Eutropha H16 Research Articles

Correction for Brigham et al., "Whole-Genome Microarray and Gene Deletion Studies Reveal Regulation of the Polyhydroxyalkanoate Production Cycle by the Stringent Response in Ralstonia eutropha H16".

Correction for Brigham et al., "Whole-Genome Microarray and Gene Deletion Studies Reveal Regulation of the Polyhydroxyalkanoate Production Cycle by the Stringent Response in Ralstonia eutropha H16".

CbbR and RegA regulate cbb operon transcription in Ralstonia eutropha H16

The biotechnologically important Gram-negative β-proteobacterium Ralstonia eutropha H16 is able to grow lithoautotrophically by utilizing CO2 and H2 as sole carbon and energy sources, respectively. CO2 is fixed by the CBB cycle, which is encoded in duplicate on the genome of R. eutropha H16. The transcription of both cbb operons is controlled by the transcription regulator CbbR dependent on intracellular PEP levels as a response to the carbon-state of the cell. As demonstrated in this study transcription control of both cbb operons appears to be more complex and additionally involves, next to CbbR, the transcription regulator RegA as part of the global transcription regulation system RegA/RegB. The identification of a highly conserved RegA/RegB homologue in R. eutropha H16 and experimental evidence gathered in this study reveal that RegA plays a crucial role in the transcription control of both cbb promoters. RegA is able to induce cbb promoter activity and controls transcription in combination with CbbR dependent on cellular PEP concentrations. These results clearly demonstrate that RegA plays an important role in cbb operon transcription regulation and may also be relevant for the control of other energy-utilizing and energy-generating pathways of R. eutropha H16. In addition to promoting a more complete understanding of the CO2 fixation mechanism of R. eutropha H16 these findings also provide crucial insights for the utilization of this bacterium in biotechnological applications with respect to CO2 fixation.

Absence of ppGpp Leads to Increased Mobilization of Intermediately Accumulated Poly(3-Hydroxybutyrate) in Ralstonia eutropha H16.

In this study, we constructed a set of Ralstonia eutropha H16 strains with single, double, or triple deletions of the (p)ppGpp synthase/hydrolase (spoT1), (p)ppGpp synthase (spoT2), and/or polyhydroxybutyrate (PHB) depolymerase (phaZa1 or phaZa3) gene, and we determined the impact on the levels of (p)ppGpp and on accumulated PHB. Mutants with deletions of both the spoT1 and spoT2 genes were unable to synthesize detectable amounts of (p)ppGpp and accumulated only minor amounts of PHB, due to PhaZa1-mediated depolymerization of PHB. In contrast, unusually high levels of PHB were found in strains in which the (p)ppGpp concentration was increased by the overexpression of (p)ppGpp synthase (SpoT2) and the absence of (p)ppGpp hydrolase. Determination of (p)ppGpp levels in wild-type R. eutropha under different growth conditions and induction of the stringent response by amino acid analogs showed that the concentrations of (p)ppGpp during the growth phase determine the amount of PHB remaining in later growth phases by influencing the efficiency of the PHB mobilization system in stationary growth. The data reported for a previously constructed ΔspoT2 strain (C. J. Brigham, D. R. Speth, C. Rha, and A. J. Sinskey, Appl Environ Microbiol 78:8033-8044, 2012, https://doi.org/10.1128/AEM.01693-12) were identified as due to an experimental error in strain construction, and our results are in contrast to the previous indication that the spoT2 gene product is essential for PHB accumulation in R. eutrophaIMPORTANCE Polyhydroxybutyrate (PHB) is an important intracellular carbon and energy storage compound in many prokaryotes and helps cells survive periods of starvation and other stress conditions. Research activities in several laboratories over the past 3 decades have shown that both PHB synthase and PHB depolymerase are constitutively expressed in most PHB-accumulating bacteria, such as Ralstonia eutropha This implies that PHB synthase and depolymerase activities must be well regulated in order to avoid a futile cycle of simultaneous PHB synthesis and PHB degradation (mobilization). Previous reports suggested that the stringent response in Rhizobium etli and R. eutropha is involved in the regulation of PHB metabolism. However, the levels of (p)ppGpp and the influence of those levels on PHB accumulation and PHB mobilization have not yet been determined for any PHB-accumulating species. In this study, we optimized a (p)ppGpp extraction procedure and a high-performance liquid chromatography-mass spectrometry (HPLC-MS)-based detection method for the quantification of (p)ppGpp in R. eutropha This enabled us to study the relationship between the concentrations of (p)ppGpp and the accumulated levels of PHB in the wild type and in several constructed mutant strains. We show that overproduction of the alarmone (p)ppGpp correlated with reduced growth and massive overproduction of PHB. In contrast, in the absence of (p)ppGpp, mobilization of PHB was dramatically enhanced.

