Increased Hydrogen Production by Genetic Engineering of Escherichia coli

Escherichia coli is capable of producing hydrogen under anaerobic growth conditions. Formate is converted to hydrogen in the fermenting cell by the formate hydrogenlyase enzyme system. The specific hydrogen yield from glucose was improved by the modification of transcriptional regulators and metabolic enzymes involved in the dissimilation of pyruvate and formate. The engineered E. coli strains ZF1 (ΔfocA; disrupted in a formate transporter gene) and ZF3 (ΔnarL; disrupted in a global transcriptional regulator gene) produced 14.9, and 14.4 µmols of hydrogen/mg of dry cell weight, respectively, compared to 9.8 µmols of hydrogen/mg of dry cell weight generated by wild-type E. coli strain W3110. The molar yield of hydrogen for strain ZF3 was 0.96 mols of hydrogen/mol of glucose, compared to 0.54 mols of hydrogen/mol of glucose for the wild-type E. coli strain. The expression of the global transcriptional regulator protein FNR at levels above natural abundance had a synergistic effect on increasing the hydrogen yield in the ΔfocA genetic background. The modification of global transcriptional regulators to modulate the expression of multiple operons required for the biosynthesis of formate hydrogenlyase represents a practical approach to improve hydrogen production

On-line analysis and in situ pH monitoring of mixed acid fermentation by Escherichia coli using combined FTIR and Raman techniques

We introduce an experimental setup allowing continuous monitoring of bacterial fermentation processes by simultaneous optical density (OD) measurements, long-path FTIR headspace monitoring of CO2, acetaldehyde and ethanol, and liquid Raman spectroscopy of acetate, formate, and phosphate anions, without sampling. We discuss which spectral features are best suited for detection, and how to obtain partial pressures and concentrations by integrations and least squares fitting of spectral features. Noise equivalent detection limits are about 2.6 mM for acetate and 3.6 mM for formate at 5 min integration time, improving to 0.75 mM for acetate and 1.0 mM for formate at 1 h integration. The analytical range extends to at least 1 M with a standard deviation of percentage error of about 8%. The measurement of the anions of the phosphate buffer allows the spectroscopic, in situ determination of the pH of the bacterial suspension via a modified Henderson-Hasselbalch equation in the 6–8 pH range with an accuracy better than 0.1. The 4 m White cell FTIR measurements provide noise equivalent detection limits of 0.21 μbar for acetaldehyde and 0.26 μbar for ethanol in the gas phase, corresponding to 3.2 μM acetaldehyde and 22 μM ethanol in solution, using Henry’s law. The analytical dynamic range exceeds 1 mbar ethanol corresponding to 85 mM in solution. As an application example, the mixed acid fermentation of Escherichia coli is studied. The production of CO2, ethanol, acetaldehyde, acids such as formate and acetate, and the changes in pH are discussed in the context of the mixed acid fermentation pathways. Formate decomposition into CO2 and H2 is found to be governed by a zeroth-order kinetic rate law, showing that adding exogenous formate to a bioreactor with E. coli is expected to have no beneficial effect on the rate of formate decomposition and biohydrogen production

Anaerobic induction of pyruvate formate-lyase gene expression is mediated by the ArcA and FNR proteins.

The pyruvate formate-lyase (pfl) gene of Escherichia coli is transcribed from seven promoters which are coordinately induced 12- to 15-fold by anaerobiosis. The FNR protein plays a major role in the anaerobic control of this system. A mutation in the fnr gene, however, only reduces anaerobic induction fivefold, indicating that FNR is not the only factor involved in the anaerobic activation process (Sawers and Böck, J. Bacteriol. 171:2485-2498, 1989). The residual anaerobic induction could be shown to be imparted by the transcriptional regulator ArcA; an arcA fnr double mutant was incapable of inducing pfl transcription anaerobically. A mutant strain unable to synthesize the membrane-associated histidine kinase (ArcB) that has been proposed to activate ArcA showed the same phenotype as an arcA mutant strain, indicating that a functional ArcB protein is also required for wild-type, anaerobic pfl transcriptional activation. Nuclease S1 analysis revealed that an arcA mutation abolished anaerobic transcription from promoter 7 and reduced expression from promoter 6 but did not affect transcription from promoters 1 to 5. On the other hand, an fnr mutation prevented anaerobic expression from promoters 6 and 7 and reduced transcription from promoters 1 to 5. These data indicate that both ArcA and FNR are essential for anaerobic activation of promoter 7 transcription, which suggests functional interaction between these proteins

Dynamics and efficiency in vivo of UGA-directed selenocysteine insertion at the ribosome.

