Bioresources and Bioprocessing ( IF 4.3 ) Pub Date : 2022-03-21 , DOI: 10.1186/s40643-022-00510-w Yang Xi 1 , Lidan Ye 1, 2 , Hongwei Yu 1
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As an important monomer for bio-based nylons PA5X, cadaverine is mainly produced by enzymatic decarboxylation of L-lysine. A key issue with this process is the instability of L-lysine decarboxylase (CadA) during the reaction due to the dissociation of CadA subunits with the accumulation of alkaline cadaverine. In this work, we attempted to improve the thermal and alkaline stability of CadA by combining directed evolution and computation-guided virtual screening. Interestingly, site 477 residue located at the protein surface and not the decamer interface was found as a hotspot in directed evolution. By combinatorial mutagenesis of the positive mutations obtained by directed evolution and virtual screening with the previously reported T88S mutation, K477R/E445Q/T88S/F102V was generated as the best mutant, delivering 37% improvement of cadaverine yield at 50 ºC and pH 8.0. Molecular dynamics simulations suggested the improved rigidity of regional structures, increased number of salt bridges, and enhancement of hydrogen bonds at the multimeric interface as possible origins of the improved stability of the mutant. Using this four-point mutant, 160.7 g/L of cadaverine was produced from 2.0 M Lysine hydrochloride at 50 °C without pH regulation, with a conversion of 78.5%, whereas the wild type produced 143.7 g/L cadaverine, corresponding to 70% conversion. This work shows the combination of directed evolution and virtual screening as an efficient protein engineering strategy.
Graphical Abstract
中文翻译：
通过结合定向进化和计算引导的虚拟筛选增强 L-赖氨酸脱羧酶 CadA 的热稳定性和碱稳定性
尸胺作为生物基尼龙PA5X的重要单体，主要由L-赖氨酸酶法脱羧产生。该过程的一个关键问题是反应过程中 L-赖氨酸脱羧酶 (CadA) 的不稳定性，这是由于 CadA 亚基随着碱性尸胺的积累而解离。在这项工作中，我们尝试通过结合定向进化和计算引导的虚拟筛选来提高 CadA 的热稳定性和碱稳定性。有趣的是，位于蛋白质表面而不是十聚体界面的位点 477 残基被发现是定向进化的热点。通过定向进化和虚拟筛选获得的阳性突变与之前报道的 T88S 突变的组合诱变，产生了 K477R/E445Q/T88S/F102V 作为最佳突变体，在 50 ℃和 pH 8.0 下尸胺产量提高了 37%。分子动力学模拟表明，区域结构刚性的提高、盐桥数量的增加以及多聚体界面氢键的增强可能是突变体稳定性提高的原因。使用该四点突变体，在 50 °C 下，不进行 pH 调节，从 2.0 M 赖氨酸盐酸盐中产生了 160.7 g/L 尸胺，转化率为 78.5%，而野生型产生了 143.7 g/L 尸胺，相当于 70%转换。这项工作展示了定向进化和虚拟筛选的结合作为一种有效的蛋白质工程策略。
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