A novel nitrilase from Ralstonia eutropha H16 and its application to nicotinic acid production.

A novel aliphatic nitrilase, REH16, was found in Ralstonia eutropha H16 and overexpressed in Escherichia coli BL21(DE3), and its enzymatic properties were studied. The temperature and pH optima were 37°C and 6.6, respectively, and the best thermostability of the nitrilase was observed at 25°C, which preserved 95% of activity after 120h of incubation. REH16 has a broad hydrolytic activity toward aliphatic and heterocyclic nitriles and showed high tolerance of 3-cyanopyridine; this enzyme could hydrolyze as high as 100mM 3-cyanopyridine completely. To improve the 3-cyanopyridine conversion efficiency in an aqueous reaction system, water-miscible organic solvents were tested, and ethanol (10% v/v) was chosen as the optimal co-solvent. Finally, under optimized conditions, using the fed-batch reaction mode, total of 1050mM 3-cyanopyridine was hydrolyzed completely in 20.8h with eight substrate feedings, yielding 129.2g/L production of nicotinic acid and thus showing a potential for industrial application.

New Insights into PhaM-PhaC-Mediated Localization of Polyhydroxybutyrate Granules in Ralstonia eutropha H16

The formation and localization of polyhydroxybutyrate (PHB) granules in Ralstonia eutropha are controlled by PhaM, which interacts both with the PHB synthase (PhaC) and with the bacterial nucleoid. Here, we studied the importance of proline and lysine residues of two C-terminal PAKKA motifs in PhaM for their importance in attaching PHB granules to DNA by in vitro and in vivo methods. Substitution of the lysine residues but not of the proline residues resulted in detachment of formed PHB granules from the nucleoid. Instead, formation of PHB granule clusters at polar regions of the rod-shaped cells and an unequal distribution of PHB granules to daughter cells were observed. The formation of PHB granules was studied by the expression of chromosomally anchored gene fusions of fluorescent proteins with PhaM and PhaC in different backgrounds. PhaM and PhaC fusions showed a distinct colocalization at formed PHB granules in the nucleoid region of the wild type. In a ΔphaC background, PhaM and the catalytically inactive PhaCC319A protein were not able to form fluorescent foci, indicating that correct positioning requires the formation of PHB. Furthermore, time-lapse experiments revealed that PhaC and PhaM proteins detach from formed PHB granules at later stages, resulting in a nonhomogeneous population of PHB granules. This could explain why growth of individual PHB granules stops under PHB-permissive conditions at a certain size.IMPORTANCE PHB granules are storage compounds for carbon and energy in many prokaryotes. Equal distribution of accumulated PHB granules during cell division is therefore important for optimal fitness of the daughter cells. In R. eutropha, PhaM is responsible for maximal activity of PHB synthase, for initiation of PHB granule formation at discrete regions in the cells, and for association of formed PHB granules with the nucleoid. Here we found that four lysine residues of C-terminal PhaM sequence motifs are essential for association of PHB granules with the nucleoid. Furthermore, we followed PHB granule formation by time-lapse microscopy and provide evidence for aging of PHB granules that is manifested by detachment of previously PHB granule-associated PhaM and PHB synthase.

Comparison of metabolite profiling of Ralstonia eutropha H16 phaBCA mutants grown on different carbon sources

Metabolite profiling was conducted to monitor the metabolic differences of the Ralstonia eutropha H16, polyhydroxybutyrate (PHB) producing strain, when grown on different sole carbon sources and to identify the effect of the deletion of the PHB production pathway. The combination of rapid filtration and boiling ethanol extraction successfully extracted intracellular metabolites. Accumulation of acetyl-CoA was detected when the strain was grown on fructose. Whereas, lower adenylate energy charge (AEC) ratio and cell mass was identified on the cells grown on acetate. No significant difference was detected between the wild type and mutant strain except metabolites in the PHB synthetic pathway. Increased PHB production is expected on fructose due to the accumulation of AcCoA, while it is more difficult to improve the production on acetate because of lower metabolic precursor level and AEC ratio.

