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 lysC fbr by 40% on glucose and 30% on fructose or sucrose. Additionally formation of the by-products glycerol and dihydroxyacetone was significantly reduced in the P EFTU fbp mutant. As revealed by 13 C 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 lysC fbr P EFTU fbp and 46% for C. glutamicum lysC fbr . The flux for NADPH supply was 180% in the P EFTU fbp 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.Corynebacterium glutamicum has been successfully used for the industrial production of lysine for more than 40 years, leading to a current market volume of about 600,000 tons, which are produced worldwide with this microorganism per annum (28). The classically derived producer strains developed and currently used in industry, however, have uncharacterized secondary mutations that are detrimental to their performance and lead to decreased sugar uptake rates, growth rates, or stress tolerance (22). This raises the question of superior strains, which exhibit a limited set of exclusively beneficial mutations.In order to rationally create such cell factories, comparative sequencing of the C. glutamicum wild-type and lysine-producing strains has recently been introduced as a powerful strategy (22). By this approach, mutations in key reactions such as pathways involved in product synthesis or supply of precursor metabolites can be identified and subsequently introduced into the wild type (21,22). This approach can be efficiently complemented by comparative metabolic profiling of the organism, which generates a detailed understanding on the quantitative physiology of the organism and, based on the knowledge obtained, also identifies promising genetic targets.In this regard, metabolic flux analysis provides detailed insight into the central metabolism of lysine producing C. glutamicum (11,16,33,35). The biosynthesis of lysine has a high requirement for NADPH, ...

Structural and Biochemical Characterization of the Type II Fructose-1,6-bisphosphatase GlpX from Escherichia coli

“…Li ϩ -sensitive phosphatases are putative targets for lithium therapy in the treatment of manic depressive patients (4), whereas FBPases are targets for the development of drugs for the treatment of noninsulindependent diabetes (5,6). In addition, FBPase is required for virulence in Mycobacterium tuberculosis and Leishmania major and plays an important role in the production of lysine and glutamate by Corynebacterium glutamicum (7,8).…”

Analysis and Engineering of Metabolic Pathway Fluxes in Corynebacterium glutamicum

“…Metabolic flux studies on glucose, fructose, and sucrose identified fructose 1,6-bisphosphatase as a non-obvious target [110,111]. Subsequent amplified expression of the fbp gene indeed increased lysine yield on glucose, fructose, and sucrose up to about 40% [128]. Hereby, the mutant with overexpression of fructose 1,6-bisphosphatase exhibited 10% enhanced PPP flux.…”

“…In C. glutamicum, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, isocitrate 13 C flux analysis: [112] (white circles), [125] (gray circles), [126] (black circles), [127] (white squares), [111] (gray squares), [111] (black squares), [128] (white triangles), [116] (gray triangles), [26] (black triangles), [129] (white diamonds), [115] (gray diamonds), [120] (black diamonds), [76] (white hexagons), and [118] (gray hexagons). The flux reversibility at the pyruvate node is defined as the ratio of the back flux to the anaplerotic net flux [130] 36 C. Wittmann dehydrogenase, and malic enzyme catalyze NADPH generating reactions, whereas NADPH consuming reactions comprise growth with a stoichiometric demand of 16.4 mmol NADPH (g biomass) À1 [34] and overproduction.…”

“…Lysine production flux (%) Fig. 6 Metabolic flux analysis linking lysine production with NADPH metabolism involving the pentose phosphate pathway (a) and isocitrate dehydrogenase (b) in different C. glutamicum strains investigated under various cultivation conditions by 13 C flux analysis: [125] (white circles), [129] (gray circles), [126] (black circles), [127] (white squares), [112] (gray squares), [126] (black squares), [128] (white triangles), [116] (gray triangles), [26,76] (black triangles), [115] (white diamonds), and [120] (gray diamonds)…”

“…For glucose based processes, disruption of glycolysis via deletion of phosphoglucose isomerase forces the cell to metabolize the substrate completely via the PPP, and indeed results in increased lysine production [108]. This strategy, however, is only applicable for glucose based processes, since other substrates such as sucrose, glycerol, fructose, xylose, or arabinose require an active phosphoglucose isomerase for channeling carbon into the PPP [111,128].…”