Expression of the pfl Gene and Resulting Metabolite Flux Distribution in nuo and ackA-pta E. coli Mutant Strains

Abstract

Our laboratory previously studied the interaction between nuo and the acetate-producing pathway encoded by ackA-pta in Escherichia coli. We examined metabolic patterns, particularly the ethanol and acetate production rates, of several mutant strains grown under anaerobic growth conditions. Since the pyruvate formate-lyase (PFL) pathway is the major route for acetyl-CoA and formate production under anaerobic conditions, we examined the effects of nuo and ackA/pta mutations on the expression of pyruvate formate-lyase (pfl) under anaerobic conditions. The ackA-pta mutant has a pfl::lacZ expression level much higher than that of the wild-type strain, and cultures also exhibit the highest ethanol production. Real-time PCR demonstrated that the adhE gene expression in the ack-pta mutant strain was approximately 100 fold that of the same gene in the ackA-pta nuo mutant strain. This result correlates with the observed ethanol production rates in cultures of the strain. However, the lack of exact correlation between the ethanol production rates and the RT-PCR data suggests additional regulation actions at the posttranslation level. In addition, the activity of the pfl gene as indicated by mRNA levels was also considerably greater in theack-pta mutant. We can conclude that deletions of nuo and ack/pta can partially affect the expression of the genes encoding adhE and pfl under anaerobic conditions.

Acetate overflow metabolism regulates a major metabolic shift after glucose depletion in Escherichia coli

“…In this study, we analyzed the underlying mechanism of the diauxic growth in the pta mutant. It is well known that pyruvate and 2‐oxoglutarate accumulate in culture media of acetate overflow‐deficient mutants [21–24]. It was also recently found that some amino acids were actively utilized even in the presence of glucose [25] and that the resultant intracellular accumulation of pyruvate and oxaloacetate reduced glucose uptake and increased acetate overflow [26].…”