Protein allocation and utilization in the versatile chemolithoautotroph Cupriavidus necator.

Bacteria must balance the different needs for substrate assimilation, growth functions, and resilience in order to thrive in their environment. Of all cellular macromolecules, the bacterial proteome is by far the most important resource and its size is limited. Here, we investigated how the highly versatile 'knallgas' bacterium Cupriavidus necator reallocates protein resources when grown on different limiting substrates and with different growth rates. We determined protein quantity by mass spectrometry and estimated enzyme utilization by resource balance analysis modeling. We found that C. necator invests a large fraction of its proteome in functions that are hardly utilized. Of the enzymes that are utilized, many are present in excess abundance. One prominent example is the strong expression of CBB cycle genes such as Rubisco during growth on fructose. Modeling and mutant competition experiments suggest that CO2-reassimilation through Rubisco does not provide a fitness benefit for heterotrophic growth, but is rather an investment in readiness for autotrophy.

Using singular perturbation theory to determine kinetic parameters in a non-standard coupled enzyme assay.

We investigate how to characterize the kinetic parameters of an aminotransaminase using a non-standard coupled (or auxiliary) enzyme assay, where the peculiarity arises for two reasons. First, one of the products of the auxiliary enzyme is a substrate for the primary enzyme and, second, we explicitly account for the reversibility of the auxiliary enzyme reaction. Using singular perturbation theory, we characterize the two distinguished asymptotic limits in terms of the strength of the reverse reaction, which allows us to determine how to deduce the kinetic parameters of the primary enzyme for a characterized auxiliary enzyme. This establishes a parameter-estimation algorithm that is applicable more generally to similar reaction networks. We demonstrate the applicability of our theory by performing enzyme assays to characterize a novel putative aminotransaminase enzyme, CnAptA (UniProtKB Q0KEZ8) from Cupriavidus necator H16, for two different omega-amino acid substrates.

Biotin Synthesis in Ralstonia eutropha H16 Utilizes Pimeloyl Coenzyme A and Can Be Regulated by the Amount of Acceptor Protein.

The biotin metabolism of the Gram-negative facultative chemolithoautotrophic bacterium Ralstonia eutropha (syn. Cupriavidus necator), which is used for biopolymer production in industry, was investigated. A biotin auxotroph mutant lacking bioF was generated, and biotin depletion in the cells and the minimal biotin demand of a biotin-auxotrophic R. eutropha strain were determined. Three consecutive cultivations in biotin-free medium were necessary to prevent growth of the auxotrophic mutant, and 40 ng/ml biotin was sufficient to promote cell growth. Nevertheless, 200 ng/ml biotin was necessary to ensure growth comparable to that of the wild type, which is similar to the demand of biotin-auxotrophic mutants among other prokaryotic and eukaryotic microbes. A phenotypic complementation of the R. eutropha ΔbioF mutant was only achieved by homologous expression of bioF of R. eutropha or heterologous expression of bioF of Bacillus subtilis but not by bioF of Escherichia coli Together with the results from bioinformatic analysis of BioFs, this leads to the assumption that the intermediate of biotin synthesis in R. eutropha is pimeloyl-CoA instead of pimeloyl-acyl carrier protein (ACP) like in the Gram-positive B. subtilis Internal biotin content was enhanced by homologous expression of accB, whereas homologous expression of accB and accC2 in combination led to decreased biotin concentrations in the cells. Although a DNA-binding domain of the regulator protein BirA is missing, biotin synthesis seemed to be influenced by the amount of acceptor protein present.IMPORTANCERalstonia eutropha is applied in industry for the production of biopolymers and serves as a research platform for the production of diverse fine chemicals. Due to its ability to grow on hydrogen and carbon dioxide as the sole carbon and energy source, R. eutropha is often utilized for metabolic engineering to convert inexpensive resources into value-added products. The understanding of the metabolic pathways in this bacterium is mandatory for further bioengineering of the strain and for the development of new strategies for biotechnological production.

A universal polyphosphate kinase: PPK2c of Ralstonia eutropha accepts purine and pyrimidine nucleotides including uridine diphosphate.

