Enhanced expression of glycolysis regulators KmGcr1p and KmGcr2p in Kluyveromyces marxianus: An efficient strategy for high-temperature ethanol production from inulin

Bioethanol production from non-food biomass has emerged as a sustainable alternative to satisfy future energy demands. Kluyveromyces marxianus stands out as a potential consolidated bioprocessing (CBP) strain for high-temperature ethanol production from inulin. Nevertheless, enhancing its fermentation efficiency remains crucial. Here, we characterized its glycolysis regulator KmGcr2p and demonstrated that KmGcr2p boosts the expression of target genes controlled by another glycolysis regulator, KmGcr1p, through their interaction. Furthermore, we engineered the strain Ogcr1 + 2 through co-overexpressing KmGCR1 and KmGCR2, leading to a 55.8 % increase in inulinase activity, improved growth at 37 °C to 45 °C and under 10 % ethanol, and enhanced ethanol production at 42 °C by 36.0 % from inulin and 39.5 % from Jerusalem artichoke tuber flour. Our results highlight that co-overexpression of glycolysis regulators KmGcr1p and KmGcr2p can simultaneously enhance inulin saccharification, thermotolerance, ethanol tolerance, and ethanol production, presenting a promising and feasible strategy for high-temperature CBP ethanol production from inulin and inulin-rich feedstocks.