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, 13C 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.

FIG. 1.

Profile of batch cultivation of lysine-producing C. glutamicum ATCC 13287. (A) Volumetric carbon dioxide production rate (Q_CO2), concentrations of glucose and citrate, and CDM. (B) Concentrations of methionine, threonine, and lysine. (C) Concentrations of glycine, alanine, and valine. (D) Concentrations of glycerol, dihydroxyacetone, and acetate. The beginning of the lysine production phase is marked by a vertical line.

FIG. 2.

Specific glucose uptake rate (q_Glc) and lysine production rate (q_Lys), shown in millimoles per gram per hour, during batch cultivation of lysine-producing C. glutamicum ATCC 13287. The beginning of the lysine production phase is marked by a vertical line. The insert in the figure displays the relationship between q_Glc and q_Lys during hours 7 to 15 of cultivation.

FIG. 3.

Intracellular and extracellular amino acid concentrations during batch cultivation of lysine-producing C. glutamicum ATCC 13287. (A) Intracellular concentrations of isoleucine, threonine, aspartate, methionine, and serine. (B) Intracellular concentrations of tyrosine, tryptophan, and phenylalanine. (C) Intracellular concentrations of alanine, leucine, and valine. (D) Intracellular concentrations of glutamine, arginine, and glutamate. (E) Intracellular concentration of lysine and extracellular concentrations of lysine and threonine. The beginning of the lysine production phase is marked by a vertical line.

FIG. 4.

Intracellular flux distribution of lysine-producing C. glutamicum after 5.8, 6.9, 8.1, and 9.2 h of cultivation (displayed in this order from top to bottom for each reaction). All fluxes are given in millimoles per gram per hour. For reversible reactions, dashed arrows indicate the direction of the net flux and the values in the shaded boxes are the obtained reversibilities of the corresponding enzymes.

FIG. 5.

Coordination of gene expression and metabolic fluxes in glycolysis (A), the TCA cycle (B), and lysine biosynthesis (C) during batch cultivation of lysine-producing C. glutamicum ATCC 13287. For substrate uptake, glucose uptake flux (Fig. 2) and the expression of PTS components (Table 4) were considered. In the other cases (B to D), the intracellular flux data obtained after 5.8, 6.9, and 8.1 h (Fig. 4) were related to expression levels at 5.6, 6.8, and 7.7 h (Table 5), respectively.