Efficient Myoinositol biosynthesis from glucose through gluconate bypass of glycolysis.

Microbial myo-inositol (MI) production typically requires deletion of conditionally essential genes such as phosphoglucose isomerase (pgi in E. coli) to increase pools of the precursor glucose-6-phosphate (G6P). This disrupts growth and has necessitated complex glycerol co-feeding strategies. Here, we address this limitation by implementing a synthetic gluconate-bypass (GBP) central metabolism that establishes an alternative route by which glucose-derived carbon enters central metabolism, allowing robust growth despite pgi deletion. However, pgi deletion alone was insufficient to improve MI production in this background. Through systematic analysis, we found that increasing G6P availability in the GBP background requires balancing carbon partitioning toward and away from this node. Specifically, by combining dynamic control of glucose dehydrogenase (Gdh), which diverts glucose away from G6P, with expression of a G6P-insensitive human hexokinase I (HK1) variant, HK1(T536A), which synthesizes G6P, we achieved a synergistic increase in G6P pools. This enabled MI titers of 233 g L⁻1 in instrumented bioreactors from glucose as the sole carbon source, representing the highest reported MI titer to date and surpassing previous glucose-glycerol co-feeding processes, with a production-phase yield of 81%. More broadly, this work demonstrates that rewiring central glucose metabolism can resolve the tradeoff between growth and precursor accumulation, providing a general strategy for efficient production of glucose-6-phosphate-derived biochemicals.