Metabolic Engineering and Transhydrogenase Effects on NADPH Availability in Escherichia coli
Master of Science thesis
The ultimate goal in the field of metabolic engineering is improving cellular processes in a rational manner using engineering design principles and molecular biology techniques. The syntheses of several industrially useful compounds are cofactor-dependent. The reducing equivalent NADPH is required in several enzymatic reactions leading up to the synthesis of high-value compounds like polymers, chiral alcohols, and antibiotics. However, it’s a highly costly compound with limited intracellular availability. This study focuses on the genetic manipulation of a whole-cell system using the two transhydrogenase isoforms pntAB and udhA. Two model systems are used: 1) the production of (S)-2-chloropropionate and 2) the production of poly(3-hydroxybutyrate). Results suggest that the presence of udhA increases product yield and NADPH availability while the presence of pntAB has the opposite effect. A maximum product yield of 1.4 mole-product/mole-glucose was achieved aerobically in a pntAB-deletion strain with udhA overexpression, a 150% improvement over the wild-type control strain.
Metabolic engineering; Escherichia coli; Nadph; Cofactor; Transhydrogenase