Abstract
Biomanufacturing of chemicals using biocatalysts is an attractive strategy for the production of valuable pharmaceuticals since it is usually more economical and has a much-reduced environmental impact. However, there are often challenges such as their thermal instability that should be overcome before a newly discovered enzyme is eventually translated into industrial processes. In this work, we describe a roadmap for the development of a robust catalyst for industrial resolution of Vince lactam, a key intermediate for the synthesis of carbocyclic-nucleoside-related pharmaceuticals. By a genome mining strategy, a new (+)-γ-lactamase (MiteL) from Microbacterium testaceum was successfully discovered and biochemically characterized. In vitro studies showed that the enzyme exhibited high activity but poor enantioselectivity (E = 6.3 ± 0.2) toward racemic Vince lactam, and thus, it is not suitable for industrial applications. Based on structural modeling and docking studies, a semi-rational engineering strategy combined with an efficient screening method was then applied to improve the enantioselectivity of MiteL. Several mutants with significant shifting stereoselectivity toward (−)-γ-lactam were obtained by site-saturation mutagenesis. Synergy effects led to the final mutant F14D/Q114R/M117L, which enabled efficient acquisition of (−)-γ-lactam with a high E value (> 200). The mutant was biochemically characterized, and the docking studies suggested a plausible mechanism for its improved selectivity. Finally, a sunflower-like nanoreactor was successfully constructed to improve the mutant’s robustness via protein supramolecular self-assembly. Thus, the synergism between semi-rational protein engineering and self-assembling immobilization enabled construction of a nanoreactor with superior properties, which can be used for resolution of Vince lactam in large scale.
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Acknowledgments
This work was supported by the National Mega-project for Innovative DrugsGreat Science and Technology Projects (20198ZX09721001-007-002), Fundamental Research Funds for the Central Universities (No. XK1802-8 and XK1803-06), and National Natural Science Foundation of China (NSFC, Grant No. 21706005).
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Li, H., Gao, S., Qiu, Y. et al. Genome mining integrating semi-rational protein engineering and nanoreactor design: roadmap for a robust biocatalyst for industrial resolution of Vince lactam. Appl Microbiol Biotechnol 104, 1109–1123 (2020). https://doi.org/10.1007/s00253-019-10275-6
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DOI: https://doi.org/10.1007/s00253-019-10275-6