L-Threonine production from acetate by metabolically engineered Corynebacterium glutamicum using a pH-controlled fed-batch process

Acetate has a considerable potential as a non-food feedstock for microbial bioproduction, yet its use for amino acid production remains underexplored. Here, we developed a process for L-threonine production from acetate as the sole carbon source with Corynebacterium glutamicum by integrating rational, genome-based metabolic engineering with pH-coupled fed-batch cultivation. The engineered strain AT-3 carried five chromosomal mutations that alleviated feedback inhibition of key enzymes and redirected carbon flux toward the aspartate pathway. Substrate supply was controlled using a custom Visual Basic-based feeding script that automatically adjusted the acetic acid feed in response to online pH measurements. Robust process performance was maintained during a tenfold scale-up from 100-mL DASbox to 1-L DASGIP bioreactors. The optimized process achieved an L-threonine titer of 10.04 g/L and a yield of 0.086 g/g acetate. This study provides a basis for further studies on the development and improvement of acetate-based amino acid production process. KEY POINTS: • Plasmid-free C. glutamicum strains produce L-threonine in acetate minimal medium • pH-controlled acetic acid feed in bioreactors improved L-threonine production • Acetate-based production of feed amino acids avoids competition with human food.