Upregulation of the gluconeogenesis pathway was observed by Kluyveromyces marxianus KDH1, mitigating glucose catabolite repression.

Kluyveromyces marxianus was engineered to mitigate carbon catabolite repression to efficient co-fermenting mixed sugars, which are primary components of cellulosic biomass. Kluyveromyces marxianus KDH1 produced ethanol with 0.42 ± 0.01 g/g yield, and 0.67 ± 0.00 g/L·h productivity for 48 h. RNA sequencing-based transcriptomic analysis showed that genes from the glycolysis pathway, gluconeogenesis pathway, and the citric acid cycle were primarily upregulated when K. marxianus KDH1 fermented mixed sugars. Furthermore, critical genes from the gluconeogenesis pathway, such as fructose-1,6-bisphosphatase and phosphoenolpyruvate carboxykinase, were upregulated by 331.72 and 47.15-fold, respectively. Citrate synthase and malate dehydrogenase, associated with the citric acid cycle, were upregulated by 2284.62 and 7.69-fold, respectively. Enzymatic assays of fructose 1, 6-bisphosphatase indicated that K. marxianus KDH1 showed 1.87-fold higher enzymatic activity than that of the parental strain. These results provide novel information on mixed sugar co-fermentation and a new glucose fermentation process that bypasses the glycolysis pathway. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10068-024-01670-5.