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Alexander Steinbuchel - One of the best experts on this subject based on the ideXlab platform.
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studies on the aerobic utilization of synthesis gas syngas by wild type and recombinant strains of Ralstonia eutropha h16
Microbial Biotechnology, 2018Co-Authors: Daniel Heinrich, Alexander Steinbuchel, Matthias RabergAbstract:The biotechnical platform strain Ralstonia eutropha H16 was genetically engineered to express a cox subcluster of the carboxydotrophic Oligotropha carboxidovoransOM5, including (i) the structural genes coxM, -S and -L, coding for an aerobic carbon monoxide dehydrogenase (CODH) and (ii) the genes coxD, -E, -F and -G, essential for the maturation of CODH. The coxOc genes expressed under control of the CO2 -inducible promoter PL enabled R. eutropha to oxidize CO to CO2 for the use as carbon source, as demonstrated by 13 CO experiments, but the recombinant strains remained dependent on H2 as external energy supply. Therefore, a synthetic metabolism, which could be described as 'carboxyhydrogenotrophic', was established in R. eutropha. With this extension of the bacterium's substrate range, growth in CO-, H2 - and CO2 -containing artificial synthesis gas atmosphere was enhanced, and poly(3-hydroxybutyrate) synthesis was increased by more than 20%.
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Ralstonia eutropha H16 in progress: Applications beside PHAs and establishment as production platform by advanced genetic tools.
Critical reviews in biotechnology, 2017Co-Authors: Matthias Raberg, Elena Volodina, Kaichien Lin, Alexander SteinbuchelAbstract:Ralstonia eutropha strain H16 is a Gram-negative non-pathogenic betaproteobacterium ubiquitously found in soils and has been the subject of intensive research for more than 50 years. Due to its rem...
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substrate and cofactor range differences of two cysteine dioxygenases from Ralstonia eutropha h16
Applied and Environmental Microbiology, 2016Co-Authors: Leonie Wenning, Alexander Steinbuchel, Nadine Stoveken, Jan Hendrik WubbelerAbstract:Cysteine dioxygenases (Cdos), which catalyze the sulfoxidation of cysteine to cysteine sulfinic acid (CSA), have been extensively studied in eukaryotes because of their roles in several diseases. In contrast, only a few prokaryotic enzymes of this type have been investigated. In Ralstonia eutropha H16, two Cdo homologues (CdoA and CdoB) have been identified previously. In vivo studies showed that Escherichia coli cells expressing CdoA could convert 3-mercaptopropionate (3MP) to 3-sulfinopropionate (3SP), whereas no 3SP could be detected in cells expressing CdoB. The objective of this study was to confirm these findings and to study both enzymes in detail by performing an in vitro characterization. The proteins were heterologously expressed and purified to apparent homogeneity by immobilized metal chelate affinity chromatography (IMAC). Subsequent analysis of the enzyme activities revealed striking differences with regard to their substrate ranges and their specificities for the transition metal cofactor, e.g., CdoA catalyzed the sulfoxidation of 3MP to a 3-fold-greater extent than the sulfoxidation of cysteine, whereas CdoB converted only cysteine. Moreover, the dependency of the activities of the Cdos from R. eutropha H16 on the metal cofactor in the active center could be demonstrated. The importance of CdoA for the metabolism of the sulfur compounds 3,3'-thiodipropionic acid (TDP) and 3,3'-dithiodipropionic acid (DTDP) by further converting their degradation product, 3MP, was confirmed. Since 3MP can also function as a precursor for polythioester (PTE) synthesis in R. eutropha H16, deletion of cdoA might enable increased synthesis of PTEs.
