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

Abstract

Aims: 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. Methods and 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 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. Conclusions: Impaired oxidative phosphorylation in T. glabrata leads to a decreased intracellular ATP production, thereby increasing the glycolytic flux. Significance and Impact of the Study: 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.

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“…The first is to supplement specific inhibitors of F 0 F 1 -ATPase, such as oligomycin and neomycin (Johnson et al, 2006). The second is genetic manipulation (Yokota et al, 1997) or mutatation of F 0 F 1 -ATPase through traditional means (Liu et al, 2006a). In prokaryotic microorganisms, such as Escherichia coli (Zhu et al, 2008) and Corynebacterium glutamicum (Sekine et al, 2001), mutants defective in the activity of F 0 F 1 -ATPase possess an accelerated glycolytic pathway.…”

Enhanced adenosine triphosphate production by Saccharomyces cerevisiae using an efficient energy regeneration system

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