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