Topology of the global regulatory network of carbon limitation in Escherichia coli
Introduction
In biotechnological processes, the cell growth often is controlled by substrate limitation. This strategy is particularly applied in fed-batch cultivations employing exponential and constant feeding profiles, which not least guarantees respectable high cell densities. Substrate-limited growth, however, results in an excessive energy consumption for the maintenance of cellular functions (Lengeler et al., 1999, Pirt, 1982) as well as in other disadvantageous stress-related effects. Enfors’ research group (Bylund et al., 1998, Larsson et al., 1996, Schweder et al., 1999, Teich et al., 1999, Xu et al., 1999a), as well as Hewitt et al., 2000, Hewitt et al., 1999, Hewitt and Nebe-Von-Caron (2001) and Lapin et al. (2006) found that in large-scale fed-batch processes the extracellular gradients of substrate concentrations have profound effects on the growth yield, product formation and viability of the cell population. Evidently, such findings are the result of the interaction of the individual cell with its abiotic environment, which determines the cell's regulatory response—and thus, its metabolic state. Consequently, the rational optimization of biotechnological processes (metabolic engineering) requires dynamic mathematical models comprising both regulation and metabolism. Ongoing research in several academic groups and industry is focused on modelling the central carbon metabolism (glycolysis, pentose phosphate pathway and TCA cycle) of Escherichia coli and its regulation during glucose-limited growth (Ellison et al., 2006, Heijnen et al., 2006). The first step in modelling is the identification of the model structure (topology), which relies on the knowledge from previous publications (bottom-up approach). Detailed information about the transcriptional regulation of the genes encoding metabolic enzymes in E. coli is available from databases (Keseler et al., 2005, Salgado et al., 2006). However, the regulators significantly affecting the central carbon metabolism during carbon limitation and consequently, the model topology of the global genetic regulatory network3 have not yet been identified. In the complementary top-down approach the model structure is deduced from the system's response to an external stimulus, applying proper systems-level experimental tools like proteomics, transcriptomics, metabolomics and metabolic flux analysis (MFA). However, the dynamic metabolic behaviour of the relevant pathways in response to carbon limitation still remains to be clarified in E. coli, which can be studied in fed-batch cultivations. When applying a constant feed rate, the substrate limitation continually increases and a succession of (physiological) quasi-steady states can be achieved (Dunn and Mor, 1975). This allows investigating the metabolic and regulatory response during the transition from exponential to carbon-limited growth.
Bacteria control metabolism and growth rate through global genetic regulatory systems (see the footnote 3), i.e. regulons and modulons (Lengeler et al., 1999, Neidhardt and Savageau, 1996). Prominent examples in E. coli are the catabolite repression (crp modulon) and the stringent response (relA/spoT modulon), two processes that are active under carbon-limiting conditions. During stringent response (reviewed in Braeken et al. (2006), Cashel et al. (1996) and Lengeler et al. (1999)), the limitation of nutrients leads to the intracellular accumulation of ppGpp (guanosine 3′,5′-bis(diphosphate)), which is supposed to bind to the RNA polymerase (Artsimovitch et al., 2004). The transcription of genes involved in the translation process – in particular of ribosomal RNA and ribosomal proteins – is negatively regulated by ppGpp. As a result, the protein biosynthesis rate declines, which in turn also leads to a reduction in growth rate (Cashel et al., 1996, Lengeler et al., 1999). During amino acid limitation, the synthesis of ppGpp or guanosine pentaphosphate (pppGpp), collectively referred to as (p)ppGpp, is mediated by RelA (GDP pyrophosphokinase/GTP pyrophosphokinase). Under amino acid-limiting conditions, the ribosome-bound RelA protein is stimulated by uncharged tRNAs at the A site of ribosomes (Wendrich et al., 2002). However, the accumulation of (p)ppGpp depends also on the dual activity of the SpoT protein as (p)ppGpp-hydrolase or (p)ppGpp-synthetase. Although it is known from a homologous protein of Streptococcus dysgalactiae subsp. equisimilis that the opposing activities of SpoT are reciprocally regulated (Hogg et al., 2004, Mechold et al., 2002), the regulation of the SpoT protein in E. coli is still hypothetical. The most important issue for understanding growth control is the signalling mechanism, which leads to accumulation of ppGpp under carbon-limiting conditions, an aspect that is still not entirely clarified.
