Construction of an alternative NADPH regeneration pathway improves ethanol production in Saccharomyces cerevisiae with xylose metabolic pathway

Full conversion of glucose and xylose from lignocellulosic hydrolysates is required for obtaining a high ethanol yield. However, glucose and xylose share flux in the pentose phosphate pathway (PPP) and glycolysis pathway (EMP), with glucose having a competitive advantage in the shared metabolic pathways. In this work, we knocked down ZWF1 to preclude glucose from entering the PPP. This reduced the [NADPH] level and disturbed growth on both glucose or xylose, confirming that the oxidative PPP, which begins with Zwf1p and ultimately leads to CO₂ production, is the primary source of NADPH in both glucose and xylose. Upon glucose depletion, gluconeogenesis is necessary to generate glucose-6-phosphate, the substrate of Zwf1p. We re-established the NADPH regeneration pathway by replacing the endogenous NAD⁺-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene TDH3 with heterogenous NADP ⁺ -GAPDH genes GDH, gapB, and GDP1. Among the resulting strains, the strain BZP1 (zwf1Δ, tdh3::GDP1) exhibited a similar xylose consumption rate before glucose depletion, but a 1.6-fold increased xylose consumption rate following glucose depletion compared to the original strain BSGX001, and the ethanol yield for total consumed sugars of BZP1 was 13.5% higher than BSGX001. This suggested that using the EMP instead of PPP to generate NADPH reduces the wasteful metabolic cycle and excess CO₂ release from oxidative PPP. Furthermore, we used a copper-repressing promoter to modulate the expression of ZWF1 and optimize the timing of turning off the ZWF1, therefore, to determine the competitive equilibrium between glucose-xylose co-metabolism. This strategy allowed fast growth in the early stage of fermentation and low waste in the following stages of fermentation.