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Investigations on three genes in Ralstonia eutropha H16 encoding putative cyanophycin metabolizing enzymes
Applied Microbiology and Biotechnology, 2013Co-Authors: Katja Adames, Karina Euting, Anna Bröker, Alexander SteinbuchelAbstract:The genome sequence of the facultative chemolithoautotrophic bacterium Ralstonia eutropha H16 exhibited two coding sequences with high homologies to cyanophycin synthetases (CphA) as well as one gene coding for a putative cyanophycinase (CphB). To investigate whether or not the genes cphA _H16 (H16_A0774), cphA ′_H16 (H16_A0775) and cphB _H16 (H16_B1013) encode active cyanophycin (CGP) metabolism proteins, several functional analyses were performed. Extensive in silico analysis revealed that all characteristic motifs are conserved within CphA_H16, whereas CphA′_H16 misses a large part of the so-called J-loop present in other active cyanophycin synthetases. Although transcription of both genes was demonstrated by RT-PCR, and heterologously expressed cphA genes led to light-scattering inclusions in recombinant cells of Escherichia coli , no CGP could be isolated from the cells or detected by HPLC analysis. For all enzyme assay experiments carried out, significant enzyme activities were determined for CphA and CphA′ in recombinant E. coli cells if crude cell extracts were applied. Homologous expression of cphA genes in cells of R. eutropha H16∆ phaC 1 did not result in the formation of light-scattering inclusions, and no CGP could be isolated from the cells or detected by HPLC analysis. No transcription of cphB encoding a putative cyanophycinase could be detected by RT-PCR analysis and no overexpression was achieved in several strains of E. coli . Furthermore, no enzyme activity was detected by using CGP overlay agar plates.
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large scale extraction of poly 3 hydroxybutyrate from Ralstonia eutropha h16 using sodium hypochlorite
AMB Express, 2012Co-Authors: Alexander Steinbuchel, Mohamed H. Madkour, Daniel Heinrich, Mansour A Alghamdi, Ibraheem I ShabbajAbstract:Isolation of polyhydroxyalkanoates (PHAs) from bacterial cell matter is a critical step in order to achieve a profitable production of the polymer. Therefore, an extraction method must lead to a high recovery of a pure product at low costs. This study presents a simplified method for large scale poly(3-hydroxybutyrate), poly(3HB), extraction using sodium hypochlorite. Poly(3HB) was extracted from cells of Ralstonia eutropha H16 at almost 96% purity. At different extraction volumes, a maximum recovery rate of 91.32% was obtained. At the largest extraction volume of 50 L, poly(3HB) with an average purity of 93.32% ± 4.62% was extracted with a maximum recovery of 87.03% of the initial poly(3HB) content. This process is easy to handle and requires less efforts than previously described processes.
Oliver Lenz - One of the best experts on this subject based on the ideXlab platform.
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Enhanced Oxygen-Tolerance of the Full Heterotrimeric Membrane- Bound [NiFe]-Hydrogenase of Ralstonia eutropha
2016Co-Authors: Valentin Radu, Oliver Lenz, Stefan Frielingsdorf, Stephen D. Evans, Lars J. C. JeukenAbstract:ABSTRACT: Hydrogenases are oxygen-sensitive en-zymes that catalyze the conversion between protons and hydrogen. Water-soluble subcomplexes of membrane-bound [NiFe]-hydrogenases (MBH) have been extensively studied for applications in hydrogen−oxygen fuel cells as they are relatively tolerant to oxygen, although even these catalysts are still inactivated in oxidative conditions. Here, the full heterotrimeric MBH of Ralstonia eutropha, including the membrane-integral cytochrome b subunit, was investigated electrochemically using electrodes modified with planar tethered bilayer lipid membranes (tBLM). Cyclic voltammetry and chronoamperometry experiments show that MBH, in equilibrium with the quinone pool in the tBLM, does not anaerobically inactivate under oxidative redox conditions. In aerobi
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Active Site of the NAD+‑Reducing Hydrogenase from Ralstonia eutropha Studied by EPR Spectroscopy
2015Co-Authors: Oliver Lenz, Julia Löwenstein, Lars Lauterbach, Christian Teutloff, Robert BittlAbstract:Pulsed ENDOR and HYSCORE measurements were carried out to characterize the active site of the oxygen-tolerant NAD+-reducing hydrogenase of Ralstonia eutropha. The catalytically active Nia-C state exhibits a bridging hydride between iron and nickel in the active site, which is photodissociated upon illumination. Its hyperfine coupling is comparable to that of standard hydrogenases. In addition, a histidine residue could be identified, which shows hyperfine and nuclear quadrupole parameters in significant variance from comparable histidine residues that are conserved in standard [NiFe] hydrogenases, and might be related to the O2 tolerance of the enzyme
