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

Metabolic Deficiences Revealed in the Biotechnologically Important Model Bacterium Escherichia coli BL21(DE3)

The Escherichia coli B strain BL21(DE3) has had a profound impact on biotechnology through its use in the production of recombinant proteins. Little is understood, however, regarding the physiology of this important E. coli strain. We show here that BL21(DE3) totally lacks activity of the four [NiFe]-hydrogenases, the three molybdenum- and selenium-containing formate dehydrogenases and molybdenum-dependent nitrate reductase. Nevertheless, all of the structural genes necessary for the synthesis of the respective anaerobic metalloenzymes are present in the genome. However, the genes encoding the high-affinity molybdate transport system and the molybdenum-responsive transcriptional regulator ModE are absent from the genome. Moreover, BL21(DE3) has a nonsense mutation in the gene encoding the global oxygen-responsive transcriptional regulator FNR. The activities of the two hydrogen-oxidizing hydrogenases, therefore, could be restored to BL21(DE3) by supplementing the growth medium with high concentrations of Ni2+ (Ni2+-transport is FNR-dependent) or by introducing a wild-type copy of the fnr gene. Only combined addition of plasmid-encoded fnr and high concentrations of MoO42− ions could restore hydrogen production to BL21(DE3); however, to only 25–30% of a K-12 wildtype. We could show that limited hydrogen production from the enzyme complex responsible for formate-dependent hydrogen evolution was due solely to reduced activity of the formate dehydrogenase (FDH-H), not the hydrogenase component. The activity of the FNR-dependent formate dehydrogenase, FDH-N, could not be restored, even when the fnr gene and MoO42− were supplied; however, nitrate reductase activity could be recovered by combined addition of MoO42− and the fnr gene. This suggested that a further component specific for biosynthesis or activity of formate dehydrogenases H and N was missing. Re-introduction of the gene encoding ModE could only partially restore the activities of both enzymes. Taken together these results demonstrate that BL21(DE3) has major defects in anaerobic metabolism, metal ion transport and metalloprotein biosynthesis

The Escherichia coli fdv gene probably encodes mutS and is located at minute 58.8 adjacent to the hyc-hyp gene cluster.

The region between min 58 and 59 of the Escherichia coli chromosome contains two gene clusters whose products are structural and regulatory components of the formate hydro-genlyase (FHL) system. The genes are organized in two divergently transcribed operons (2, 5, 6). The hyc operon, transcribed in the counterclockwise direction, contains hy-drogenase 3 structural genes (2), whereas the products of the clockwise-transcribed hyp genes pleiotropically influence the activities of all three hydrogenases of E. coli (6, 10). The last gene of the hyp transcriptional unit (6), IhIA, codes for a regulatory protein which activates the expression of the structural genes of the FHL complex under fermentative conditions (11). The product of the gene followingJIhA in the same transcriptional direction (termed fdv) has been pro-posed to also play a role in the FHL system (7, 9). Determination and analysis of the nucleotide sequence of fdv revealed that it is separated fromJhlA by a 676-bp DNA segment. The large distance between the genes and the presence of a potential transcriptional terminator behind IA make it unlikely that fdv is part of the hyp transcrip-tional unit. Analysis of the deduced amino acid sequence of Fdv showed approximately 90 % similarity (identical resi-dues and conservative substitutions) to that of the MutS protein of Salmonella typhimurium (3). Both proteins have 853 amino acids and therefore molecular weights of approx-imately 95,000. Large regions of the primary structures of the two proteins are nearly identical, while three smal

HNF1A recruits KDM6A to activate differentiated acinar cell programs that suppress pancreatic cancer

