Construction and evolution of an Escherichia coli strain relying on nonoxidative glycolysis for sugar catabolism

Abstract

SignificanceWe constructed an Escherichia coli strain that does not use glycolysis for sugar catabolism. Instead, it uses the synthetic nonoxidative glycolysis cycle to directly synthesize stoichiometric amounts of the two-carbon building block (acetyl-CoA), which is then converted to three-carbon metabolites to support growth. The resulting strain grows aerobically in glucose minimal medium and can achieve near-complete carbon conservation in the production of acetyl-CoA–derived products during anaerobic fermentation. This strain improves the theoretical carbon yield from 66.7% to 100% in acetyl-CoA–derived product formation.

A hybrid Embden–Meyerhof–Parnas pathway provides a synthetic link between sugar and phosphate metabolism

“…Further, the evolution of the engineered E. coli led to aerobic cell growth in the minimal glucose medium. However, NOG-evolved strains showed much slower growth rates in the minimal glucose medium than that of the WT due weakened C3 synthesis 10 . Therefore, we designed a hybrid EMP pathway consisting of three phases: (i) an NOG pathway phase that converts F6P to AcP ( eq.…”

“…It is possible to preserve half the CO 2 produced using hybrid EMP compared to native EMP. In case of pyruvate synthesis from glucose, 1.5 CO 2 per glucose can be produced using NOG and a single ATP is required 10 . Whereas in the hybrid EMP, ATP/NADH can be generated without CO 2 loss during pyruvate synthesis and 1.5 mole acetate can be obtained instead of CO 2 .…”

“…Unlike the E. coli NOG strains 10 , adaptive laboratory evolution of C. glutamicum does not require growth in minimal glucose medium. To investigate whether NOG is an active pathway in the hybrid EMP pathway, zwf (encoding for glucose 6-phosphate dehydrogenase) was deleted to block the oxidative phosphate pathway that was only a possible catabolic pathway in YL1.…”

“…Non-oxidative glycolysis (NOG) has provided a biotechnological basis for increasing the theoretical yield by reducing CO 2 evolution. To fulfill carbon conservation in Escherichia coli , adaptive evolution has been performed in a minimal glucose medium after rational metabolic engineering 10 , which results in slower growth of the NOG strains than their parental strain. This could be due to insufficient reducing equivalents generated via NOG.…”

“…( A ) a simplified scheme of EMP (or glycolysis) ( B ) a simplified scheme of non-oxidative glycolysis 9,10 ( C ) a simplified hybrid EMP where the preparatory phase was replaced by the NOG pathway. ( D ) Metabolic networks in the engineered C. glutamicum with the hybrid EMP.…”