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Increased productivity of l-2-aminobutyric acid and total turnover number of NAD+/NADH in a one-pot system through enhanced thermostability of l-threonine deaminase

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Abstract

Objective

To strengthen NADH regeneration in the biosynthesis of l-2-aminobutyric acid (l-ABA).

Results

l-Threonine deaminase (l-TD) from Escherichia coli K12 was modified by directed evolution and rational design to improve its endurance to heat treatment. The half-life of mutant G323D/F510L/T344A at 42 °C increased from 10 to 210 min, a 20-fold increase compared to the wild-type l-TD, and the temperature at which the activity of the enzyme decreased by 50% in 15 min increased from 39 to 53 °C. The mutant together with thermostable l-leucine dehydrogenase from Bacillus sphaericus DSM730 and formate dehydrogenase from Candida boidinii constituted a one-pot system for l-ABA biosynthesis. Employing preheat treatment in the one-pot system, the biosynthesis of l-ABA and total turnover number of NAD+/NADH were 0.993 M and 16,469, in contrast to 0.635 M and 10,531 with wild-type l-TD, respectively.

Conclusions

By using the engineered l-TD during endured preheat treatment, the one-pot system has achieved a higher productivity of l-ABA and total turnover number of coenzyme.

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Acknowledgements

This work was funded by the National Hi-Tech Research and Development Program of China (2014AA022105).

Supporting information

Supplementary Table 1—Bacterial strains and plasmids used in this study.

Supplementary Table 2—Primers used in this study.

Supplementary Table 3—Biotransformation of l-ABA by the original and revised systems.

Supplementary Fig. 1—Electronic interaction between substrate l-threonine and (a) wild-type l-TD or (b) G323D/F510L/T344A.

Author information

Authors and Affiliations

  1. Key Laboratory of Biomass Chemical Engineering of Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, 38 Zhe-da Road, Hangzhou, 310007, Zhejiang, People’s Republic of China

    Ying Wang, Guo-Si Li, Pei Qiao, Ling Lin, Hai-Long Xue, Li Zhu, Mian-Bin Wu, Jian-Ping Lin & Li-Rong Yang

Authors
  1. Ying Wang
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  2. Guo-Si Li
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  3. Pei Qiao
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  4. Ling Lin
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  5. Hai-Long Xue
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  6. Li Zhu
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  7. Mian-Bin Wu
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  8. Jian-Ping Lin
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  9. Li-Rong Yang
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Corresponding author

Correspondence to Jian-Ping Lin.

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Wang, Y., Li, GS., Qiao, P. et al. Increased productivity of l-2-aminobutyric acid and total turnover number of NAD+/NADH in a one-pot system through enhanced thermostability of l-threonine deaminase. Biotechnol Lett 40, 1551–1559 (2018). https://doi.org/10.1007/s10529-018-2607-3

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  • DOI: https://doi.org/10.1007/s10529-018-2607-3

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