Conversion of cysteine to 3-mercaptopyruvic acid by bacterial aminotransferases.

3-Mercaptopyruvate (3MPy), a structural analog of 3-mercaptopropionic acid, is a precursor compound for biosynthesis of polythioesters in bacteria. The cost-effectiveness and sustainability of the whole process could be greatly improved by using the cysteine degradation pathway for an intracellular supply of 3MPy. Transamination of cysteine to its corresponding α-keto acid 3MPy is catalyzed by cysteine aminotransferases (CAT). However, CAT activity has so far not been described for bacterial aminotransferases (AT), and it was unknown whether they can be applied for the conversion of cysteine to 3MPy. In this study, we selected eight bacterial aminotransferases based on sequence homology to CAT of Rattus norvegicus (Got1). The aminotransferases included four aspartate aminotransferases (AATs) and four aromatic amino acid aminotransferases (ArATs) from Advenella mimigardefordensis DPN7, Escherichia coli MG1655, Shimwellia blattae ATCC 33430, Ralstonia eutropha H16 and Paracoccus denitrificans PD1222. For a more detailed characterization, all selected AAT or ArAT encoding genes were heterologously expressed in E. coli and purified. CAT activity was detected for all aminotransferases when a novel continuous coupled enzyme assay was applied. Kinetic studies revealed the highest catalytic efficiency of 5.1mM/s for AAT from A. mimigardefordensis. Formation of 3MPy from cysteine could additionally be verified by an optimized approach using derivatization of 3MPy with the Girard T reagent and liquid chromatography-mass spectrometry analyses.

Generation of poly-β-hydroxybutyrate from acetate in higher plants: Detection of acetoacetyl CoA reductase- and PHB synthase- activities in rice

It has been reported that Poly-β-hydroxybutyrate (PHB) is generated from acetate in the rice root. However, no information is available about the biosynthetic pathway of PHB from acetate in plant cells. In the bacterium Ralstonia eutropha H16 (R. eutropha), PHB is synthesized from acetyl CoA by the consecutive reaction of three enzymes: β-ketothiolase (EC: 2.3.1.9), acetoacetyl CoA reductase (EC: 1.1.1.36) and PHB synthase (EC: 2.3.1.-). Thus, in this study, we examined whether the above three enzymatic activities were also detected in rice seedlings. The results clearly showed that the activities of the above three enzymes were all detected in rice. In particular, the PHB synthase activity was detected specifically in the sonicated particulate fractions (2000g 10min precipitate (ppt) and the 8000g 30min ppt) of rice roots and leaves. In addition to these enzyme activities, several new experimental results were obtained on PHB synthesis in higher plants: (a) 14C-PHB generated from 2-14C-acetate was mainly localized in the 2000g 10min ppt and the 8000g 30min ppt of rice root. (b) Addition of acetate (0.1–10mM) to culture medium of rice seedlings did not increase the content of PHB in the rice root or leaf. (c) In addition to C3 plants, PHB was generated from acetate in a C4 plant (corn) and in a CAM plant (Bryophyllum pinnatum). d) Washing with ethylenediaminetetraacetic acid (EDTA) strongly suggested that the PHB synthesized from acetate was of plant origin and was not bacterial contamination.

Microbial production of ethanol from acetate by engineered Ralstonia eutropha

This study was performed to produce ethanol from acetate using a genetically engineered Ralstonia eutropha. In order to genetically modify R. eutropha H16, phaCAB operon encoding metabolic pathway genes from acetyl-CoA to polyhydroxybutyrate (PHB) was deleted and adhE encoding an alcohol dehydrogenase from Escherichia coli was overexpressed for conversion of acetyl-CoA to ethanol. The resulting strain produced ethanol up to 170 mg/L when cultivated in minimal media supplemented with 5 g/L of acetate as a sole carbon source. Growth and ethanol production were optimized by adjusting nitrogen source (NH4Cl) content and repetitive feeding of acetate into the bacterial culture, by which the ethanol production was reached to approximately 350 mg/L for 84 h.

Oxidized Polyethylene Wax as a Potential Carbon Source for PHA Production.

