Global transcription and metabolic flux analysis of Escherichia coli in glucose-limited fed-batch cultivations

Abstract

A time series of whole-genome transcription profiling of Escherichia coli K-12 W3110 was performed during a carbon-limited fed-batch process. The application of a constant feed rate led to the identification of a dynamic sequence of diverse carbon limitation responses (e.g., the hunger response) and at the same time provided a global view of how cellular and extracellular resources are used: the synthesis of high-affinity transporters guarantees maximal glucose influx, thereby preserving the phosphoenolpyruvate pool, and energy-dependent chemotaxis is reduced in order to provide a more economic "work mode." sigma(S)-mediated stress and starvation responses were both found to be of only minor relevance. Thus, the experimental setup provided access to the hunger response and enabled the differentiation of the hunger response from the general starvation response. Our previous topological model of the global regulation of the E. coli central carbon metabolism through the crp, cra, and relA/spoT modulons is supported by correlating transcript levels and metabolic fluxes and can now be extended. The substrate is extensively oxidized in the tricarboxylic acid (TCA) cycle to enhance energy generation. However, the general rate of oxidative decarboxylation within the pentose phosphate pathway and the TCA cycle is restricted to a minimum. Fine regulation of the carbon flux through these pathways supplies sufficient precursors for biosyntheses. The pools of at least three precursors are probably regulated through activation of the (phosphoenolpyruvate-)glyoxylate shunt. The present work shows that detailed understanding of the genetic regulation of bacterial metabolism provides useful insights for manipulating the carbon flux in technical production processes.

FIG. 1.

Glucose-limited fed-batch cultivation of E. coli K-12 W3110 with a constant feed rate and sampling for transcriptome analysis. The vertical line at t = 0 indicates glucose limitation (as judged from the dissolved oxygen concentration time course [data not shown]). The concentrations of biomass (▪), glucose (▾), and acetate (□) are given, as well as the time course of the specific growth rate μ) (broken line). Arrows above the graph indicate the times when the samples were removed for microarray analysis (R, reference; T1 to T8, time series samples).

FIG. 2.

Global regulation of the E. coli K-12 W3110 metabolism during carbon-limited growth, derived from a genome-wide transcriptome and metabolic-flux analysis. The regulatory processes that are most relevant for utilization of available intra- and extracellular resources are proposed. mRNA/flux levels: +, increase; −, decrease; =, invariable. Short dashed lines, transcriptional regulation; long dashed lines, signaling processes (regulation of protein activities: arrowhead, positive; blunt end, negative). (I) A cluster of high-affinity transporters is synthesized (mglBA, galP, and lamB), while the activity of medium-affinity transporters is maintained. This is due mainly to their regulation by the Crp-cAMP complex but also to the effect of the transcriptional regulatory proteins MalT, YeeI, and Mlc (Fig. 3b and c). The glucose flux entering the cell is directed via transporters that do not use pep for phosphorylation. This preserves the pool of this metabolite (homeostasis) and affects the EIIA^Glc∼P-dependent activation of cAMP synthesis through the enzyme adenylate cyclase (CyaA). (II) These transport systems in particular depend on a membrane proton gradient for proper function. The expression of the proton gradient-dependent chemotaxis system is reduced, thereby enabling the transport system to effectively utilize the energy available. (III) The flux through the upper part of glycolysis is favored, whereas the flux through the PPP is minimized, which is most likely due to the reduced synthesis of gnd mRNA. The flux entering the PPP is used for biosynthesis at the expense of the reflux into the glycolysis pathway, which might be regulated by the RpiA/Rpe split ratio. The reaction rates in the lower glycolysis decrease due to decreasing mRNA levels (cra modulon; signaling through fbp), thereby providing a sufficient, though minimal, efflux into the PPP. The carbon flux entering the TCA cycle (influx is enhanced via gltA expression) is split into the GS, the PEP-GS, and the full TCA cycle. The GS and PEP-GS provide a better pep, pyruvate (pyr), and oac precursor supply. It is assumed that the global regulation via the crp and cra modulons is the most relevant in this respect. (IV) Cellular growth is regulated predominantly by the stringent response (alarmone ppGpp, relA/spoT modulon). (V) No extensive induction of the general rpoS-dependent response could be observed (opposing regulation via the crp and relA/spoT modulons). It is expected that slow substrate concentration changes do not trigger a strong starvation response. However, other stress responses were detected. Details are presented in the text.

FIG. 3.

Dynamic changes in transporter mRNA levels and their regulation in E. coli K-12 W3110 during glucose-limited fed-batch growth with a constant feed rate. (a) Sugar transport systems. (b) Regulation of transporter gene expression. (c) Expression of proteins relevant for the regulation of transporters. The time courses of the transcript levels are given for samples T1 to T8 relative to the reference sample in the batch phase (R) (Fig. 1). Green, mRNA level lower than in the reference state. Red, higher mRNA level. Statistical significance (P ≤ 0.05) is indicated by asterisks. Glc, glucose; P, phosphoryl group. Node symbols (states): rectangle, gene; parallelogram, RNA; rounded rectangle, protein; black rounded rectangle, regulator protein; gray rounded rectangle, protein with differentially expressed mRNA. Arrow symbols: solid line, regulatory interaction; dash-dot-dot-dash line, transcription; dash-dot-dash-dot line, translation. Arrowhead symbols: filled arrow, transformation; blunt-end arrow, inhibition or repression; open arrow, activation; filled arrow with crossbar: transport.

FIG. 4.

Time series of DNA microarray and metabolic-flux analyses of the central carbon metabolism in E. coli K-12 W3110 during glucose-limited fed-batch growth with a constant feed rate. The time courses of the transcript levels are given for samples T1 to T8 relative to the reference sample in the batch phase (R) (Fig. 1). Green, mRNA level lower than in the reference state. Red, higher mRNA level. Statistical significance (P ≤ 0.05) is indicated by asterisks. The metabolic fluxes are given for the −0.3 h (batch), 3.9 h, and 7.7 h (fed batch) (Fig. 1). Fluxes are mean values from the stoichiometric metabolite balancing of five independent cultivations and are given as molar percentages of the glucose influx. Notation is according to reference .