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Enhanced Oxygen-Tolerance of the Full Heterotrimeric Membrane-Bound [NiFe]-Hydrogenase of Ralstonia eutropha
2015Co-Authors: Valentin Radu, Oliver Lenz, Stefan Frielingsdorf, Stephen D. Evans, Lars J. C. JeukenAbstract:Hydrogenases are oxygen-sensitive enzymes that catalyze the conversion between protons and hydrogen. Water-soluble subcomplexes of membrane-bound [NiFe]-hydrogenases (MBH) have been extensively studied for applications in hydrogen–oxygen fuel cells as they are relatively tolerant to oxygen, although even these catalysts are still inactivated in oxidative conditions. Here, the full heterotrimeric MBH of Ralstonia eutropha, including the membrane-integral cytochrome b subunit, was investigated electrochemically using electrodes modified with planar tethered bilayer lipid membranes (tBLM). Cyclic voltammetry and chronoamperometry experiments show that MBH, in equilibrium with the quinone pool in the tBLM, does not anaerobically inactivate under oxidative redox conditions. In aerobic environments, the MBH is reversibly inactivated by O2, but reactivation was found to be fast even under oxidative redox conditions. This enhanced resistance to inactivation is ascribed to the oligomeric state of MBH in the lipid membrane
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enhanced oxygen tolerance of the full heterotrimeric membrane bound nife hydrogenase of Ralstonia eutropha
Journal of the American Chemical Society, 2014Co-Authors: Valentin Radu, Oliver Lenz, Stefan Frielingsdorf, Stephen D. Evans, Lars J. C. JeukenAbstract:Hydrogenases are oxygen-sensitive enzymes that catalyze the conversion between protons and hydrogen. Water-soluble subcomplexes of membrane-bound [NiFe]-hydrogenases (MBH) have been extensively studied for applications in hydrogen–oxygen fuel cells as they are relatively tolerant to oxygen, although even these catalysts are still inactivated in oxidative conditions. Here, the full heterotrimeric MBH of Ralstonia eutropha, including the membrane-integral cytochrome b subunit, was investigated electrochemically using electrodes modified with planar tethered bilayer lipid membranes (tBLM). Cyclic voltammetry and chronoamperometry experiments show that MBH, in equilibrium with the quinone pool in the tBLM, does not anaerobically inactivate under oxidative redox conditions. In aerobic environments, the MBH is reversibly inactivated by O2, but reactivation was found to be fast even under oxidative redox conditions. This enhanced resistance to inactivation is ascribed to the oligomeric state of MBH in the lipid ...
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novel oxygen insensitive group 5 nife hydrogenase in Ralstonia eutropha
Applied and Environmental Microbiology, 2013Co-Authors: Caspar Schafer, Barbel Friedrich, Oliver LenzAbstract:Recently, a novel group of [NiFe]-hydrogenases has been defined that appear to have a great impact in the global hydrogen cycle. This so-called group 5 [NiFe]-hydrogenase is widespread in soil-living actinobacteria and can oxidize molecular hydrogen at atmospheric levels, which suggests a high affinity of the enzyme toward H2. Here, we provide a biochemical characterization of a group 5 hydrogenase from the betaproteobacterium Ralstonia eutropha H16. The hydrogenase was designated an actinobacterial hydrogenase (AH) and is catalytically active, as shown by the in vivo H2 uptake and by activity staining in native gels. However, the enzyme does not sustain autotrophic growth on H2. The AH was purified to homogeneity by affinity chromatography and consists of two subunits with molecular masses of 65 and 37 kDa. Among the electron acceptors tested, nitroblue tetrazolium chloride was reduced by the AH at highest rates. At 30°C and pH 8, the specific activity of the enzyme was 0.3 μmol of H2 per min and mg of protein. However, an unexpectedly high Michaelis constant (Km) for H2 of 3.6 ± 0.5 μM was determined, which is in contrast to the previously proposed low Km of group 5 hydrogenases and makes atmospheric H2 uptake by R. eutropha most unlikely. Amperometric activity measurements revealed that the AH maintains full H2 oxidation activity even at atmospheric oxygen concentrations, showing that the enzyme is insensitive toward O2.
Barbel Friedrich - One of the best experts on this subject based on the ideXlab platform.
