Liu Liming, Li Yin, Du Guocheng, Chen Jian
School of Biotechnology, Southern Yangtze University, 170 , Huihe Road, Wuxi, 214036, People's Republic of China.
Appl Microbiol Biotechnol. 2006 Sep;72(2):377-85. doi: 10.1007/s00253-005-0284-3. Epub 2006 Jan 11.
This study aimed at increasing the pyruvate productivity of a multi-vitamin auxotrophic yeast Torulopsis glabrata by redirecting NADH oxidation from adenosine triphosphate (ATP)-production pathway (oxidative phosphorylation pathway) to non-ATP production pathway (fermentative pathway). Two respiratory-deficient mutants, RD-17 and RD-18, were screened and selected after ethidium bromide (EtBr) mutagenesis of the parent strain T. glabrata CCTCC M202019. Compared with the parent strain, cytochrome aa (3) and b in electron transfer chain (ETC) of RD-18 and cytochrome b in RD-17 were disrupted. As a consequence, the activities of key ETC enzymes of the mutant RD-18, including F(0)F(1)-ATP synthase, complex I, complex I + III, complex II + III, and complex IV, decreased by 22.2, 41.6, 53.1, 23.6, and 84.7%, respectively. With the deficiency of cytochromes in ETC, a large amount of excessive cytosolic NADH was accumulated, which hampered the further increase of the glycolytic flux. An exogenous electron acceptor, acetaldehyde, was added to the strain RD-18 culture to oxidize the excessive NADH. Compared with the parent strain, the concentration of pyruvate and the glucose consumption rate of strain RD-18 were increased by 26.5 and 17.6%, respectively, upon addition of 2.1 mM of acetaldehyde. The strategy for increasing the glycolytic flux in T. glabrata by redirecting the NADH oxidation pathway may provide an alternative approach to enhance the glycolytic flux in yeast.
本研究旨在通过将烟酰胺腺嘌呤二核苷酸（NADH）氧化从三磷酸腺苷（ATP）生成途径（氧化磷酸化途径）重定向至非ATP生成途径（发酵途径），提高多种维生素营养缺陷型酵母光滑球拟酵母的丙酮酸产量。在对亲本菌株光滑球拟酵母CCTCC M202019进行溴化乙锭（EtBr）诱变后，筛选并选出了两个呼吸缺陷型突变体RD - 17和RD - 18。与亲本菌株相比，RD - 18的电子传递链（ETC）中的细胞色素aa(3)和b以及RD - 17中的细胞色素b被破坏。因此，突变体RD - 18的关键ETC酶的活性，包括F(0)F(1)-ATP合酶、复合体I、复合体I + III、复合体II + III和复合体IV，分别下降了22.2%、41.6%、53.1%、23.6%和84.7%。由于ETC中细胞色素的缺乏，大量过量的胞质NADH积累，这阻碍了糖酵解通量的进一步增加。向菌株RD - 18培养物中添加外源电子受体乙醛以氧化过量的NADH。与亲本菌株相比，添加2.1 mM乙醛后，菌株RD - 18的丙酮酸浓度和葡萄糖消耗率分别提高了26.5%和17.6%。通过重定向NADH氧化途径来增加光滑球拟酵母中糖酵解通量的策略可能为提高酵母中的糖酵解通量提供一种替代方法。