Polyphosphosphate kinases (PPKs) catalyse the reversible transfer of the γ-phosphate group of a nucleoside-triphosphate to a growing chain of polyphosphate. Most known PPKs are specific for ATP, but some can also use GTP as a phosphate donor. In this study, we describe the properties of a PPK2-type PPK of the ß-proteobacterium Ralstonia eutropha. The purified enzyme (PPK2c) is highly unspecific and accepts purine nucleotides as well as the pyridine nucleotides including UTP as substrates. The presence of a polyP primer is not necessary for activity. The corresponding nucleoside diphosphates and microscopically detectable polyphosphate granules were identified as reaction products. PPK2c also catalyses the formation of ATP, GTP, CTP, dTTP and UTP from the corresponding nucleoside diphosphates, if polyP is present as a phosphate donor. Remarkably, the nucleoside-tetraphosphates AT(4)P, GT(4)P, CT(4)P, dTT(4)P and UT(4)P were also detected in substantial amounts. The low nucleotide specificity of PPK2c predestines this enzyme in combination with polyP to become a powerful tool for the regeneration of ATP and other nucleotides in biotechnological applications. As an example, PPK2c and polyP were used to replace ATP and to fuel the hexokinase-catalysed phosphorylation of glucose with only catalytic amounts of ADP. KEY POINTS: • PPK2c of R. eutropha can be used for regeneration of any NTP or dNTP. • PPK2c is highly unspecific and accepts all purine and pyrimidine nucleotides. • PPK2c forms polyphosphate granules in vitro from any NTP.

Characterization of highly active 2-keto-3-deoxy-L-arabinonate and 2-keto-3-deoxy-D-xylonate dehydratases in terms of the biotransformation of hemicellulose sugars to chemicals.

2-keto-3-L-arabinonate dehydratase (L-KdpD) and 2-keto-3-D-xylonate dehydratase (D-KdpD) are the third enzymes in the Weimberg pathway catalyzing the dehydration of respective 2-keto-3-deoxy sugar acids (KDP) to α-ketoglutaric semialdehyde (KGSA). The Weimberg pathway has been explored recently with respect to the synthesis of chemicals from L-arabinose and D-xylose. However, only limited work has been done toward characterizing these two enzymes. In this work, several new L-KdpDs and D-KdpDs were cloned and heterologously expressed in Escherichia coli. Following kinetic characterizations and kinetic stability studies, the L-KdpD from Cupriavidus necator (CnL-KdpD) and D-KdpD from Pseudomonas putida (PpD-KdpD) appeared to be the most promising variants from each enzyme class. Magnesium had no effect on CnL-KdpD, whereas increased activity and stability were observed for PpD-KdpD in the presence of Mg2+. Furthermore, CnL-KdpD was not inhibited in the presence of L-arabinose and L-arabinonate, whereas PpD-KdpD was inhibited with D-xylonate (I50 of 75 mM), but not with D-xylose. Both enzymes were shown to be highly active in the one-step conversions of L-KDP and D-KDP. CnL-KdpD converted > 95% of 500 mM L-KDP to KGSA in the first 2 h while PpD-KdpD converted > 90% of 500 mM D-KDP after 4 h. Both enzymes in combination were able to convert 83% of a racemic mixture of D,L-KDP (500 mM) after 4 h, with both enzymes being specific toward the respective stereoisomer. Key points • L-KdpDs and D-KdpDs are specific toward L- and D-KDP, respectively. • Mg2+affected activity and stabilities of D-KdpDs, but not of L-KdpDs. • CnL-KdpD and PpD-KdpD converted 0.5 M of each KDP isomer reaching 95 and 90% yield. • Both enzymes in combination converted 0.5 M racemic D,L-KDP reaching 83% yield.

Crystallographic and kinetic analyses of the FdsBG subcomplex of the cytosolic formate dehydrogenase FdsABG from Cupriavidus necator.

