Cytochrome c peroxidases (CCP) play a key role in cellular detoxification by catalyzing the reduction of hydrogen peroxide to water. The di-heme CCP from Rhodobacter capsulatus is the fastest enzyme (1060 s ؊1 ), when tested with its physiological cytochrome c substrate, among all di-heme CCPs characterized to date and has, therefore, been an attractive target to investigate structure-function relationships for this family of enzymes. Here, we combine for the first time structural studies with site-directed mutagenesis and spectroscopic studies of the mutant enzymes to investigate the roles of amino acid residues that have previously been suggested to be important for activity. The crystal structure of R. capsulatus at 2.7 Å in the fully oxidized state confirms the overall molecular scaffold seen in other di-heme CCPs but further reveals that a segment of about 10 amino acids near the peroxide binding site is disordered in all four molecules in the asymmetric unit of the crystal. Structural and sequence comparisons with other structurally characterized CCPs suggest that flexibility in this part of the molecular scaffold is an inherent molecular property of the R. capsulatus CCP and of CCPs in general and that it correlates with the levels of activity seen in CCPs characterized, thus, far. Mutagenesis studies support the spin switch model and the roles that Met-118, Glu-117, and Trp-97 play in this model. Our results help to clarify a number of aspects of the debate on structure-function relationships in this family of bacterial CCPs and set the stage for future studies.

E. coli cells produce acetate as an extracellular coproduct of aerobic cultures. Acetate is undesirable because it retards growth and inhibits protein formation. Most process designs or genetic modifications to minimize acetate formation aim at balancing growth rate and oxygen consumption. In this research, three genetic approaches to reduce acetate formation were investigated: (1) direct reduction of the carbon flow to acetate (ackA-pta, poxB knock-out); (2) anticipation on the underlying metabolic and regulatory mechanisms that lead to acetate (constitutive ppc expression mutant); and (3) both (1) and (2). Initially, these mutants were compared to the wild-type E. coli via batch cultures under aerobic conditions. Subsequently, these mutants were further characterized using metabolic flux analysis on continuous cultures. It is concluded that a combination of directly reducing the carbon flow to acetate and anticipating on the underlying metabolic and regulatory mechanism that lead to acetate, is the most promising approach to overcome acetate formation and improve recombinant protein production. These genetic modifications have no significant influence on the metabolism when growing the micro-organisms under steady state at relatively low dilution rates (less than 0.4 h(-1)).

The main requirement for metabolic flux analysis (MFA) is that the cells are in a pseudo-steady state, that there is no accumulation or depletion of intracellular metabolites. In the past, the applications of MFA were limited to the analysis of continuous cultures. This contribution introduces the concept of dynamic MFA and extends MFA so that it is applicable to transient cultures. Time series of concentration measurements are transformed into flux values. This transformation involves differentiation, which typically increases the noisiness of the data. Therefore, a noise-reducing step is needed. In this work, polynomial smoothing was used. As a test case, dynamic MFA is applied on Escherichia coli cultivations shifting from carbon limitation to nitrogen limitation and vice versa. After switching the limiting substrate from N to C, a lag phase was observed accompanied with an increase in maintenance energy requirement. This lag phase did not occur in the C- to N-limitation case.

Current climate issues and the ongoing depletion of oil reserves have led to increased attention for biobased production processes. Not only has the production of bio-energy gained interest, but also the production of biochemicals. Succinate is one of those biochemicals. In the presented work, the dicarboxylic acid transport system of Escherichia coli was modified to enhance production of succinate, a highly attractive chemical building block. The engineering comprised the elimination of succinate uptake and the overexpression of succinate export. However, succinate export in Escherichia coli is normally only active under anaerobic conditions and import only under aerobic conditions. Therefore, the gene responsible for succinate import, dctA, was knocked out and the gene coding for succinate export, dcuC, was overexpressed with a constitutive artificial promoter. In the applied batch cultivations, these modifications increased succinate yield and specific production rate more than 50% in a Delta dhA Delta sdhB background (0.16 C-mole/C-mole glucose and 0.17 C-mole/C-mole biomass/h, respectively), but also revealed alternative succinate import proteins, YdjN and YbhI. Mutations in the genes coding for these proteins led to increased growth rates and specific production rates, however, they did not increase succinate yield (e. g. the deletion of ybhI resulted in a growth rate of 0.54 h(-1) and a specific production rate of 0.23 C-mole/C-mole biomass/h)

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