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Glc) and catabolite repression mediated by the global regulator cyclic AMP (cAMP)-cAMP receptor protein (CRP). We measured in a systematic way the relation between cellular growth rates and the key parameters of catabolite repression, i.e., the phosphorylated EIIA Crr (EIIA Crr ϳP) level and the cAMP level, using in vitro and in vivo assays. Different growth rates were obtained by using either various carbon sources or by growing the cells with limited concentrations of glucose, sucrose, and mannitol in continuous bioreactor experiments. The ratio of EIIA Crr to EIIA Crr ϳP and the intracellular cAMP concentrations, deduced from the activity of a cAMP-CRP-dependent promoter, correlated well with specific growth rates between 0.3 h ؊1 and 0.7 h ؊1 , corresponding to generation times of about 138 and 60 min, respectively. Below and above this range, these parameters were increasingly uncoupled from the growth rate, which perhaps indicates an increasing role executed by other global control systems, in particular the stringent-relaxed response system.In Escherichia coli, the phosphoenolpyruvate (PEP)-dependent phosphotransferase systems (PTSs) represent important uptake systems for a number of carbohydrates which mediate transport and concomitant phosphorylation of their respective substrates (10,44). In addition to their transport function, all components of the various PTSs of a cell form an important signal transduction system. The signal transduction properties of the PTS depend on the phosphorylation state of its proteins (26,49). The PTSs usually consist of two general proteins, i.e., the PEP-dependent protein kinase enzyme I (EI), and the histidine-containing protein (HPr), and up to 20 different, substrate-specific enzymes II (EII). EII usually comprise two soluble domains EIIA and EIIB involved in phosphotransfer and the membrane-bound transporter domain EIIC (44). The major regulatory output signal of the PTS depends on the phosphorylation level of EIIA Crr (according to its genetic nomenclature), also designated EIIA Glc due to its function as the EIIA domain for the glucose-specific PTS (9, 23, 52). EIIA Crr inhibits the activity of a number of non-PTS transporters and enzymes (8,32,33,35,36), a process referred to as inducer exclusion. Furthermore, the phosphorylated form of EIIA Crr (EIIA Crr ϳP) activates adenylate cyclase (1, 13, 41, 57), which in turn synthesizes cyclic AMP (cAMP) (59). The indicator molecule or alarmone cAMP is the coactivator of the important global transcription factor CRP (cAMP receptor protein). Together, they regulate in a process called cAMP-CRP-dependent catabolite repression efficient transcription of different genes involved in the synthesis of a large number of catabolic enzymes (4,39,43). The central role of EIIA Crr ϳP in the activation of adenylate cyclase is largely based on mutant analysis (13,23,33).The phosphorylation state of the PTS and hence the intracellular cAMP concentrations are postulated to depend largely on two major factors: (i) the uptake rate of any P...

