Metabolic engineering of Corynebacterium glutamicum for production of myo-Inositol and D-chiro-Inositol.

D-chiro-inositol (DCI), a key signaling molecule in glucose and lipid metabolism and steroid synthesis, plays important roles in ameliorating human metabolic disorders. To achieve de novo DCI biosynthesis in Corynebacterium glutamicum, the inositol catabolism-blocked chassis strain DCI‑1 was engineered by dynamically downregulating pgi, pfkA, and zwf genes. The most effective strain DPa‑1 produced 2.89 g/L myo-inositol (MI), the direct precursor of DCI, and 0.83 g/L DCI, with the total inositol over four times the control. Subsequently, to promote the conversion of MI to DCI and the supply of precursor glucose-6-phosphate, key enzymes for DCI synthesis (ino1, iolI2, and iolG) and glucose transport‑phosphorylation were overexpressed. In combination with the knockout of ndnR to enhance NAD(H) supply, the resulting strain DPaZbN‑3 accumulated 2.82 g/L DCI and 2.93 g/L MI. Further regulation of the mycothiol synthetic pathway by σᴮ-recognized promoter in the final engineered strain DPaZbNMh‑3 enabled efficient accumulation of 3.39 g/L DCI and 4.12 g/L MI, representing the highest reported level of microbial DCI production from glucose to date. This research provided effective metabolic engineering strategies and a robust microbial platform for the green biomanufacturing of DCI.