Formate oxidation to carbon dioxide is a key reaction in one-carbon compound metabolism, and its reverse reaction represents the first step in carbon assimilation in the acetogenic and methanogenic branches of many anaerobic organisms. The molybdenum-containing dehydrogenase FdsABG is a soluble NAD+-dependent formate dehydrogenase and a member of the NADH dehydrogenase superfamily. Here, we present the first structure of the FdsBG subcomplex of the cytosolic FdsABG formate dehydrogenase from the hydrogen-oxidizing bacterium Cupriavidus necator H16 both with and without bound NADH. The structures revealed that the two iron-sulfur clusters, Fe4S4 in FdsB and Fe2S2 in FdsG, are closer to the FMN than they are in other NADH dehydrogenases. Rapid kinetic studies and EPR measurements of rapid freeze-quenched samples of the NADH reduction of FdsBG identified a neutral flavin semiquinone, FMNH•, not previously observed to participate in NADH-mediated reduction of the FdsABG holoenzyme. We found that this semiquinone forms through the transfer of one electron from the fully reduced FMNH-, initially formed via NADH-mediated reduction, to the Fe2S2 cluster. This Fe2S2 cluster is not part of the on-path chain of iron-sulfur clusters connecting the FMN of FdsB with the active-site molybdenum center of FdsA. According to the NADH-bound structure, the nicotinamide ring stacks onto the re-face of the FMN. However, NADH binding significantly reduced the electron density for the isoalloxazine ring of FMN and induced a conformational change in residues of the FMN-binding pocket that display peptide-bond flipping upon NAD+ binding in proper NADH dehydrogenases.

Application of organic waste glycerol to produce crude extracts of bacterial cells and microbial hydrogenase-the anode enzymes of bio-electrochemical systems.

Glycerol is an organic waste material that can be used for the production of microbial biomass, consequently providing valuable biocatalysts promoting the generation of electrical current in microbial fuel cells (MFCs). [NiFe]-Hydrogenases (Hyds) of Escherichia coli and Ralstonia eutropha may be applied as potential anode biocatalysts in MFCs. In this study, E. coli K12 whole cells or crude extracts and R. eutropha HF649 synthesizing Strep-tagged membrane-bound Hyds (MBH) were evaluated as anode enzymes in a bioelectrochemical system. The samples were immobilized on the sensors with polyvinyl acetate support. Mediators like ferrocene and its derivatives (ferrocene-carboxy-aldehyde, ferrocene-carboxylic acid, methyl-ferrocene-methanol) were employed. The maximal level of bioelectrocatalytic activity of Hyds was demonstrated at 500 mV voltage. Depending on the mediator and biocatalyst, current strength varied from 5 to 42 µA. Introduction of ferrocene-carboxylic acid enhanced current strength; moreover, the current flow was directly correlated with H2 concentration. The maximal value (up to 150 µA) of current strength was achieved with a 2-fold hydrogen supply. It may be inferred that Hyds are efficiently produced by E. coli and R. eutropha grown on glycerol, while ferrocene derivatives act as agents mediating the electrochemical activity of Hyds.

Mechanism and Application of the Catalytic Reaction of [NiFe] Hydrogenase: Recent Developments.

Hydrogenases (H2 ase) catalyze the oxidation of dihydrogen and the reduction of protons with remarkable efficiency, thereby attracting considerable attention in the energy field due to their biotechnological potential. For this simple reaction, [NiFe] H2 ase has developed a sophisticated but intricate mechanism with the heterolytic cleavage of dihydrogen, where its Ni-Fe active site exhibits various redox states. Recently, new spectroscopic and crystal structure studies of [NiFe] H2 ases have been reported, providing significant insights into the catalytic reaction mechanism, hydrophobic gas-access tunnel, proton-transfer pathway, and electron-transfer pathway of [NiFe] H2 ases. In addition, [NiFe] H2 ases have been shown to play an important role in biofuel cell and solar dihydrogen production. This concept provides an overview of the biocatalytic reaction mechanism and biochemical application of [NiFe] H2 ases based on the new findings.

Biosynthesis of polyhydroxyalkanoates from sucrose by metabolically engineered Escherichia coli strains.

