Synergetic utilization of glucose and glycerol for efficient myo‐inositol biosynthesis

myo‐Inositol (MI) as a dietary supplement can provide various health benefits. One major challenge to its efficient biosynthesis is to achieve proper distribution of carbon flux between growth and production. Herein, this challenge was overcome by synergetic utilization of glucose and glycerol. Specifically, glycerol was catabolized to support cell growth while glucose was conserved as the building block for MI production. Growth and production were coupled via the phosphotransferase system, and both modules were optimized to achieve efficient production. First, the optimal enzyme combination was established for the production module. It was observed that enhancing the production module resulted in both increased MI production and better cell growth. In addition, glucose was shown to inhibit glycerol utilization via carbon catabolite repression and the inhibition was released by over‐expressing glycerol kinase. Furthermore, the inducible promoter was replaced by strong constitutive promoters to avoid inducer use. With these efforts, the final strain produced MI with both high titer and yield. In fed‐batch cultivation, 76 g/L of MI was produced, showing scale‐up potential. This study provides a promising strategy to achieve rational distribution of carbon flux. A synergetic carbon utilization strategy was used for myo‐inositol biosynthesis, in which glycerol was catabolized to support cell growth while glucose was conserved as the building block. The two modules were coupled via the phosphotransferase system. With this strategy, efficient production was achieved. This work provides a promising solution to rational distribution of carbon flux.