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novel oxygen insensitive group 5 nife hydrogenase in Ralstonia eutropha
Applied and Environmental Microbiology, 2013Co-Authors: Caspar Schafer, Barbel Friedrich, Oliver LenzAbstract:Recently, a novel group of [NiFe]-hydrogenases has been defined that appear to have a great impact in the global hydrogen cycle. This so-called group 5 [NiFe]-hydrogenase is widespread in soil-living actinobacteria and can oxidize molecular hydrogen at atmospheric levels, which suggests a high affinity of the enzyme toward H2. Here, we provide a biochemical characterization of a group 5 hydrogenase from the betaproteobacterium Ralstonia eutropha H16. The hydrogenase was designated an actinobacterial hydrogenase (AH) and is catalytically active, as shown by the in vivo H2 uptake and by activity staining in native gels. However, the enzyme does not sustain autotrophic growth on H2. The AH was purified to homogeneity by affinity chromatography and consists of two subunits with molecular masses of 65 and 37 kDa. Among the electron acceptors tested, nitroblue tetrazolium chloride was reduced by the AH at highest rates. At 30°C and pH 8, the specific activity of the enzyme was 0.3 μmol of H2 per min and mg of protein. However, an unexpectedly high Michaelis constant (Km) for H2 of 3.6 ± 0.5 μM was determined, which is in contrast to the previously proposed low Km of group 5 hydrogenases and makes atmospheric H2 uptake by R. eutropha most unlikely. Amperometric activity measurements revealed that the AH maintains full H2 oxidation activity even at atmospheric oxygen concentrations, showing that the enzyme is insensitive toward O2.
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autotrophic production of stable isotope labeled arginine in Ralstonia eutropha strain h16
Applied and Environmental Microbiology, 2012Co-Authors: Steffen Lutte, Anne Pohlmann, Edward Schwartz, Evgeny Zaychikov, Johannes R Becher, Hermann Heumann, Barbel FriedrichAbstract:With the aim of improving industrial-scale production of stable-isotope (SI)-labeled arginine, we have developed a system for the heterologous production of the arginine-containing polymer cyanophycin in recombinant strains of Ralstonia eutropha under lithoautotrophic growth conditions. We constructed an expression plasmid based on the cyanophycin synthetase gene (cphA) of Synechocystis sp. strain PCC6308 under the control of the strong PcbbL promoter of the R. eutropha H16 cbbc operon (coding for autotrophic CO2 fixation). In batch cultures growing on H2 and CO2 as sole sources of energy and carbon, respectively, the cyanophycin content of cells reached 5.5% of cell dry weight (CDW). However, in the absence of selection (i.e., in antibiotic-free medium), plasmid loss led to a substantial reduction in yield. We therefore designed a novel addiction system suitable for use under lithoautotrophic conditions. Based on the hydrogenase transcription factor HoxA, this system mediated stabilized expression of cphA during lithoautotrophic cultivation without the need for antibiotics. The maximum yield of cyanophycin was 7.1% of CDW. To test the labeling efficiency of our expression system under actual production conditions, cells were grown in 10-liter-scale fermentations fed with 13CO2 and 15NH4Cl, and the 13C/15N-labeled cyanophycin was subsequently extracted by treatment with 0.1 M HCl; 2.5 to 5 g of [13C/15N]arginine was obtained per fed-batch fermentation, corresponding to isotope enrichments of 98.8% to 99.4%.
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h2 conversion in the presence of o2 as performed by the membrane bound nife hydrogenase of Ralstonia eutropha
ChemPhysChem, 2010Co-Authors: Oliver Lenz, Marcus Ludwig, Torsten Schubert, Ingmar Burstel, Stefanie Ganskow, Tobias Goris, Alexander Schwarze, Barbel FriedrichAbstract:[NiFe]-hydrogenases catalyze the oxidation of H(2) to protons and electrons. This reversible reaction is based on a complex interplay of metal cofactors including the Ni-Fe active site and several [Fe-S] clusters. H(2) catalysis of most [NiFe]-hydrogenases is sensitive to dioxygen. However, some bacteria contain hydrogenases that activate H(2) even in the presence of O(2). There is now compelling evidence that O(2) affects hydrogenase on three levels: 1) H(2) catalysis, 2) hydrogenase maturation, and 3) H(2)-mediated signal transduction. Herein, we summarize the genetic, biochemical, electrochemical, and spectroscopic properties related to the O(2) tolerance of hydrogenases resident in the facultative chemolithoautotroph Ralstonia eutropha H16. A focus is given to the membrane-bound [NiFe]-hydogenase, which currently represents the best-characterized member of O(2)-tolerant hydrogenases.