Sucrose utilization has been established in Escherichia coli strains by expression of Mannheimia succiniciproducens ß-fructofuranosidase (SacC), which hydrolyzes sucrose into glucose and fructose. Recombinant E. coli strains that can utilize sucrose were examined for their abilities to produce poly(3-hydroxybutyrate) [P(3HB)] and poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)] from sucrose. When recombinant E. coli strains expressing Ralstonia eutropha PhaCAB and SacC were cultured in MR medium containing 20 g/L of sucrose, all recombinant E. coli strains could produce P(3HB) from sucrose. Also, recombinant E. coli strains expressing Pseudomonas sp. MBEL 6-19 PhaC1437, Clostridium propionicum Pct540, R. eutropha PhaAB enzymes along with SacC could produce P(3HB-co-LA) from sucrose. Among the examined E. coli strains, recombinant E. coli XL1-Blue produced the highest contents of P(3HB) (53.60 ± 2.55 wt%) and P(3HB-co-LA) (29.44 ± 0.39 wt%). In the batch fermentations, recombinant E. coli XL1-Blue strains completely consumed 20 g/L of sucrose as the sole carbon source and supported the production of 3.76 g/L of P(3HB) and 1.82 g/L of P(3HB-co-LA) with 38.21 wt% P(3HB) and 20.88 wt% P(3HB-co-LA) contents, respectively. Recombinant E. coli strains developed in this study can be used to establish a cost-efficient biorefinery for the production of polyhydroxyalkanoates (PHAs) from sucrose, which is an abundant and inexpensive carbon source.

Enhanced tolerance to inhibitors of Escherichia coli by heterologous expression of cyclopropane-fatty acid-acyl-phospholipid synthase (cfa) from Halomonas socia.

Bacteria have evolved a defense system to resist external stressors, such as heat, pH, and salt, so as to facilitate survival in changing or harsh environments. However, the specific mechanisms by which bacteria respond to such environmental changes are not completely elucidated. Here, we used halotolerant bacteria as a model to understand the mechanism conferring high tolerance to NaCl. We screened for genes related to halotolerance in Halomonas socia, which can provide guidance for practical application. Phospholipid fatty acid analysis showed that H. socia cultured under high osmotic pressure produced a high portion of cyclopropane fatty acid derivatives, encoded by the cyclopropane-fatty acid-acyl phospholipid synthase gene (cfa). Therefore, H. socia cfa was cloned and introduced into Escherichia coli for expression. The cfa-overexpressing E. coli strain showed better growth, compared with the control strain under normal cultivation condition as well as under osmotic pressure (> 3% salinity). Moreover, the cfa-overexpressing E. coli strain showed 1.58-, 1.78-, 3.3-, and 2.19-fold higher growth than the control strain in the presence of the inhibitors furfural, 4-hydroxybenzaldehyde, vanillin, and acetate from lignocellulosic biomass pretreatment, respectively. From a practical application perspective, cfa was co-expressed in E. coli with the polyhydroxyalkanoate (PHA) synthetic operon of Ralstonia eutropha using synthetic and biosugar media, resulting in a 1.5-fold higher in PHA production than that of the control strain. Overall, this study demonstrates the potential of the cfa gene to boost cell growth and production even in heterologous strains under stress conditions.

Kinetics aspects of Gamma-hydroxybutyrate dehydrogenase.

Two groups of metabolically related enzymes, the Group III family of Fe2+-dependent alcohol dehydrogenases (ADHs) and the separate subfamily of nucleoside diphosphates linked to x (nudix) hydrolases that activate Group III ADHs are under-characterized. Here we report the steady-state initial-velocity forward direction (alcohol → aldehyde) reaction of a Group III ADH, namely gamma-hydroxybutyrate dehydrogenase (GHBDH, UniProt: Q59104), cloned from Cupriavidus necator as a fusion protein. We also report the effects of nudix hydrolases on the GHBDH reaction. At optimal pH 9.0, the GHBDH reaction is activated ~2-fold by two different saturating purified nudix hydrolases, namely Bacillus methanolicus activator (ACT, UniProt: I3EA59) and Escherichia coli NudF (UniProt Q93K97) proteins. At physiological pH values of ~7.0, ACT activates by >3.5-fold. Initial-rate characterization at pH 9.0 of the forward direction un-activated and ACT-activated reactions show for both cases competitive inhibition by the product succinic semialdehyde versus GHB, and noncompetitive inhibitions by the three other substrate-product combinations. This pattern is consistent with NAD+ binding first in Mono-Iso Theorell-Chance kinetics. Mutants of some possibly important residues in GHBDH also were characterized. H265, conserved among all Group III ADHs and previously proposed to be a critical general base, is only ~4-fold helpful for GHBDH activity relevant to H265A. The four previously proposed conserved Fe2+ chelators (D193, H197, H261 and H280) each are essential for GHBDH activity. A 2-step explanation for cross-species stimulation by sub-stoichiometric ACT in the forward direction and confirmed lack of ACT stimulation in the reverse direction reaction is proposed.

