Increasing glycolytic flux in Torulopsis glabrata by redirecting ATP production from oxidative phosphorylation to substrate-level phosphorylation.
OBJECTIVE
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.


RESULTS
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.


CONCLUSIONS
Impaired oxidative phosphorylation in T. glabrata leads to a decreased intracellular ATP production, thereby increasing the glycolytic flux.


CONCLUSIONS
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.
Liu
L M
LM
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Southern Yangtze University, Wuxi, China.
Li
Y
Y
Du
G C
GC
Chen
J
J
D000255:Adenosine Triphosphate / Q000096:biosynthesis*; D000935:Antifungal Agents / Q000494:pharmacology; D041221:Candida glabrata / Q000187:drug effects / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D003470:Culture Media; D004305:Dose-Response Relationship, Drug; D025141:Drug Resistance, Fungal; D006019:Glycolysis / Q000187:drug effects; D016296:Mutagenesis; D009840:Oligomycins / Q000494:pharmacology; D010085:Oxidative Phosphorylation; D010766:Phosphorylation
D000935:Antifungal Agents; D003470:Culture Media; D009840:Oligomycins; D000255:Adenosine Triphosphate