Efficient Fermentative Production of d-Alanine and Other d-Amino Acids by Metabolically Engineered Corynebacterium glutamicum

d-Amino acids (d-AAs) have wide applications in industries such as pharmaceutical, food, and cosmetics due to their unique properties. Currently, the production of d-AAs has relied on chemical synthesis or enzyme catalysts, and it is challenging to produce d-AAs via direct fermentation from glucose. We observed that Corynebacterium glutamicum exhibits a remarkable tolerance to high concentrations of d-Ala, a crucial characteristic for establishing a successful fermentation process. By optimizing meso-diaminopilmelate dehydrogenases in different C. glutamicum strains and successively deleting l-Ala biosynthetic pathways, we developed an efficient d-Ala fermentation system. The d-Ala titer was enhanced through systems metabolic engineering, which involved strengthening glucose assimilation and pyruvate supply, reducing the formation of organic acid byproducts, and attenuating the TCA cycle. During fermentation in a 5-L bioreactor, a significant accumulation of l-Ala was observed in the broth, which was subsequently diminished by introducing an l-amino acid deaminase. Ultimately, the engineered strain DA-11 produced 85 g/L d-Ala with a yield of 0.30 g/g glucose, accompanied by an optical purity exceeding 99%. The fermentation platform has the potential to be extended for the synthesis of other d-AAs, as demonstrated by the production of d-Val and d-Glu.