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

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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.

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Acknowledgements

This work was financially supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (No. NRF-2020R1A5A1019631).

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Author notes

  1. Thai Le, Bassey Friday Bassey and Thuan Phu Nguyen-Vo: co-first authors.

Authors and Affiliations

  1. School of Energy and Chemical Engineering, Ulsan National Institute of Science and Technology, Ulsan, 44919, Republic of Korea

    Thai Le, Bassey Friday Bassey, Thuan Phu Nguyen-Vo & Sunghoon Park

  2. Department of Chemical and Biochemical Engineering, North Carolina State University, Raleigh, NC, 27606, USA

    Thuan Phu Nguyen-Vo

  3. Institute of Systems Biotechnology, Saarland University, Saarbrücken, Germany

    Thai Le

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Contributions

Thai Le: conceptualization, experimentation, data curation. Bassey Friday Bassey: conceptualization, experimentation, data curation and visualization, writing—original draft. Thuan Phu Nguyen-Vo: conceptualization, experimentation, data curation and visualization. Sunghoon Park: conceptualization, funding acquisition, supervision, validation, writing—review and editing.

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Correspondence to Sunghoon Park.

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Le, T., Bassey, B.F., Nguyen-Vo, T.P. et al. Achieving high titer and yield in the bioconversion of l-threonine to 2-hydroxybutyric acid with Escherichia coli BL21. Syst Microbiol and Biomanuf 4, 708–715 (2024). https://doi.org/10.1007/s43393-023-00224-w

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