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spectroscopic insights into the oxygen tolerant membrane associated nife hydrogenase of Ralstonia eutropha h16
Journal of Biological Chemistry, 2009Co-Authors: Miguel Saggu, Marcus Ludwig, Barbel Friedrich, Oliver Lenz, Ingo Zebger, Peter Hildebrandt, Friedhelm LendzianAbstract:This study provides the first spectroscopic characterization of the membrane-bound oxygen-tolerant [NiFe] hydrogenase (MBH) from Ralstonia eutropha H16 in its natural environment, the cytoplasmic membrane. The H2-converting MBH is composed of a large subunit, harboring the [NiFe] active site, and a small subunit, capable in coordinating one [3Fe4S] and two [4Fe4S] clusters. The hydrogenase dimer is electronically connected to a membrane-integral cytochrome b. EPR and Fourier transform infrared spectroscopy revealed a strong similarity of the MBH active site with known [NiFe] centers from strictly anaerobic hydrogenases. Most redox states characteristic for anaerobic [NiFe] hydrogenases were identified except for one remarkable difference. The formation of the oxygen-inhibited Niu-A state was never observed. Furthermore, EPR data showed the presence of an additional paramagnetic center at high redox potential (+290 mV), which couples magnetically to the [3Fe4S] center and indicates a structural and/or redox modification at or near the proximal [4Fe4S] cluster. Additionally, significant differences regarding the magnetic coupling between the Nia-C state and [4Fe4S] clusters were observed in the reduced form of the MBH. The spectroscopic properties are discussed with regard to the unusual oxygen tolerance of this hydrogenase and in comparison with those of the solubilized, dimeric form of the MBH.
Edward Schwartz - One of the best experts on this subject based on the ideXlab platform.
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autotrophic production of stable isotope labeled arginine in Ralstonia eutropha strain h16
Applied and Environmental Microbiology, 2012Co-Authors: Steffen Lutte, Anne Pohlmann, Edward Schwartz, Evgeny Zaychikov, Johannes R Becher, Hermann Heumann, Barbel FriedrichAbstract:With the aim of improving industrial-scale production of stable-isotope (SI)-labeled arginine, we have developed a system for the heterologous production of the arginine-containing polymer cyanophycin in recombinant strains of Ralstonia eutropha under lithoautotrophic growth conditions. We constructed an expression plasmid based on the cyanophycin synthetase gene (cphA) of Synechocystis sp. strain PCC6308 under the control of the strong PcbbL promoter of the R. eutropha H16 cbbc operon (coding for autotrophic CO2 fixation). In batch cultures growing on H2 and CO2 as sole sources of energy and carbon, respectively, the cyanophycin content of cells reached 5.5% of cell dry weight (CDW). However, in the absence of selection (i.e., in antibiotic-free medium), plasmid loss led to a substantial reduction in yield. We therefore designed a novel addiction system suitable for use under lithoautotrophic conditions. Based on the hydrogenase transcription factor HoxA, this system mediated stabilized expression of cphA during lithoautotrophic cultivation without the need for antibiotics. The maximum yield of cyanophycin was 7.1% of CDW. To test the labeling efficiency of our expression system under actual production conditions, cells were grown in 10-liter-scale fermentations fed with 13CO2 and 15NH4Cl, and the 13C/15N-labeled cyanophycin was subsequently extracted by treatment with 0.1 M HCl; 2.5 to 5 g of [13C/15N]arginine was obtained per fed-batch fermentation, corresponding to isotope enrichments of 98.8% to 99.4%.
