Achieving high titer and yield in the bioconversion of l-threonine to 2-hydroxybutyric acid with Escherichia coli BL21

Abstract

The study investigated the enhanced production of 2-hydroxybutyric acid (2-HBA) from threonine using a two-step whole-cell bioconversion by recombinant Escherichia coli BL21 (DE3) overexpressing threonine dehydratase and keto-reductase. To address the rate-limiting step posed by NADH regeneration for the keto-reductase reaction converting 2-ketobutyric acid (2-KBA) to 2-HBA, formate dehydrogenase from Candida boidinii was overexpressed under the T7 promoter, resulting in a high titer of 1015 mM and a yield of 0.70 mol/mol. Furthermore, the yield was improved by disrupting three enzymes responsible for the degradation of the intermediate (2-KBA), pyruvate-formate lyase (PflB), pyruvate oxidase (PoxB), and pyruvate dehydrogenase complex (PDHc), leading to an impressive yield of 0.99 mol/mol, closely approaching the theoretical maximum of 1.00 mol/mol. The triple mutant, designed to prevent 2-KBA degradation, achieved a remarkable titer of 1,400 mM and volumetric productivity of 58 mmol/L/h. To the best of our knowledge, this achievement represents the highest reported titer and yield for 2-HBA production to date.

Keywords

2-hydroxybutyric acid (2-HBA) / Whole-cell bioconversion / Threonine / Degradation pathway / High titer

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Funding

National Research Foundation of Korea (NRF)(NRF-2020R1A5A1019631)