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

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 mi-crobial 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 degrees 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 alterna-tive pathway. Molecular mechanism of the D-alanine dependence is revealed via mutational analysis. This study demonstrates a novel technology for high -temper-ature L-alanine production and shows that activating dormant metabolic path-way(s) is an effective strategy of metabolic engineering.