Amplified expression of fructose 1,6-bisphosphatase in Corynebacterium glutamicum increases in vivo flux through the pentose phosphate pathway and lysine production on different carbon sources

Abstract

The overexpression of fructose 1,6-bisphosphatase (FBPase) in Corynebacterium glutamicum leads to significant improvement of lysine production on different sugars. Amplified expression of FBPase via the promoter of the gene encoding elongation factor TU (EFTU) increased the lysine yield in the feedback-deregulated lysine-producing strain C. glutamicum lysCfbr by 40% on glucose and 30% on fructose or sucrose. Additionally formation of the by-products glycerol and dihydroxyacetone was significantly reduced in the PEFTUfbp mutant. As revealed by 13C metabolic flux analysis on glucose the overexpression of FBPase causes a redirection of carbon flux from glycolysis toward the pentose phosphate pathway (PPP) and thus leads to increased NADPH supply. Normalized to an uptake flux of glucose of 100%, the relative flux into the PPP was 56% for C. glutamicum lysCfbr PEFTUfbp and 46% for C. glutamicum lysCfbr. The flux for NADPH supply was 180% in the PEFTUfbp strain and only 146% in the parent strain. Amplification of FBPase increases the production of lysine via an increased supply of NADPH. Comparative studies with another mutant containing the sod promoter upstream of the fbp gene indicate that the expression level of FBPase relates to the extent of the metabolic effects. The overexpression of FBPase seems useful for starch- and molasses-based industrial lysine production with C. glutamicum. The redirection of flux toward the PPP should also be interesting for the production of other NADPH-demanding compounds as well as for products directly stemming from the PPP.

FIG. 1.

In vitro activity of fructose 1,6-bisphosphatase in different strains of Corynebacterium glutamicum. The data are given for Corynebacterium glutamicum ATCC 13032 lysC^fbr and two mutants, Corynebacterium glutamicum ATCC 13032 lysC^fbr P_SODfbp and Corynebacterium glutamicum ATCC 13032 lysC^fbr P_EFTUfbp. The strains were grown on minimal medium containing glucose, fructose, or sucrose as the sole carbon source. All measurements were carried out in triplicate; corresponding deviations are given.

FIG. 2.

In vivo carbon flux distribution in the central metabolism of glucose-grown Corynebacterium glutamicum ATCC 13032 lysC^fbr during lysine production. The strain contains a feedback-resistant aspartokinase. Fluxes were estimated from the best fit to the experimental results using a comprehensive approach of combined metabolite balancing and isotopomer modeling for a ¹³C tracer experiment during growth on [1-¹³C]glucose and measurement of labeling of amino acids from the cell protein and of trehalose from the culture supernatant by GC-MS. Net fluxes are given in square symbols; for reversible reactions the direction of the net flux is indicated by an arrow beside the corresponding black box. Numbers in brackets below the fluxes of transaldolase, transketolase, and glucose 6-phosphate isomerase indicate flux reversibility. Fluxes toward anabolism are displayed in gray boxes. All fluxes are expressed as a molar percentage of the mean specific glucose uptake rate (4.9 mmol g⁻¹ h⁻¹), which was set to 100%.

FIG. 3.

In vivo carbon flux distribution in the central metabolism of glucose-grown Corynebacterium glutamicum ATCC 13032 lysC^fbr P_EFTUfbp during lysine production. The strain contains a feedback-resistant aspartokinase and exhibits amplified expression of fructose 1,6-bisphosphatase. The fluxes were estimated from the best fit to the experimental results using a comprehensive approach of combined metabolite balancing and isotopomer modeling for a ¹³C tracer experiment during growth on [1-¹³C]glucose and measurement of labeling of amino acids from the cell protein and of trehalose from the culture supernatant by GC-MS. Net fluxes are given in square symbols; for reversible reactions the direction of the net flux is indicated by an arrow beside the corresponding black box. Numbers in brackets below the fluxes of transaldolase, transketolase, and glucose 6-phosphate isomerase indicate flux reversibility. Fluxes toward anabolism are displayed in gray boxes. All fluxes are expressed as a molar percentage of the mean specific glucose uptake rate (4.8 mmol g⁻¹ h⁻¹), which was set to 100%.