We report on the ability of bacteria to produce biodegradable polyhydroxyalkanoates (PHA) using oxidized polyethylene wax (O-PEW) as a novel carbon source. The O-PEW was obtained in a process that used air or oxygen as an oxidizing agent. R. eutropha H16 was grown for 48 h in either tryptone soya broth (TSB) or basal salts medium (BSM) supplemented with O-PEW and monitored by viable counting. Study revealed that biomass and PHA production was higher in TSB supplemented with O-PEW compared with TSB only. The biopolymers obtained were preliminary characterized by nuclear magnetic resonance (NMR), gel permeation chromatography (GPC), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). The detailed structural evaluation at the molecular level was performed by electrospray ionization tandem mass spectrometry (ESI-MS/MS). The study revealed that, when TSB was supplemented with O-PEW, bacteria produced PHA which contained 3-hydroxybutyrate and up to 3 mol % of 3-hydroxyvalerate and 3-hydroxyhexanoate co-monomeric units. The ESI-MS/MS enabled the PHA characterization when the content of 3-hydroxybutyrate was high and the appearance of other PHA repeating units was very low.

Characterization of two novel alcohol short-chain dehydrogenases/reductases from Ralstonia eutropha H16 capable of stereoselective conversion of bulky substrates

Biocatalysis has significant advantages over organic synthesis in the field of chiral molecule production and several types of stereoselective enzymes are already in use in industrial biotechnology. However, there is still a high demand for new enzymes capable of transforming bulky molecules with sufficient operability. In order to reveal novel high-potential biocatalysts, the complete genome of the β-proteobacterium Ralstonia eutropha H16 was screened for potential short-chain dehydrogenases/reductases (SDRs). We were able to identify two (S)-enantioselective SDRs named A5 and B3. These showed clear preference towards long-chain and aromatic secondary alcohols, aldehydes and ketones, with diaryl diketone benzil as one of the best substrates. In addition the phylogenetic analysis of all enzyme types, which are known to facilitate benzil reduction, revealed at least two separate evolutionary clusters. Our results indicate the biotechnological potential of SDRs A5 and B3 for the production of chiral compounds with potential commercial value.

Substrate and Cofactor Range Differences of Two Cysteine Dioxygenases from Ralstonia eutropha H16.

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.

Strain and process development for poly(3HB-co-3HP) fermentation by engineered Shimwellia blattae from glycerol.

Poly(3-hydroxybytyrate-co-3-hydroxypropionate), poly(3HB-co-3HP), is a possible alternative to synthetic polymers such as polypropylene, polystyrene and polyethylene due to its low crystallinity and fragility. We already reported that recombinant strains of Shimwellia blattae expressing 1,3-propanediol dehydrogenase DhaT as well as aldehyde dehydrogenase AldD of Pseudomonas putida KT2442, propionate-CoA transferase Pct of Clostridium propionicum X2 and PHA synthase PhaC1 of Ralstonia eutropha H16 are able to accumulate up to 14.5% (wtPHA/wtCDW) of poly(3-hydroxypropionate), poly(3HP), homopolymer from glycerol as a sole carbon source (Appl Microbiol Biotechnol 98:7409-7422, 2014a). However, the cell density was rather low. In this study, we optimized the medium aiming at a more efficient PHA synthesis, and we engineered a S. blattae strain accumulating poly(3HB-co-3HP) with varying contents of the constituent 3-hydroxypropionate (3HP) depending on the cultivation conditions. Consequently, 7.12, 0.77 and 0.32 gPHA/L of poly(3HB-co-3HP) containing 2.1, 8.3 and 18.1 mol% 3HP under anaerobic/aerobic (the first 24 hours under anaerobic condition, thereafter, aerobic condition), low aeration/agitation (the minimum stirring rate required in medium mixing and small amount of aeration) and anaerobic conditions (the minimum stirring rate required in medium mixing without aeration), respectively, were synthesized from glycerol by the genetically modified S. blattae ATCC33430 strains in optimized culture medium.

