Metabolic engineering of Escherichia coli for improved cofactor regeneration in lactate to acetoin via whole-cell conversion

Abstract

Background

Acetoin is a crucial intermediate in asymmetric syntheses of high-value chemicals and pharmaceuticals. However, its production still relies on traditional fossil-based processes. Developing efficient microbial cell factories for green and low-cost acetoin production is urgently needed.

Methods

Acetoin was produced from inexpensive and shortcut lactate substrate using whole-cell Escherichia coli through overexpression of highly active α-acetolactate synthetase and decarboxylase from Bacillus subtilis (annotated as SD). Precise stepwise optimization of pathway and enzymatic reaction was executed by (1) harboring the most efficient cofactor-regenerating system, (2) tuning expression design, (3) disrupting byproduct pathway, and (4) optimizing a series of biotransformation parameters.

Significant Findings

The recombinant E. coli successfully produced acetoin. The titer was gradually increased by expressing a pyruvate-producing gene from NAD⁺ dependent or independent system and its cofactor regeneration systems. Co-expressing lactate oxidase (lox) and catalase (cat) achieved a conversion efficiency of 50 % and eliminated NAD⁺ usage. The conversion efficiency was further pulled by knocking out acetate-generating genes (pta and poxB), thus boosting acetoin conversion to 92.4 %. Under optimized whole-cell biotransformation parameters, the highest acetoin titer reached 20.6 g/L within 30 h. This work provides an economical biomanufacturing process for acetoin from lactate via whole-cell bioconversion with remarkable yield.