Construction of an alternative NADPH regeneration pathway improves ethanol production in 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 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 CO 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 with heterogenous NADP  -GAPDH genes , , and . Among the resulting strains, the strain BZP1 (, ::) 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 CO release from oxidative PPP. Furthermore, we used a copper-repressing promoter to modulate the expression of and optimize the timing of turning off the , 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.