Cascade biocatalysis for production of enantiopure (S)-2-hydroxybutyric acid using recombinant Escherichia coli with a tunable multi-enzyme-coordinate expression system

Racemize 2-hydroxybutyric acid is usually synthesized by organic methods and needs additional deracemization to obtain optically pure enantiomers for industrial application. Here we present a cascade biocatalysis system in Escherichia coli BL21 which employed L-threonine deaminase (TD), NAD-dependent L-lactate dehydrogenase (LDH) alcohol (ADH) producing (S)-2-hydroxybutyric ((S)-2-HBA) from bulk chemical L-threonine. To solve the mismatch conversion rate consumption of intermediate 2-oxobutyric (2-OBA) formed multi-enzyme catalysis reaction, ribosome binding site regulation strategy was explored control TD expression levels, achieving an eightfold alteration 2-OBA. With optimized activity ratio three enzymes using ADH NADH regeneration, recombinant strain ADH-r53 showed increased production (S)-2-HBA with highest titer 129 g/L molar yield 93% within 24 h, approximately 1.65 times that reported so far. Moreover, could easily be purified distillation, making it have great potential Additionally, our results indicated constructing tunable multi-enzyme-coordinate single cell had significance hydroxyl acids.