Contrasting effects of isocitrate dehydrogenase deletion on fluxes through enzymes of central metabolism in Escherichia coli

Abstract

Flux analysis is central to understanding cellular metabolism and successful manipulation of metabolic fluxes in microbial cell-factories. Isocitrate dehydrogenase (ICDH) deletion conferred contrasting effects on fluxes through substrate-level phosphorylation (SLP) reactions. While significantly increasing flux through pyruvate kinase, it diminishes flux through succinyl CoA synthetase and upregulates phosphotransacetylase (PTA) and acetate kinase (AK). In addition to acetate, the ICDH-less strain excretes pyruvate, citrate and isocitrate. While efflux to acetate excretion by the Escherichia coli parental strain and its ICDH-less derivative is a reflection of high throughput of glycolytic intermediates, excretion of pyruvate is a reflection of high throughput via pyruvate kinase. On the other hand, citrate and isocitrate excretion is a reflection of truncating the Krebs cycle at the level of ICDH. Furthermore, another striking finding is the inability of the ICDH-less cultures to utilize acetate as a source of carbon despite the availability of an adequate supply of extracellular glutamate (for biosynthesis) and elevated levels of AK and PTA (for acetate uptake). This striking observation is now explicable in the light of the newly proposed hypothesis that the expression of the ace operon enzymes is controlled in response to a minimum threshold signal (ATP), which could not be achieved in the ICDH-less strain.

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

“…It was previously suggested that limited availability of HS‐CoA in a cell could be a constraint for the metabolic flux because HS‐CoA is a common cofactor for PDHc and 2‐oxoglutarate dehydrogenase complex (OGDHc) [24]. Together with this, our previous study showed the requirement of the OGDHc activity for the growth after glucose depletion because knockout mutants of OGDHc subunits showed complete growth arrest after glucose depletion [16].…”

“…It has been known that pyruvate accumulated in the cells is easily excreted into the culture medium [24,41]. Thus, we analyzed the pyruvate accumulation in the medium and it was observed that the pta mutant accumulated extracellular pyruvate after glucose depletion (Fig.…”

“…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].…”