College of Life Science, Hebei Normal University, Shijiazhuang, 050024, China.
Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China.
Microb Cell Fact. 2024 Jun 4;23(1):164. doi: 10.1186/s12934-024-02439-5.
Optically active D-amino acids are widely used as intermediates in the synthesis of antibiotics, insecticides, and peptide hormones. Currently, the two-enzyme cascade reaction is the most efficient way to produce D-amino acids using enzymes DHdt and DCase, but DCase is susceptible to heat inactivation. Here, to enhance the enzymatic activity and thermal stability of DCase, a rational design software "Feitian" was developed based on k prediction using the deep learning approach.
According to empirical design and prediction of "Feitian" software, six single-point mutants with high k value were selected and successfully constructed by site-directed mutagenesis. Out of six, three mutants (Q4C, T212S, and A302C) showed higher enzymatic activity than the wild-type. Furthermore, the combined triple-point mutant DCase-M3 (Q4C/T212S/A302C) exhibited a 4.25-fold increase in activity (29.77 ± 4.52 U) and a 2.25-fold increase in thermal stability as compared to the wild-type, respectively. Through the whole-cell reaction, the high titer of D-HPG (2.57 ± 0.43 mM) was produced by the mutant Q4C/T212S/A302C, which was about 2.04-fold of the wild-type. Molecular dynamics simulation results showed that DCase-M3 significantly enhances the rigidity of the catalytic site and thus increases the activity of DCase-M3.
In this study, an efficient rational design software "Feitian" was successfully developed with a prediction accuracy of about 50% in enzymatic activity. A triple-point mutant DCase-M3 (Q4C/T212S/A302C) with enhanced enzymatic activity and thermostability was successfully obtained, which could be applied to the development of a fully enzymatic process for the industrial production of D-HPG.
光学活性 D-氨基酸被广泛用作抗生素、杀虫剂和肽激素合成的中间体。目前，使用 DHdt 和 DCase 两种酶的级联反应是生产 D-氨基酸最有效的方法，但 DCase 易受热失活。在这里，为了提高 DCase 的酶活性和热稳定性，我们基于深度学习方法的 k 预测，开发了一个名为“飞天”的理性设计软件。
根据经验设计和“飞天”软件的预测，选择了六个具有高 k 值的单点突变体，并通过定点突变成功构建。在这六个突变体中，有三个突变体（Q4C、T212S 和 A302C）的酶活性高于野生型。此外，三重突变体 DCase-M3（Q4C/T212S/A302C）的酶活性提高了 4.25 倍（29.77±4.52 U），热稳定性提高了 2.25 倍，分别比野生型高。通过全细胞反应，突变体 Q4C/T212S/A302C 产生了高浓度的 D-HPG（2.57±0.43 mM），约为野生型的 2.04 倍。分子动力学模拟结果表明，DCase-M3 显著增强了催化位点的刚性，从而提高了 DCase-M3 的活性。
本研究成功开发了一种高效的理性设计软件“飞天”，其酶活性预测准确率约为 50%。成功获得了酶活性和热稳定性增强的三重突变体 DCase-M3（Q4C/T212S/A302C），可应用于 D-HPG 工业生产的全酶法工艺的开发。