Biocatalytic hydrogenations on carbon supports.

We describe the use of carbon as a versatile support for H2-driven redox biocatalysis for NADH-dependent CX bond reductions in batch and flow reactions. In each case, carbon is providing an electronic link between enzymes for H2 oxidation and reduction of the biological cofactor NAD+, as well as a support for a multi-enzyme biocatalysis system. Carbon nanopowders offer high surface areas for enzyme immobilization and good dispersion in aqueous solution for heterogeneous batch reactions. Difficulties in handling multi-wall carbon nanotubes in aqueous solution are overcome by growing them on quartz tubes to form carbon nanotube column reactors, and we show that these facilitate simple translation of H2-driven biocatalysis into flow processes. Using this flow reactor design, high conversions (90%) and total enzyme turnover numbers up to 54,000 could be achieved. Use of an entirely heterogeneous biocatalysis system simplifies recovery and re-use of the enzymes; combined with highly atom-efficient cofactor recycling, this means that high product purity can be achieved. We demonstrate these methods as platform approaches for overcoming challenges with NADH-dependent biocatalysis.

Characterization of an l-Ascorbate Catabolic Pathway with Unprecedented Enzymatic Transformations.

l-Ascorbate (vitamin C) is ubiquitous in both our diet and the environment. Here we report that Ralstonia eutropha H16 (Cupriavidus necator ATCC 17699) uses l-ascorbate as sole carbon source via a novel catabolic pathway. RNaseq identified eight candidate catabolic genes, sequence similarity networks, and genome neighborhood networks guided predictions for function of the encoded proteins, and the predictions were confirmed by in vitro assays and in vivo growth phenotypes of gene deletion mutants. l-Ascorbate, a lactone, is oxidized and ring-opened by enzymes in the cytochrome b561 and gluconolactonase families, respectively, to form 2,3-diketo-l-gulonate. A protein predicted to have a WD40-like fold catalyzes an unprecedented benzilic acid rearrangement involving migration of a carboxylate group to form 2-carboxy-l-lyxonolactone; the lactone is hydrolyzed by a member of the amidohydrolase superfamily to yield 2-carboxy-l-lyxonate. A member of the PdxA family of oxidative decarboxylases catalyzes a novel decarboxylation that uses NAD+ catalytically. The product, l-lyxonate, is catabolized to α-ketoglutarate by a previously characterized pathway. The pathway is found in hundreds of bacteria, including the pathogens Pseudomonas aeruginosa and Acinetobacter baumannii.

Expanded amino acid sequence of the PhaC box in the active center of polyhydroxyalkanoate synthases.

Polyhydroxyalkanoate (PHA) synthases catalyze the polymerization reaction of the acyl moiety of hydroxyacyl-coenzyme A into polyester. The catalytic subunit PhaC of PHA synthase has the PhaC box sequence at the active site that is typically described as G-X-C-X-G-G (X is an arbitrary amino acid), and cysteine is an active center. In this study, an amino acid replacement was introduced into the PhaC box of the PHA synthase derived from Ralstonia eutropha (PhaCRe ) to investigate the importance of highly conserved residues in polymerizing activity. Point mutagenesis revealed that PhaCRe mutants with the expanded PhaC box sequence ([GAST]-X-C-X-[GASV]-[GA]) are functional PHA synthases. These findings highlight the low mutational robustness of the last glycine residue in the PhaC box as well as that of the active center cysteine.