A dynamic mathematical model was developed to describe the uptake of various carbohydrates (glucose, lactose, glycerol, sucrose, and galactose) in Escherichia coli. For validation a number of isogenic strains with defined mutations were used. By considering metabolic reactions as well as signal transduction processes influencing the relevant pathways, we were able to describe quantitatively the phenomenon of catabolite repression in E. coli. We verified model predictions by measuring time courses of several extraand intracellular components such as glycolytic intermediates, EII-A Crr phosphorylation level, both LacZ and PtsG concentrations, and total cAMP concentrations under various growth conditions. The entire data base consists of 18 experiments performed with nine different strains. The model describes the expression of 17 key enzymes, 38 enzymatic reactions, and the dynamic behavior of more than 50 metabolites. The different phenomena affecting the phosphorylation level of EIIA Crr , the key regulation molecule for inducer exclusion and catabolite repression in enteric bacteria, can now be explained quantitatively.Catabolite repression in Escherichia coli designates the observation that if different carbohydrates are present in a medium under unlimited conditions, one of them is usually taken up preferentially. Although the fundamental biochemical principles of the regulatory network have been revealed, a quantitative description of this growth behavior is still missing. The center of the regulatory network is formed by the phosphoenolpyruvate (PEP) 4 :carbohydrate phosphotransferase systems (PTS). These systems are involved in both transport and phosphorylation of a large number of carbohydrates, in movement toward these carbon sources (chemotaxis), and in regulation of a number of metabolic pathways (1-3). The PTS in E. coli consist of two common cytoplasmatic proteins, EI (enzyme I) and HPr (histidine-containing protein), as well as an array of carbohydrate-specific EII (enzyme II) complexes. Because all components of the PTS, depending on their phosphorylation status, can interact with various key regulator proteins, the output of the PTS is represented by the degree of phosphorylation of the proteins involved in phosphoryl group transfer, e.g. unphosphorylated EIIA Crr inhibits the uptake of other non-PTS carbohydrates by a process called inducer exclusion. Phosphorylated EIIA Crr activates the adenylate cyclase (CyaA) and leads to an increase in the intracellular cAMP level.Understanding the regulation of carbohydrate uptake requires a quantitative description of the PTS. In this context it is important that the degree of phosphorylation of EIIA Crr is proportional to the PEP/ pyruvate ratio, when no carbohydrates are transported (4) and the respective equilibrium constant is an upper boundary when the PTS is active (5). The PTS should therefore not be regarded as a measure for the transport of PTS substrates but more as a general measure for carbohydrate availability. One feature of our contribution...

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A benchmark problem is described for the reconstruction and analysis of biochemical networks given sampled experimental data. The growth of the organisms is described in a bioreactor in which one substrate is fed into the reactor with a given feed rate and feed concentration. Measurements for some intracellular components are provided representing a small biochemical network. Problems of reverse engineering, parameter estimation, and identifiability are addressed. The contribution mainly focuses on the problem of model discrimination. If two or more model variants describe the available experimental data, a new experiment must be designed to discriminate between the hypothetical models. For the problem presented, the feed rate and feed concentration of a bioreactor system are available as control inputs. To verify calculated input profiles an interactive Web site (http://www.sysbio.de/ projects/benchmark/) is provided. Several solutions based on linear and nonlinear models are discussed.

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BackgroundOne of the major challenges for the present and future generations is to find suitable substitutes for the fossil resources we rely on today. Cyanobacterial carbohydrates have been discussed as an emerging renewable feedstock in industrial biotechnology for the production of fuels and chemicals, showing promising production rates when compared to crop-based feedstock. However, intrinsic capacities of cyanobacteria to produce biotechnological compounds are limited and yields are low.ResultsHere, we present an approach to circumvent these problems by employing a synthetic bacterial co-culture for the carbon-neutral production of polyhydroxyalkanoates (PHAs) from CO2. The co-culture consists of two bio-modules: Bio-module I, in which the cyanobacterial strain Synechococcus elongatus cscB fixes CO2, converts it to sucrose, and exports it into the culture supernatant; and bio-module II, where this sugar serves as C-source for Pseudomonas putida cscAB and is converted to PHAs that are accumulated in the cytoplasm. By applying a nitrogen-limited process, we achieved a maximal PHA production rate of 23.8 mg/(L day) and a maximal titer of 156 mg/L. We will discuss the present shortcomings of the process and show the potential for future improvement.ConclusionsThese results demonstrate the feasibility of mixed cultures of S. elongatus cscB and P. putida cscAB for PHA production, making room for the cornucopia of possible products that are described for P. putida. The construction of more efficient sucrose-utilizing P. putida phenotypes and the optimization of process conditions will increase yields and productivities and eventually close the gap in the contemporary process. In the long term, the co-culture may serve as a platform process, in which P. putida is used as a chassis for the implementation of synthetic metabolic pathways for biotechnological production of value-added products.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-017-0875-0) contains supplementary material, which is available to authorized users.