# De novo synthesis of L-2-aminobutyric acid in Escherichia coli based on multi-layered metabolic engineering strategies

## TL;DR

Scientists engineered E. coli to efficiently produce a non-protein amino acid used in pharmaceuticals by using advanced metabolic engineering techniques.

## Contribution

A multi-layered metabolic engineering strategy was developed to enable high-yield de novo synthesis of L-2-ABA in E. coli without antibiotics or inducers.

## Key findings

- A quorum-sensing-based circuit improved precursor toxicity and flux coordination.
- The engineered strain ABA40 produced 45.3 g/L L-2-ABA with a yield of 0.31 g/g glucose.
- Dynamic and integrated strategies proved effective for biosynthesis of non-natural amino acids.

## Abstract

L-2-Aminobutyric acid (L-2-ABA) is a non-proteinogenic amino acid and an important chiral intermediate widely used in pharmaceuticals and fine chemicals. However, its fermentative production is limited by intermediate toxicity and imbalanced metabolic flux. In this study, Escherichia coli was systematically engineered for efficient de novo synthesis of L-2-ABA using a multi-layer metabolic engineering strategy. A quorum-sensing–based dynamic control circuit was introduced to decouple cell growth from 2-oxobutyric acid formation, thereby alleviating precursor toxicity and improving flux coordination. Combined with optimization of the L-2-ABA conversion pathway, model-guided carbon flux redistribution, cofactor regeneration, and tuning of global transcriptional regulation, a high-performance production strain was obtained without the need for antibiotics or inducers. The final engineered strain ABA40 achieved 45.3 g/L L-2-ABA with a yield of 0.31 g/g glucose in a 72 h fed-batch fermentation. This study demonstrates the effectiveness of dynamic and integrated metabolic engineering strategies for the biosynthesis of non-natural amino acids.

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