Liu L M, Li Y, Du G C, Chen J
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Southern Yangtze University, Wuxi, China.
J Appl Microbiol. 2006 May;100(5):1043-53. doi: 10.1111/j.1365-2672.2006.02871.x.
This study aimed at further increasing the pyruvate productivity of a multi-vitamin auxotrophic yeast Torulopsis glabrata by redirecting ATP production from oxidative phosphorylation to substrate-level phosphorylation.
We examined two strategies to decrease the activity of F0F1-ATPase. The strategies were to inhibit F0F1-ATPase activity by addition of oligomycin, or to disrupt F0F1-ATPase by screening neomycin-resistant mutant. The addition of 0.05 mmol l(-1) oligomycin to the culture broth of T. glabrata CCTCC M202019 resulted in a significantly decreased intracellular ATP level (35.7%) and a significantly increased glucose consumption rate (49.7%). A neomycin-resistant mutant N07 was screened and selected after nitrosoguanidine mutagenesis of the parent strain T. glabrata CCTCC M202019. Compared with the parent strain, the F0F1-ATPase activity of the mutant N07 decreased about 65%. As a consequence, intracellular ATP level of the mutant N07 decreased by 24%, which resulted in a decreased growth rate and growth yield. As expected, glucose consumption rate and pyruvate productivity of the mutant N07 increased by 34% and 42.9%, respectively. Consistently, the activities of key glycolytic enzymes of the mutant N07, including phosphofructokinase, pyruvate kinase and glyceraldehyde-3-phosphate dehydrogenase, increased by 63.7%, 28.8% and 14.4%, respectively. In addition, activities of the key enzymes involved in electron transfer chain of the mutant N07 also increased.
Impaired oxidative phosphorylation in T. glabrata leads to a decreased intracellular ATP production, thereby increasing the glycolytic flux.
The strategy of redirecting ATP production from oxidative phosphorylation to substrate-level phosphorylation provides an alternative approach to enhance the glycolytic flux in eukaryotic micro-organisms.
本研究旨在通过将ATP生成从氧化磷酸化重定向至底物水平磷酸化，进一步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。
我们研究了两种降低F0F1 - ATP酶活性的策略。策略一是通过添加寡霉素抑制F0F1 - ATP酶活性，二是通过筛选新霉素抗性突变体破坏F0F1 - ATP酶。向光滑球拟酵母CCTCC M202019的培养液中添加0.05 mmol l(-1)寡霉素，导致细胞内ATP水平显著降低（35.7%），葡萄糖消耗速率显著增加（49.7%）。对亲本菌株光滑球拟酵母CCTCC M202019进行亚硝基胍诱变后，筛选并选出新霉素抗性突变体N07。与亲本菌株相比，突变体N07的F0F1 - ATP酶活性降低了约65%。结果，突变体N07的细胞内ATP水平降低了24%，导致生长速率和生长产量下降。正如预期的那样，突变体N07的葡萄糖消耗速率和丙酮酸产量分别提高了34%和42.9%。同样，突变体N07的关键糖酵解酶活性，包括磷酸果糖激酶、丙酮酸激酶和甘油醛 - 3 - 磷酸脱氢酶，分别提高了63.7%、28.8%和14.4%。此外，突变体N07中参与电子传递链的关键酶活性也有所增加。
光滑球拟酵母中氧化磷酸化受损导致细胞内ATP生成减少，从而增加糖酵解通量。
将ATP生成从氧化磷酸化重定向至底物水平磷酸化的策略为增强真核微生物中的糖酵解通量提供了一种替代方法。
