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

## TL;DR

Scientists engineered a way to temporarily disable an enzyme in E. coli to boost the production of a valuable metabolic intermediate.

## Contribution

A protease-based system was developed to transiently inactivate TpiA, enabling controlled DHAP biosynthesis.

## Key findings

- Conditional degradation of TpiA using PPV-NIa protease significantly increased DHAP levels.
- The engineered system minimally disrupted other metabolic intermediates.
- This approach offers a strategy for transient enzyme inactivation in metabolic engineering.

## Abstract

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.