are: (1) alter regulation to increase the flux to chorismate while limiting the side reactions from chorismate to amino acids, (2) model and map the fluxes in the folic acid-producing candidate strain of B. subtilis using the investigators software and NMR analyses, (3) test the strains in batch and continuous cultivation, and (4) based on the results of steps 1-3, further fine tune the pathways. After achieving success on the chorismate-side of the folic acid biosynthetic pathway, subsequent work can explore whether other precursors (e.g. GTP) have become limiting.