To efficiently produce myo-inositol from glucose, the PGI1, ZWF1, ITR2, and MIOX5 genes in Kluyveromyces marxianus were knocked out to block glucose metabolism via the Embden-Meyerhof-Parnas (EMP) and pentose phosphate pathways (PPP), prevent myo-inositol oxidative degradation. The metabolically engineered KM-JC4 strain, introduced with myo-inositol synthesis genes, produced 80.7 g/L in a 5 L bioreactor using glucose and glycerol as carbon sources. Subsequently, the starch-fermenting and inositol-producing strain KM-JC5 was constructed by co-expressing BadGlA, an α-glucoamylase from Blastobotrys adeninivorans with high ability to release glucose from soluble starch, and the myo-inositol synthesis enzymes. Using 5 % soluble starch and liquefied starch, the myo-inositol yields reached 32.2 g/L and 40.6 g/L, with the starch-to-myo-inositol conversion rates of 64.4 % and 81.1 %, respectively. This study provides an effective strategy for bioproduction by balancing glycolysis and PPP metabolism in yeast, and the metabolically engineered strain represents a promising platform for inositol production.
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