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a proteomic view of the facultatively chemolithoautotrophic lifestyle of Ralstonia eutropha h16
Proteomics, 2009Co-Authors: Edward Schwartz, Anne Pohlmann, Oliver Lenz, Alexander Schwarze, Birgit Voigt, Daniela Zuhlke, Dirk Albrecht, Yvonne Kohlmann, Cornelia Krause, Michael HeckerAbstract:Ralstonia eutropha H16 is an H2-oxidizing, facultative chemolithoautotroph. Using 2-DE in conjunction with peptide mass spectrometry we have cataloged the soluble proteins of this bacterium during growth on different substrates: (i) H2 and CO2, (ii) succinate and (iii) glycerol. The first and second conditions represent purely lithoautotrophic and purely organoheterotrophic nutrition, respectively. The third growth regime permits formation of the H2-oxidizing and CO2-fixing systems concomitant to utilization of an organic substrate, thus enabling mixotrophic growth. The latter type of nutrition is probably the relevant one with respect to the situation faced by the organism in its natural habitats, i.e. soil and mud. Aside from the hydrogenase and Calvin-cycle enzymes, the protein inventories of the H2-CO2- and succinate-grown cells did not reveal major qualitative differences. The protein complement of the glycerol-grown cells resembled that of the lithoautotrophic cells. Phosphoenolpyruvate (PEP) carboxykinase was present under all three growth conditions, whereas PEP carboxylase was not detectable, supporting earlier findings that PEP carboxykinase is alone responsible for the anaplerotic production of oxaloacetate from PEP. The elevated levels of oxidative stress proteins in the glycerol-grown cells point to a significant challenge by ROS under these conditions. The results reported here are in agreement with earlier physiological and enzymological studies indicating that R. eutropha H16 has a heterotrophic core metabolism onto which the functions of lithoautotrophy have been grafted.
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Genome sequence of the bioplastic-producing “Knallgas” bacterium Ralstonia eutropha H16
Nature Biotechnology, 2006Co-Authors: Anne Pohlmann, Christian Ewering, Markus Pötter, Frank Reinecke, Wolfgang Florian Fricke, Thomas Eitinger, Bernhard Kusian, Rainer Cramm, Heiko Liesegang, Edward SchwartzAbstract:The H(2)-oxidizing lithoautotrophic bacterium Ralstonia eutropha H16 is a metabolically versatile organism capable of subsisting, in the absence of organic growth substrates, on H(2) and CO(2) as its sole sources of energy and carbon. R. eutropha H16 first attracted biotechnological interest nearly 50 years ago with the realization that the organism's ability to produce and store large amounts of poly[R-(-)-3-hydroxybutyrate] and other polyesters could be harnessed to make biodegradable plastics. Here we report the complete genome sequence of the two chromosomes of R. eutropha H16. Together, chromosome 1 (4,052,032 base pairs (bp)) and chromosome 2 (2,912,490 bp) encode 6,116 putative genes. Analysis of the genome sequence offers the genetic basis for exploiting the biotechnological potential of this organism and provides insights into its remarkable metabolic versatility.
Anthony J Sinskey - One of the best experts on this subject based on the ideXlab platform.
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2001. Mechanistic studies on class I polyhydroxybutyrate (PHB) synthase from Ralstonia eutropha: class I and class III synthases share a similar catalytic mechanism. Biochemistry 40:1011–1019
2015Co-Authors: Yong Jia, Anthony J Sinskey, Wei Yuan, Jola Wodzinska, Chung Park, Joanne StubbeAbstract:ABSTRACT: The Class I and III polyhydroxybutyrate (PHB) synthases from Ralstonia eutropha and Chromatium Vinosum, respectively, catalyze the polymerization of â-hydroxybutyryl-coenzyme A (HBCoA) to generate PHB. These synthases have different molecular weights, subunit composition, and kinetic properties. Recent studies with the C. Vinosum synthase suggested that it is structurally homologous to bacterial lipases and allowed identification of active site residues important for catalysis [Jia, Y., Kappock
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periplasmic α carbonic anhydrase plays an essential role in Ralstonia eutropha co2 metabolism
