Precision Proteolysis of Triosephosphate Isomerase of Escherichia coli Boosts Dihydroxyacetone Phosphate Biosynthesis

Dihydroxyacetone phosphate (DHAP), a key metabolic intermediate of the Embden–Meyerhof–Parnas pathway of Escherichia coli, has a considerable value as a precursor of high-added-value compounds. While eliminating the triosephosphate isomerase (tpiA) gene should theoretically channel 50% of the glycolytic flux into dead-end production of DHAP, the permanent loss of this activity triggers alternative routes that decrease (rather than increase) DHAP levels. To address this limitation and establish transient regimes of high DHAP biosynthesis, we harnessed the unusual structural tolerance of TpiA for designing a variant of the enzyme that can be rapidly degraded, thus temporarily adopting a null phenotype. This was achieved through conditional expression of the highly specific viral protease PPV-NIa, which cleaves a cognate recognition sequence strategically engineered into an exposed, permissive loop on the protein surface. Optimization of such an in vivo proteolytic device resulted in fully active TpiA variants that become nearly instantly destroyed upon induction of NIa in trans, which was itself engineered as an ON/OFF switch. Metabolomic data of an engineered E. coli strain genomically encoding the cognate genetic device showed that precise post-transcriptional targeting of TpiA leads to a substantial transitory increase of DHAP with minimal disturbance of other typical intermediates. The general value of targeting enzymes in central carbon metabolism, such as TpiA, is discussed in light of systems metabolic engineering.