Han Xiao, Liu Jiongqin, Wu Yutong, Yang Yuhan, Tao Fei, Xu Ping
State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China.
iScience. 2023 Mar 15;26(4):106397. doi: 10.1016/j.isci.2023.106397. eCollection 2023 Apr 21.
l-Alanine is an important amino acid widely used in food, medicine, materials, and other fields. Here, we develop as an efficient l-alanine microbial cell factory capable of realizing high-temperature fermentation. By enhancing the glycolytic pathway, knocking out the by-product pathways and overexpressing the thermostable alanine dehydrogenase, the engineered . strain BLA3 produced 93.7 g/L optically pure l-alanine at 50°C. Subsequently, d-alanine dependence of an alanine racemase-deficient strain is relieved by adaptive laboratory evolution, implying that a dormant alternative pathway for d-alanine synthesis is activated in the evolved strain. The d-amino acid aminotransferase Dat1 is shown to be a key enzyme in the dormant alternative pathway. Molecular mechanism of the d-alanine dependence is revealed via mutational analysis. This study demonstrates a novel technology for high-temperature l-alanine production and shows that activating dormant metabolic pathway(s) is an effective strategy of metabolic engineering.
L-丙氨酸是一种重要的氨基酸，广泛应用于食品、医药、材料等领域。在此，我们开发了一种能够实现高温发酵的高效L-丙氨酸微生物细胞工厂。通过增强糖酵解途径、敲除副产物途径并过表达耐热丙氨酸脱氢酶，工程化的菌株BLA3在50°C下产生了93.7 g/L的光学纯L-丙氨酸。随后，通过适应性实验室进化缓解了丙氨酸消旋酶缺陷菌株对D-丙氨酸的依赖性，这意味着在进化菌株中激活了一条用于D-丙氨酸合成的休眠替代途径。D-氨基酸转氨酶Dat1被证明是休眠替代途径中的关键酶。通过突变分析揭示了D-丙氨酸依赖性的分子机制。本研究展示了一种高温生产L-丙氨酸的新技术，并表明激活休眠代谢途径是代谢工程的一种有效策略。
