In-Depth Profiling of Lysine-Producing Corynebacterium glutamicum by Combined Analysis of the Transcriptome, Metabolome, and Fluxome

Abstract

An in-depth analysis of the intracellular metabolite concentrations, metabolic fluxes, and gene expression (metabolome, fluxome, and transcriptome, respectively) of lysine-producing Corynebacterium glutamicum ATCC 13287 was performed at different stages of batch culture and revealed distinct phases of growth and lysine production. For this purpose, 13 C flux analysis with gas chromatography-mass spectrometry-labeling measurement of free intracellular amino acids, metabolite balancing, and isotopomer modeling were combined with expression profiling via DNA microarrays and with intracellular metabolite quantification. The phase shift from growth to lysine production was accompanied by a decrease in glucose uptake flux, the redirection of flux from the tricarboxylic acid (TCA) cycle towards anaplerotic carboxylation and lysine biosynthesis, transient dynamics of intracellular metabolite pools, such as an increase of lysine up to 40 mM prior to its excretion, and complex changes in the expression of genes for central metabolism. The integrated approach was valuable for the identification of correlations between gene expression and in vivo activity for numerous enzymes. The glucose uptake flux closely corresponded to the expression of glucose phosphotransferase genes. A correlation between flux and expression was also observed for glucose-6-phosphate dehydrogenase, transaldolase, and transketolase and for most TCA cycle genes. In contrast, cytoplasmic malate dehydrogenase expression increased despite a reduction of the TCA cycle flux, probably related to its contribution to NADH regeneration under conditions of reduced growth. Most genes for lysine biosynthesis showed a constant expression level, despite a marked change of the metabolic flux, indicating that they are strongly regulated at the metabolic level. Glyoxylate cycle genes were continuously expressed, but the pathway exhibited in vivo activity only in the later stage. The most pronounced changes in gene expression during cultivation were found for enzymes at entry points into glycolysis, the pentose phosphate pathway, the TCA cycle, and lysine biosynthesis, indicating that these might be of special importance for transcriptional control in C. glutamicum.

Analysis and Engineering of Metabolic Pathway Fluxes in Corynebacterium glutamicum

“…The most effective sulfur source was methanethiol, allowing an almost complete conversion of glucose into methionine with a yield of 0.91 C-mol given as relative molar fluxes to the glucose uptake, whereby the relative flux of a reaction is reflected by the thickness of the corresponding arrow. The data are taken from recent simulation studies [5,76] Analysis and Engineering of Metabolic Pathway Fluxes in Corynebacterium glutamicumC-mol À1 . Summarizing, stoichiometric modeling studies for C. glutamicum provide a sound basis for strain or process optimization.…”

“…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)…”

“…Among the achievements obtained from flux analysis are the identification of novel pathways, the elucidation of metabolic control, identification of targets for rational strain engineering in biotechnology, and first insights into design principles of the metabolic network. Hereby, metabolic flux analysis provided quantitative data which directly reflect the phenotype of the investigated C. glutamicum strain, whereas other omics approaches often do not allow a direct conclusion on the active pathways determining the phenotypic behavior [76,175]. Recent developments provide the next level of systems biology studies, the parallel investigation of C. glutamicum on levels of gene expression, proteins, metabolites, and fluxes providing important links between the different functional components of cellular physiology.…”

“…Recent developments provide the next level of systems biology studies, the parallel investigation of C. glutamicum on levels of gene expression, proteins, metabolites, and fluxes providing important links between the different functional components of cellular physiology. First examples of such systems-oriented studies already reveal a great potential [76,119,176]. Such approaches are especially promising for the targeted multidimensional alteration of complex regulatory networks towards better tolerance of production strains to high temperature or salt levels, or extreme pH values [137], but also reveal a great potential for efficient design of novel bioprocesses.…”