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

• Published in: Journal of applied microbiology 2006-05, Vol.100 (5), p.1043-1053
• Main Authors: Liu, L.M, Li, Y, Du, G.C, Chen, J
• Format: Article
• Language: English
• Subjects: adenosine triphosphate; Adenosine Triphosphate - biosynthesis; Antifungal Agents - pharmacology; Biological and medical sciences; Candida glabrata; Candida glabrata - drug effects; Candida glabrata - genetics; Candida glabrata - growth & development; Candida glabrata - metabolism; Culture Media; Dose-Response Relationship, Drug; Drug Resistance, Fungal; F0F1‐ATPase; Fundamental and applied biological sciences. Psychology; glucose; glycolysis; Glycolysis - drug effects; glycolytic flux; H-transporting ATP synthase; Microbiology; Mutagenesis; mutants; neomycin‐resistant mutant; oligomycin; Oligomycins - pharmacology; Oxidative Phosphorylation; phosphofructokinases; Phosphorylation; pyruvate kinase; pyruvic acid; screening; Torulopsis glabrata; yeasts
• ISSN: 1364-5072; 1365-2672
• 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[superscript [-]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 glyceraldyde-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.