Besides various effects on growth-related functions (Cashel et al., 1996), the alarmone ppGpp is known to be involved in the regulation of the sigma S factor concentration (σS; rpoS gene) on the transcriptional and posttranscriptional level (Hengge-Aronis, 2002). As an alternative subunit of RNA polymerase, σS is involved in the regulation of transcription in the general stress response in E. coli (also designated as ‘stationary phase response’). It is assumed that elevated levels of σS negatively regulate σD-dependent housekeeping genes, such as the TCA cycle genes (Patten et al., 2004). Moreover, ppGpp influences the competition between different stress-related sigma factors in the binding of the RNA polymerase core enzyme at the expense of the sigma factor σD (Jishage et al., 2002) and the RNA polymerase availability (Barker et al., 2001a, Barker et al., 2001b, Cashel et al., 1996, Jensen and Pedersen, 1990, Traxler et al., 2006). The crp modulon belongs to a group of global genetic regulatory systems, which can be subsumed under the term catabolite control. One basic feature of these systems is that the presence or absence of an extracellular carbon source is indicated by an intracellular metabolite (catabolite) that serves as a signal for derepression (catabolite activation) or deactivation (catabolite repression) of catabolic genes (Saier et al., 1996). The crp modulon includes catabolic operons for the utilization of various carbon sources and is regulated by the Crp–cAMP complex. The synthesis of the alarmone cAMP (cyclic 3′,5′-AMP) by the enzyme adenylate cyclase (CyaA) is stimulated by the phophorylated EIIAGlc protein, a component of the E. coli phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) (reviewed in Lengeler et al. (1999) and Postma et al. (1993)). It is assumed that a low glucose uptake rate by the PTS and a high ratio of phosphoenolpyruvate and pyruvate concentrations (cPEP/cPyr) lead to the phosphorylation of the EIIAGlc protein (Hogema et al., 1998). Consequently, limited glucose availability leads to the synthesis of cAMP and the transcriptional regulator complex Crp–cAMP is formed. Catabolite control is also exerted by the catabolite repressor/activator protein Cra (formerly designated FruR), which regulates numerous genes involved in the carbon and energy metabolism (the cra modulon) (reviewed in Ramseier (1996), Saier and Ramseier (1996) and Saier et al. (1996)). The regulator protein Cra is inactivated by the catabolites fructose 1-phosphate and fructose 1,6-bis(phosphate) (Saier and Ramseier, 1996).
The present study demonstrates the signal formation and dynamic metabolic responses of E. coli K-12 W3110 exposed to an increasing carbon limitation during fed-batch cultivation applying a constant feed rate. The observed decrease in the biomass yield is shown to result from a substantial carbon flow into the TCA cycle and from the subsequent oxidation of the carbon source. Simultaneously, most of the fluxes in the central carbon metabolism and in the biosynthesis pathways decreased significantly. This rearrangement is supposed to re-establish a balance between anabolism and catabolism after nutrient limitation. The genetic regulatory systems responsible for the illustrated metabolic responses are proposed and assembled to a global regulatory network. A new method for determining the qualitative time course of the intracellular cAMP concentration is presented. For the first time the profile of the intracellular cAMP level is shown in fed-batch cultivations of E. coli wild-type cells. Most importantly, the resetting of the cAMP signal could be demonstrated. The suggested network takes account of the observed signal resetting (cAMP and ppGpp) and of a probable stringent response-signalling pathway during carbon limitation. The provided network topology is novel inasmuch as it comprehensively explains the obtained systems-level data of the metabolic transition from exponential to carbon-limited growth typical of fed-batch processes.
Section snippets
Bacterial strain and fed-batch cultivation
The fed-batch cultivations were carried out with the bacterial strain E. coli K-12 W3110 (DSM 5911, German Collection of Microorganisms and Cell Cultures) in a 30-l bioreactor (Bioengineering AG, Wald, Switzerland). Minimal medium supplemented with glucose as the carbon source was used. The batch medium (batch volume VR,0 = 17 l) consisted of 8.8 g l−1 glucose·H2O, 2.0 g l−1 Na2SO4·10H2O, 2.68 g l−1 (NH4)2SO4, 1.0 g l−1 NH4Cl, 14.6 g l−1 K2HPO4, 4.02 g l−1 NaH2PO4·2H2O, 0.01 g l−1 thiamine HCl; 0.3 mM CaCl2·2H2
Fed-batch cultivation
Five independent fed-batch cultivation experiments applying a constant feed rate were carried out to study the response of the central carbon metabolism of E. coli concerning glucose limitation. The time profiles of the extracellular concentrations of glucose, acetate and biomass are presented in Fig. 1a. Acetate was produced during the consumption of glucose in the batch phase and was used up after 1 h of glucose-limited growth (Fig. 1a). In response to the limited availability of the carbon
Topology of the network regulating the central carbon metabolism
The work presented here demonstrates the formation and resetting of the intracellular signals of carbon limitation, cAMP and ppGpp, and the redistribution of metabolic fluxes during glucose-limited fed-batch growth. In order to explain the observed dynamic behaviour the topology of the global genetic regulatory network of the regulation of central carbon metabolism is identified. This involves the examination of global genetic regulatory systems (regulons and modulons) that are known to be
Acknowledgments
The authors would like to thank Insilico Biotechnology AG for providing the software tool Insilico Discovery. We would also like to thank Petra Schlack, Andreas Freund and Achim Hauck for excellent technical assistance in the analysis of nucleotides and during the cultivations; Heiko Schwarz for the support in the development of the analytical procedure for the determination of nucleotides; Volker Windeisen for the in vitro synthesis of ppGpp; Jochen Rebell, Institut für Organische Chemie
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- 1
Present address: Institute of Biochemical Engineering, University of Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany.
- 2
Present address: Fraunhofer Institute for Manufacturing Engineering and Automation, Department Cleanroom Manufacturing, Nobelstrasse 12, 70569 Stuttgart, Germany.