BMC Proceedings, 2014Co-Authors: Claudia S Gai, Christopher J Brigham, Amanda Bernardi, Anthony J SinskeyAbstract:Background Carbonic anhydrase (CA) enzymes catalyze the interconversion of CO2 and bicarbonate. These enzymes play important roles in cellular metabolism such as CO2 transport, ion transport, and internal pH regulation. Understanding the roles of CAs in the chemolithotropic betaproteobacteria Ralstonia eutropha is important for the development of fermentation processes based on the bacterium’s capacity for carbon fixation using the CalvinBenson-Bassham cycle. Of the five classes of CA, the alpha-CA is the best-characterized thus far. The gene encoding a periplasmic alpha-CA (caa, H16 B2403) has been identified in the R. eutropha H16 genome, along with three others CA from different classes. In this study, we evaluated the importance of Caa in the metabolism of R. eutropha by examination of CA activity and growth in caa gene deletion, complementation, and overexpression strains. Localization of Caa in the cell was accessed by fluorescent microscopy. Methods
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effects of intracellular poly 3 hydroxybutyrate reserves on physiological biochemical properties and growth of Ralstonia eutropha
Research in Microbiology, 2013Co-Authors: Tatiana G. Volova, Christopher J Brigham, Galina S. Kalacheva, Natalia O Zhila, Anthony J SinskeyAbstract:Microbial polyhydroxyalkanoates (PHAs), because of their well studied complex physiology and commercial potential, are vehicles for carbon and potential storage reduction for many microbial species. Even with the wealth of studies about microbial PHAs in the scientific literature, polymer accumulation and degradation are still not comprehensively understood. Poly(3-hydroxybutyrate) (P3HB) granule formation and polymer mobility were studied here in the bacterium Ralstonia eutropha strain B5786 in autotrophic cultures. Electron microscopy studies revealed decreasing cell size concomitant with enlargement of size and number of intracellular granules, and inhibition of cell division during intracellular polymer production. Activities of key P3HB biosynthetic enzymes demonstrated correlations with each other during polymer accumulation, suggesting an intricately regulated P3HB cycle in autotrophically grown R. eutropha cells. 2012 Published by Elsevier Masson SAS on behalf of Institut Pasteur.
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engineering Ralstonia eutropha for production of isobutanol from co2 h2 and o2
2013Co-Authors: Christopher J Brigham, Jingnan Lu, Daan R Speth, Mark R Worden, Anthony J SinskeyAbstract:Isobutanol (IBT) can be used as a 100% replacement for gasoline in existing automobile engines, has >90% of the energy density of gasoline and is compatible with established fuel distribution infrastructure. The facultatively autotrophic bacterium Ralstonia eutropha can utilize H2 for energy and CO2 for carbon and is also employed in industrial processes that produce biodegradable plastics. Using a carefully designed production pathway, R. eutropha, a genetically tractable organism, can be modified to produce biofuels from autotrophic growth. Microbial production of IBT can be achieved by directing the flow of carbon through a synthetic production pathway involving the branched-chain amino acid biosynthesis pathway, a heterologously expressed ketoisovalerate decarboxylase, and a broad substrate specificity alcohol dehydrogenase. We discuss the motivations and the methods used to engineer R. eutropha to produce the liquid transportation fuel IBT from CO2, H2, and O2.
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Growth and polyhydroxybutyrate production by Ralstonia eutropha in emulsified plant oil medium
Applied Microbiology and Biotechnology, 2011Co-Authors: Charles F. Budde, Stefan Risch, Chokyun Rha, Florian Hubner, Sebastian L. Riedel, Milan K Popovic, Anthony J SinskeyAbstract:Polyhydroxyalkanoates (PHAs) are natural polyesters synthesized by bacteria for carbon and energy storage that also have commercial potential as bioplastics. One promising class of carbon feedstocks for industrial PHA production is plant oils, due to the high carbon content of these compounds. The bacterium Ralstonia eutropha accumulates high levels of PHA and can effectively utilize plant oil. Growth experiments that include plant oil, however, are difficult to conduct in a quantitative and reproducible manner due to the heterogeneity of the two-phase medium. In order to overcome this obstacle, a new culture method was developed in which palm oil was emulsified in growth medium using the glycoprotein gum arabic as the emulsifying agent. Gum arabic did not influence R. eutropha growth and could not be used as a nutrient source by the bacteria. R. eutropha was grown in the emulsified oil medium and PHA production was measured over time. Additionally, an extraction method was developed to monitor oil consumption. The new method described in this study allows quantitative, reproducible R. eutropha experiments to be performed with plant oils. The method may also prove useful for studying growth of different bacteria on plant oils and other hydrophobic carbon sources.
