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

作　　者：Yali Qiu Wei Liu Meiling Wu Haodong Bao Xinhua Sun Qin Dou Hongying Jia Weifeng Liu Yu Shen 

机构地区：[1]State Key Laboratory of Microbial Technology,Institute of Microbial Technology,Shandong University,Qingdao,266237,China [2]Advanced Medical Research Institute,Shandong University,Jinan,250012,China

出　　处：《Synthetic and Systems Biotechnology》2024年第2期269-276,共8页合成和系统生物技术（英文）

基　　金：This work was supported by the National Key Research and Development Program of China(2021YFC2101303);the National Natural Science Foundation of China(32170039).

摘　　要：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 CO_(2) 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 CO_(2)^(++) 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.

关 键 词：Saccharomyces cerevisiae XYLOSE ETHANOL NADPH Glyceraldehyde-3-phosphate dehydrogenase 

分 类 号：Q815[生物学—生物工程]