Model-based control to maximise biomass and PHB in the autotrophic cultivation of Ralstonia eutropha

This paper presents a realisation of a closed-loop controlled autotrophic fed-batch cultivation of Ralstonia eutropha H16 with the aim to maximise the amount of biomass and internal storage compound PHB. The special control unit is a model-based on-line trajectory planning with a sigma-point Kalman filter for state estimation and an additional gas phase controller. For the control, a medium-sized structured model describing the process is proposed and compared to cultivation data. The model considers the internal storage of PHB by R. eutropha and pays special attention to the production of the oxygen-tolerant membranebound hydrogenase. This enzyme is in the center of biological research as a component of a biological fuel cell. It is shown that on-line optimisation works for maximising the amounts of biomass and PHB.

Comparative proteome analysis reveals four novel polyhydroxybutyrate (PHB) granule-associated proteins in Ralstonia eutropha H16.

Identification of proteins that were present in a polyhydroxybutyrate (PHB) granule fraction isolated from Ralstonia eutropha but absent in the soluble, membrane, and membrane-associated fractions revealed the presence of only 12 polypeptides with PHB-specific locations plus 4 previously known PHB-associated proteins with multiple locations. None of the previously postulated PHB depolymerase isoenzymes (PhaZa2 to PhaZa5, PhaZd1, and PhaZd2) and none of the two known 3-hydroxybutyrate oligomer hydrolases (PhaZb and PhaZc) were significantly present in isolated PHB granules. Four polypeptides were found that had not yet been identified in PHB granules. Three of the novel proteins are putative α/β-hydrolases, and two of those (A0671 and B1632) have a PHB synthase/depolymerase signature. The third novel protein (A0225) is a patatin-like phospholipase, a type of enzyme that has not been described for PHB granules of any PHB-accumulating species. No function has been ascribed to the fourth protein (A2001), but its encoding gene forms an operon with phaB2 (acetoacetyl-coenzyme A [CoA] reductase) and phaC2 (PHB synthase), and this is in line with a putative function in PHB metabolism. The localization of the four new proteins at the PHB granule surface was confirmed in vivo by fluorescence microscopy of constructed fusion proteins with enhanced yellow fluorescent protein (eYFP). Deletion of A0671 and B1632 had a minor but detectable effect on the PHB mobilization ability in the stationary growth phase of nutrient broth (NB)-gluconate cells, confirming the functional involvement of both proteins in PHB metabolism.

Development of a feeding strategy for high cell and PHA density fed-batch fermentation of Ralstonia eutropha H16 from organic acids and their salts

The utilization of inexpensive carbon sources, including waste streams, for production of value added products has been explored for over two decades. Palm oil mill effluent (POME), a waste water stream from the palm oil industry, can be used to produce organic acids and their salts by anaerobic digestion. These organic acids are suitable as the main carbon source for growth and production of polyhydroxyalkanoates (PHAs) by organisms like Ralstonia eutropha. The presence of propionate/propionic acid results in the biosynthesis of the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(HB-co-HV)], which is desired for its favourable thermal and mechanical properties compared to the polyhydroxybutyrate (PHB) homopolymer. A specially-designed fed-batch strategy is necessary to decrease the toxic effects of organic acids in cultures of R. eutropha. A pH-stat fed-batch culture technique, using organic acids as the main carbon source, was adopted in combination with an additional pO2-dependent feed for delivering organic acid salts. The developed strategy is highly reproducible and shows a high productivity of >2g PHA/L/h. After 44h of fermentation, 112.4±2.3g/L cell dry weight (CDW) with 83.3±1.1% P(HB-co-HV)/CDW containing 5.6±0.4mol% 3-hydroxyvalerate (3HV) were achieved. The described productivity, CDW and P(HB-co-HV)/CDW values are by far the highest reported results for R. eutropha cultivated on mixed organic acids as the main carbon source.

NADH-dependent lactate dehydrogenase from Alcaligenes eutrophus H16 reduces 2-oxoadipate to 2-hydroxyadipate

Adipic acid is an important monomer for the production of nylon-6,6 polyamide. One novel biological route for the synthesis of adipic acid, which combines the lysine synthetic pathway and glutaconic acid production pathway, has been suggested, but this route has suffered from the lack of an efficient 2-oxoadipate reductase connecting the two pathways or converting 2-oxoadipate to 2-hydroxyadipate. In this study, we report that the lactate dehydrogenase of Alcaligenes eutrophus H16 is a promising catalyst for this reaction. The lactate dehydrogenase gene (Ae-ldhO) was cloned, expressed in Escherichia coli, purified, and characterized. The recombinant enzyme, having a molecular weight of 36.7 kDa, exhibited broad substrate specificity for various 2-oxoacids. NADH was the preferred coenzyme over NADPH for all 2-oxoacids tested. The maximum specific activity of Ae-LdhO on 2-oxoadipate was 454.5 ± 20.1 U/mg protein at pH 7.0 and 30℃. The Km values for 2-oxoadipic acid and NADH were 0.32 ± 0.02 and 0.09 ± 0.002 mM, respectively. The activity of Ae-LdhO was enhanced in the presence of some metal ions, such as Mg2+, Co2+ or Ni2+, whereas it was completely inhibited by Hg2+, Ag+, Cu2+ and DTT.