Defects in transcriptional regulators of pancreatic exocrine differentiation have been implicated in pancreatic tumorigenesis, but the molecular mechanisms are poorly understood. The locus encoding the transcription factor HNF1A harbors susceptibility variants for pancreatic ductal adenocarcinoma (PDAC), while KDM6A, encoding Lysine-specific demethylase 6A, carries somatic mutations in PDAC. Here, we show that pancreas-specific Hnf1a null mutant transcriptomes phenocopy those of Kdm6a mutations, and both defects synergize with KrasG12D to cause PDAC with sarcomatoid features. We combine genetic, epigenomic, and biochemical studies to show that HNF1A recruits KDM6A to genomic binding sites in pancreatic acinar cells. This remodels the acinar enhancer landscape, activates differentiated acinar cell programs, and indirectly suppresses oncogenic and epithelial-mesenchymal transition genes. We also identify a subset of non-classical PDAC samples that exhibit the HNF1A/KDM6A-deficient molecular phenotype. These findings provide direct genetic evidence that HNF1A deficiency promotes PDAC. They also connect the tumor-suppressive role of KDM6A deficiency with a cell-specific molecular mechanism that underlies PDAC subtype definition.This research was supported by the National Institute for Health Research (NIHR) Imperial Biomedical Research Centre. Work was funded by grants from the Wellcome Trust (WT101033 to J.F.), Medical Research Council (MR/L02036X/1 to J.F.), European Research Council Advanced Grant (789055 to J.F.), Ministerio de Ciencia e Innovación (BFU2014‐54284‐R, RTI2018‐095666‐B‐I00 to J.F., SAF2011‐29530 and SAF2015‐70553‐R to F.X.R.) and RTICC from Instituto de Salud Carlos III (RD12/0036/0034, RD12/0036/0050) to F.X.R. M.K. was supported by a Juvenile Diabetes Research Foundation postdoctoral fellowship (3‐PDF‐2014‐192‐A‐N). I.M. was supported by a Fellowship from Fundació Bancaria La Caixa (ID 100010434) (grant number LCF/BQ/ES18/11670009). Work in CRG was supported by the CERCA Programme, Generalitat de Catalunya, and support from Ministerio de Ciencia e Innovación to the EMBL partnership. Work at CRG and CNIO was supported by Centro de Excelencia Severo Ochoa grants SEV‐2012‐0208, SEV‐2016‐0510

Nanogap Dielectric Spectroscopy for Aptamer-Based Protein Detection

Among the various label-free methods for monitoring biomolecular interactions, capacitive sensors stand out due to their simple instrumentation and compatibility with multiplex formats. However, electrode polarization due to ion gradient formation and noise from solution conductance limited early dielectric spectroscopic measurements to high frequencies only, which in turn limited their sensitivity to biomolecular interactions, as the applied excitation signals were too fast for the charged macromolecules to respond. To minimize electrode polarization effects, capacitive sensors with 20 nm electrode separation were fabricated using silicon dioxide sacrificial layer techniques. The nanoscale separation of the capacitive electrodes in the sensor results in an enhanced overlapping of electrical double layers, and apparently a more ordered “ice-like” water structure. Such effects in turn reduce low frequency contributions from bulk sample resistance and from electrode polarization, and thus markedly enhance sensitivity toward biomolecular interactions. Using these nanogap capacitive sensors, highly sensitive, label-free aptamer-based detection of protein molecules is achieved

Estimation of the annual primary production of the lichen Cetrariella delisei in a glacier foreland in the High Arctic, Ny-Ølesund, Svalbard

The fruticose lichen Cetrariella delisei is among the dominant lichen species in the deglaciated High Arctic areas of Svalbard. As part of a study of carbon cycling in the High Arctic, we aimed to estimate the primary production of lichen in a deglaciated area in Ny-Ålesund, Svalbard (79° N), by examining the effects of abiotic factors on the net photosynthesis (Pn) and dark respiration (R) rates of C. delisei. Experiments were conducted in the snow-free season of 2000 using an open-fl ow gas exchange system with an infrared gas analyser. Positive photosynthetic activities were observed on rainy days or soon after rainfall when the thallus water content was high, whereas photosynthetic activities dropped below the detectable limit on clear days because of the low thallus water content. Under a suffi ciently high thallus water content and close to light saturation, Pn was nearly constant over a wide temperature range of 4 - 20 °C, while R increased with increasing temperature. We constructed a model for estimating the net primary production (NPP) of lichen based on the relationships between abiotic factors and the CO2 exchange rate. The mean, minimum and maximum NPP values in the snow-free season, estimated using meteorological data obtained from 1995–2003, were 5.1, 1.0 and 8.4 g dry wt. m–2 snow-free season–1, respectively. These results suggest that NPP is highly variable and the contribution of lichen to carbon input is small compared with that of vascular plants and mosses in the study site