Abstract
(R)-Selective ω-transaminase (ω-TA) is a key enzyme for the asymmetric reductive amination of carbonyl compounds to produce chiral amines which are essential parts of many therapeutic compounds. However, its practical industrial applications are hindered by the low catalytic efficiency and poor thermostability of naturally occurring enzymes. In this work, we report the molecular modification of (R)-selective ω-TA from Aspergillus terreus (AtTA) to allow asymmetric reductive amination of 4-hydroxy-2-butanone, producing (R)-3-amino-1-butanol. Based on substrate docking analysis, 4 residues in the substrate tunnel and binding pocket of AtTA were selected as mutation hotspots. The screening procedure was facilitated by the construction of a “small-intelligent” library and the use of thin-layer chromatography for preliminary screening. The resulting mutant AtTA-M5 exhibited a 9.6-fold higher kcat/Km value and 9.4 °C higher \( {\mathrm{T}}_{1/2}^{10\min } \) than that of wild-type AtTA. Furthermore, the conversion of 20 and 50 g L−1 4-hydroxy-2-butanone by AtTA-M5 reached 90.8% and 79.1%, suggesting significant potential for production of (R)-3-amino-1-butanol. Under the same conditions, wild-type AtTA achieved less than 5% conversion. Moreover, the key mutation (S215P in AtTA) was validated in 7 other (R)-selective ω-TAs, indicating its general applicability in improving the catalytic efficiency of homologous (R)-selective ω-TAs.
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Funding
This work was financially supported by the Natural Science Fund for Colleges and Universities in Jiangsu Province (18KJB416006 and 17KJB350011), the Natural Science Foundation of Jiangsu Province (BK20160575), the National Natural Science Foundation of China (81703363), the Scientific Research Starting Foundation of Taizhou University (TZXY2017QDJJ008), and the Social Development Program of Taizhou (TS201916).
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Xinxing Gao and Xin Zhang performed the experiments. Xinxing Gao, Nianqing Zhu, and Yi Mou conceived and designed the research. Hailing Zhang and Xin Liu analyzed the data and supervised the projected. Xinxing Gao wrote the initial draft of the paper, and Xin Zhang and Pinghe Wei revised the manuscript.
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Key points
• A “small-intelligent” library was constructed to reduce the screening library size.
• Thin-layer chromatography was applied for high-throughput preliminary screening.
• AtTA-M5 displayed significantly enhanced catalytic efficiency and thermostability.
• The identified mutation was validated in other homologous (R)-selective ω-TAs.
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Gao, X., Zhang, X., Zhu, N. et al. Reshaping the substrate binding region of (R)-selective ω-transaminase for asymmetric synthesis of (R)-3-amino-1-butanol. Appl Microbiol Biotechnol 104, 3959–3969 (2020). https://doi.org/10.1007/s00253-020-10539-6
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DOI: https://doi.org/10.1007/s00253-020-10539-6