Impact of Ralstonia eutropha's poly(3-Hydroxybutyrate) (PHB) Depolymerases and Phasins on PHB storage in recombinant Escherichia coli.

The model organism for polyhydroxybutyrate (PHB) biosynthesis, Ralstonia eutropha H16, possesses multiple isoenzymes of granules coating phasins as well as of PHB depolymerases, which degrade accumulated PHB under conditions of carbon limitation. In this study, recombinant Escherichia coli BL21(DE3) strains were used to study the impact of selected PHB depolymerases of R. eutropha H16 on the growth behavior and on the amount of accumulated PHB in the absence or presence of phasins. For this purpose, 20 recombinant E. coli BL21(DE3) strains were constructed, which harbored a plasmid carrying the phaCAB operon from R. eutropha H16 to ensure PHB synthesis and a second plasmid carrying different combinations of the genes encoding a phasin and a PHB depolymerase from R. eutropha H16. It is shown in this study that the growth behavior of the respective recombinant E. coli strains was barely affected by the overexpression of the phasin and PHB depolymerase genes. However, the impact on the PHB contents was significantly greater. The strains expressing the genes of the PHB depolymerases PhaZ1, PhaZ2, PhaZ3, and PhaZ7 showed 35% to 94% lower PHB contents after 30 h of cultivation than the control strain. The strain harboring phaZ7 reached by far the lowest content of accumulated PHB (only 2.0% [wt/wt] PHB of cell dry weight). Furthermore, coexpression of phasins in addition to the PHB depolymerases influenced the amount of PHB stored in cells of the respective strains. It was shown that the phasins PhaP1, PhaP2, and PhaP4 are not substitutable without an impact on the amount of stored PHB. In particular, the phasins PhaP2 and PhaP4 seemed to limit the degradation of PHB by the PHB depolymerases PhaZ2, PhaZ3, and PhaZ7, whereas almost no influence of the different phasins was observed if phaZ1 was coexpressed. This study represents an extensive analysis of the impact of PHB depolymerases and phasins on PHB accumulation and provides a deeper insight into the complex interplay of these enzymes.

Reprint of “Versatile and stable vectors for efficient gene expression in Ralstonia eutropha H16”

The Gram-negative β-proteobacterium Ralstonia eutropha H16 is primarily known for polyhydroxybutyrate (PHB) production and its ability to grow chemolithoautotrophically by using CO2 and H2 as sole carbon and energy sources. The majority of metabolic engineering and heterologous expression studies conducted so far rely on a small number of suitable expression systems. Particularly the plasmid based expression systems already developed for the use in R. eutropha H16 suffer from high segregational instability and plasmid loss after a short time of fermentation. In order to develop efficient and highly stable plasmid expression vectors for the use in R. eutropha H16, a new plasmid design was created including the RP4 partitioning system, as well as various promoters and origins of replication. The application of minireplicons derived from broad-host-range plasmids RSF1010, pBBR1, RP4 and pSa for the construction of expression vectors and the use of numerous, versatile promoters extend the range of feasible expression levels considerably. In particular, the use of promoters derived from the bacteriophage T5 was described for the first time in this work, characterizing the j5 promoter as the strongest promoter yet to be applied in R. eutropha H16. Moreover, the implementation of the RP4 partition sequence in plasmid design increased plasmid stability significantly and enables fermentations with marginal plasmid loss of recombinant R. eutropha H16 for at least 96h. The utility of the new vector family in R. eutropha H16 is demonstrated by providing expression data with different model proteins and consequently further raises the value of this organism as cell factory for biotechnological applications including protein and metabolite production.

Periplasmic α-carbonic anhydrase plays an essential role in Ralstonia eutropha CO2 metabolism

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