Dual-channel glycolysis balances cofactor supply for l-homoserine biosynthesis in Corynebacterium glutamicum

l-Homoserine is an important platform compound that is widely used to produce many valuable bio-based products, but production of l-homoserine in Corynebacterium glutamicum remains low. In this study, an efficient l-homoserine-producing strain was constructed. Native pentose phosphate pathway (PPP) was enhanced and heterologous Entner-Doudoroff (ED) pathway was carefully introduced into l-homoserine-producing strain, which increased the l-homoserine titer. Coexpression of NADH-dependent aspartate-4-semialdehyde dehydrogenase and aspartate dehydrogenase could increase the titer from 11.3 to 13.3 g/L. Next, NADP⁺-dependent glyceraldehyde-3-phosphate dehydrogenase (NADP-GPD) was coexpressed with that of NAD⁺-dependent (NAD-GPD) to construct dual-channel glycolysis for balance of intracellular cofactors, which increased the l-homoserine titer by 48.6 % to 16.8 g/L. Finally, engineered strain Cg18-1 accumulated 63.5 g/L l-homoserine after 96 h in a 5 L bioreactor, the highest titer reported to date for C. glutamicum. This dual-channel glycolysis strategy provides a reference for automatic cofactor regulation to promote efficient biosynthesis of other target products.