Ni Zifu, Zhang Linshang, Nie Azhen, Wang Huan, Wu Xiaoling
National Engineering Research Center of Wheat and Corn Further Processing, Henan University of Technology, Zhengzhou 450001, China.
College of Biological Engineering, Henan University of Technology, Zhengzhou 450001, China.
Biomolecules. 2024 Dec 5;14(12):1553. doi: 10.3390/biom14121553.
As the only naturally occurring β-amino acid, β-alanine has important application prospects in many fields. Driven by the huge demand, biosynthesis is becoming more and more popular as a potential alternative to the chemical synthesis of β-alanine. Although the direct pathway from L-aspartic acid to β-alanine, catalyzed by L-aspartic acid-α-decarboxylase (PanD), is ideal for β-alanine synthesis, it is hindered by the high cost of the substrate and limited economic viability. In this work, a cell-free dual enzyme cascade system based on methylaspartate lyase (EcMAL) and panD was constructed to safely and efficiently synthesize β-alanine using fumarate as a substrate. Taking the previously engineered EcMAL as the target, CgPanD was finally screened as the best candidate through gene mining, sequence alignment, and enzyme property analysis. Finally, under the optimal conditions of 35 °C, pH 8.0, and EcMAL: CgPanD concentration ratio of 1:5, the yield of β-alanine reached 80% theoretical yield within 120 min. This study provides a potential strategy for the biosynthesis of β-alanine, paving the way for future industrial-scale production.
作为唯一天然存在的β-氨基酸，β-丙氨酸在许多领域具有重要的应用前景。在巨大需求的推动下，生物合成作为β-丙氨酸化学合成的潜在替代方法正变得越来越流行。尽管由L-天冬氨酸-α-脱羧酶（PanD）催化的从L-天冬氨酸到β-丙氨酸的直接途径是β-丙氨酸合成的理想途径，但它受到底物成本高和经济可行性有限的阻碍。在这项工作中，构建了一种基于甲基天冬氨酸裂解酶（EcMAL）和PanD的无细胞双酶级联系统，以富马酸为底物安全高效地合成β-丙氨酸。以先前工程改造的EcMAL为靶点，通过基因挖掘、序列比对和酶性质分析，最终筛选出CgPanD作为最佳候选酶。最后，在35℃、pH 8.0以及EcMAL与CgPanD浓度比为1:5的最佳条件下，β-丙氨酸的产量在120分钟内达到理论产量的80%。本研究为β-丙氨酸的生物合成提供了一种潜在策略，为未来的工业规模生产铺平了道路。
