Activating a dormant metabolic pathway for high-temperature l-alanine production in Bacillus licheniformis

Xiao Han; Jiongqin Liu; Yutong Wu; Yuhan Yang; Fei Tao; Ping Xu

doi:10.1016/j.isci.2023.106397

Activating a dormant metabolic pathway for high-temperature l-alanine production in Bacillus licheniformis

iScience. 2023 Mar 15;26(4):106397. doi: 10.1016/j.isci.2023.106397. eCollection 2023 Apr 21.

Authors

Xiao Han¹, Jiongqin Liu¹, Yutong Wu¹, Yuhan Yang¹, Fei Tao¹, Ping Xu¹

Affiliation

¹ 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.

Abstract

l-Alanine is an important amino acid widely used in food, medicine, materials, and other fields. Here, we develop Bacillus licheniformis 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 B. licheniformis 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.

Keywords: Applied microbiology; Microbial biotechnology; Microbiology.