The kinetics and efficiency of decoding of the UGA of a bacterial selenoprotein mRNA with selenocysteine has been studied in vivo. A gst-lacZ fusion, with the fdhF SECIS element ligated between the two fusion partners, gave an efficiency of read-through of 4-5%; overproduction of the selenocysteine insertion machinery increased it to 7-10%. This low efficiency is caused by termination at the UGA and not by translational barriers at the SECIS. When the selenocysteine UGA codon was replaced by UCA, and tRNASec with anticodon UGA was allowed to compete with seryl-tRNASer1 for this codon, selenocysteine was found in 7% of the protein produced. When a non-cognate SelB-tRNASec complex competed with EF-Tu for a sense codon, no effects were seen, whereas a non-cognate SelB-tRNASec competing with EF-Tu-mediated Su7-tRNA nonsense suppression of UGA interfered strongly with suppression. The induction kinetics of beta-galactosidase synthesis from fdhF'-'lacZ gene fusions in the absence or presence of SelB and/or the SECIS element, showed that there was a translational pause in the fusion containing the SECIS when SelB was present. The results show that decoding of UGA is an inefficient process and that using the third dimension of the mRNA to accommodate an additional amino acid is accompanied by considerable quantitative and kinetic costs

The soluble cytoplasmic N‐terminal domain of the FocA channel gates bidirectional formate translocation

FocA belongs to the pentameric FNT (formate-nitrite transporter) superfamily of anion channels, translocating formate bidirectionally across the cytoplasmic membrane of Escherichia coli and other microorganisms. While the membrane-integral core of FocA shares considerable amino acid sequence conservation with other FNT family members, the soluble cytoplasmic N-terminal domain does not. To analyze the potential biochemical function of FocA’s N-terminal domain in vivo, we constructed truncation derivatives and amino acid-exchange variants, and determined their ability to translocate formate across the membrane of E. coli cells by monitoring intracellular formate levels using a formate-sensitive reporter system. Analysis of strains synthesizing these FocA variants provided insights into formate efflux. Strains lacking the ability to generate formate intracellularly allowed us to determine whether these variants could import formate or its toxic chemical analog hypophosphite. Our findings reveal that the N-terminal domain of FocA is crucial for bidirectional FocA-dependent permeation of formate across the membrane. Moreover, we show that an amino acid sequence motif and secondary structural features of the flexible N-terminal domain are important for formate translocation, and efflux/influx is influenced by pyruvate formate-lyase. The soluble N-terminal domain is, therefore, essential for bidirectional formate translocation by FocA, suggesting a “gate-keeper” function controlling anion accessibility.Publikationsfonds ML

Identification and functional validation of novel autoantigens in equine uveitis.

The development, progression, and recurrence of autoimmune diseases are frequently driven by a group of participatory autoantigens. We identified and characterized novel autoantigens by analyzing the autoantibody binding pattern from horses affected by spontaneous equine recurrent uveitis to the retinal proteome. Cellular retinaldehyde-binding protein (cRALBP) had not been described previously as autoantigen, but subsequent characterization in equine recurrent uveitis horses revealed B and T cell autoreactivity to this protein and established a link to epitope spreading. We further immunized healthy rats and horses with cRALBP and observed uveitis in both species with typical tissue lesions at cRALBP expression sites. The autoantibody profiling outlined here could be used in various autoimmune diseases to detect autoantigens involved in the dynamic spreading cascade or serve as predictive markers. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc