Comparison of dynamic responses of cellular metabolites in Escherichia coli to pulse addition of substrates

Abstract

Abstract:We conducted an integrated study of cell growth parameters, product formation, and the dynamics of intracellular metabolite concentrations using Escherichia coli with genes knocked out in the glycolytic and oxidative pentose phosphate pathway (PPP) for glucose catabolism. We investigated the same characteristics in the wild-type strain, using acetate or pyruvate as the sole carbon source. Dramatic effects on growth parameters and extracellular and intracellular metabolite concentrations were observed after blocking either glycolytic breakdown of glucose by inactivation of phosphoglucose isomerase (disruption of pgi gene) or pentose phosphate breakdown of glucose by inactivation of glucose-6-phosphate dehydrogenase (disruption of zwf gene). Reducing power (NADPH) was mainly produced through PPP when the pgi gene was knocked out, while NADPH was produced through the tricarboxylic acid (TCA) cycle by isocitrate dehydrogenase or NADP-linked malic enzyme when the zwf gene was knocked out. As expected, when the pgi gene was knocked out, intracellular concentrations of PPP metabolites were high and glycolytic and concentrations of TCA cycle pathway metabolites were low. In the zwf gene knockout, concentrations of PPP metabolites were low and concentrations of intracellular glycolytic and TCA cycle metabolites were high.

Improved production of the non-native cofactor F420 in Escherichia coli

“…We also examined whether alternative substrates for FbiD could be used to remove the reliance on PEP, showing that although 3PG is a viable substrate and is more abundant in E. coli, the use of the P. rhizoxinica FbiD did not result in higher 3PG-F 420 yields (compared with F 420 ), presumably because of the downstream enzymes, which are sourced from M. smegmatis , have a preference for F 420 -metabolites over 3PG-F 420 metabolites. Replacing other enzymes in the pathway with those from P. rhizoxinica may be a worthwhile strategy to improve 3PG-F 420 yield, as the theoretical yield of 3PG-F 420 is similar to that of PEP-derived F 420 and strategies have already been developed to improve 3PG availability by increasing glycolytic flux by knocking out the zwf gene involved in first step of pentose phosphate pathway 55 . Other strategies that we tested to increase F 420 yield included increasing PEP production through over expression of PPS; this produced the highest productivity with pyruvate.…”

Metabolic Engineering of E. coli for Xylose Production from Glucose as the Sole Carbon Source

“…The pgi gene is the second enzyme of the glycolytic pathway and can promote G6P catabolism in the glycolytic pathway and the subsequent TCA cycle instead of the PPP . Therefore, we chose to additionally prevent glucose-6-phosphate (G6P) catabolism through the glycolytic pathway by deleting the gene pgi , which encodes the G6P isomerase.…”

“…The pgi gene is the second enzyme of the glycolytic pathway and can promote G6P catabolism in the glycolytic pathway and the subsequent TCA cycle instead of the PPP. 35 Therefore, we chose to additionally prevent glucose-6-phosphate (G6P) catabolism through the glycolytic pathway by deleting the gene pgi, which encodes the G6P isomerase. As expected, when we engineered a Δpgi strain in the E. coli BL21(DE3) ΔxylB background, we found that strain GX6 performed better than GX1, producing more xylose.…”