Growth of the facultative chemolithoautotroph Ralstonia eutropha on organic waste materials: growth characteristics, redox regulation and hydrogenase activity.

BACKGROUND: The chemolithoautotrophic ß-proteobacterium Ralstonia eutropha H16 (Cupriavidus necator) is one of the most studied model organisms for growth on H2 and CO2. R. eutropha H16 is also a biologically significant bacterium capable of synthesizing O2-tolerant [NiFe]-hydrogenases (Hyds), which can be used as anode biocatalysts in enzyme fuel cells. For heterotrophic growth of R. eutropha, various sources of organic carbon and energy can be used. RESULTS: Growth, bioenergetic properties, and oxidation-reduction potential (ORP) kinetics were investigated during cultivation of R. eutropha H16 on fructose and glycerol or lignocellulose-containing brewery spent grain hydrolysate (BSGH). BSGH was used as carbon and energy source by R. eutropha H16, and the activities of the membrane-bound hydrogenase (MBH) and cytoplasmic, soluble hydrogenase (SH) were measured in different growth phases. Growth of R. eutropha H16 on optimized BSGH medium yielded ~ 0.7 g cell dry weight L-1 with 3.50 ± 0.02 (SH) and 2.3 ± 0.03 (MBH) U (mg protein)-1 activities. Upon growth on fructose and glycerol, a pH drop from 7.0 to 6.7 and a concomitant decrease of ORP was observed. During growth on BSGH, in contrast, the pH and ORP stayed constant. The growth rate was slightly stimulated through addition of 1 mM K3[Fe(CN)6], whereas temporarily reduced growth was observed upon addition of 3 mM dithiothreitol. The overall and N,N'-dicyclohexylcarbodiimide-sensitive ATPase activities of membrane vesicles were ~ 4- and ~ 2.5-fold lower, respectively, upon growth on fructose and glycerol (FGN) compared with only fructose utilization (FN). Compared to FN, ORP was lower upon bacterial growth on FGN, GFN, and BSGH. CONCLUSIONS: Our results suggest that reductive conditions and low ATPase activity might be signals for energy depletion, which, in turn, leads to increased hydrogenase biosynthesis to overcome this unfavorable situation. Addition of fructose or microelements have no, or a negative, influence on hydrogenase activity. Organic wastes (glycerol, BSGH) are promising carbon and energy sources for the formation of biomass harboring significant amounts of the biotechnologically relevant hydrogenases MBH and SH. The results are valuable for using microbial cells as producers of hydrogenase enzymes as catalysts in enzymatic fuel cells.

In vivo immobilization of an organophosphorus hydrolyzing enzyme on bacterial polyhydroxyalkanoate nano-granules.

BACKGROUND: Polyhydroxyalkanoate (PHA) are nano-granules naturally produced by bacteria. Two types of proteins, PHA synthase (PhaC) and phasins (PhaPs), are attached to the PHA surface by covalent and hydrophobic interactions. Utilizing these anchored proteins, functionalized PHA nano-granules displaying proteins of interest can be easily prepared by fermentation. RESULTS: In this study, a one-step fabrication method was developed for stable and efficient immobilization of an organophosphorus degrading enzyme on PHA nano-granules. The nano-biocatalysts were produced in recombinant Escherichia coli cells into which the polyhydroxyalkanoate synthesis pathway from Cupriavidus necator had been introduced. Two different strategies, covalent attachment and hydrophobic binding, were investigated by fusing bacterial organophosphorus anhydride hydrolase (OPAA4301) with PhaC and PhaP, respectively. Using both methods, the tetrameric enzyme successfully self-assembled and was displayed on the PHA surface. The display density of the target fused enzyme was enhanced to 6.8% of total protein on decorated PHA by combination of covalent and non-covalent binding modes. Immobilization of the enzyme on PHA granules resulted in higher catalytic efficiency, increased stability and excellent reusability. The kcat values of the immobilized enzymes increased by threefold compared to that of the free enzyme. The pH stability under acidic conditions was significantly enhanced, and the immobilized enzyme was stable at pH 3.0-11.0. Furthermore, more than 80% of the initial enzyme activity retained after recycling ten times. CONCLUSIONS: This study provides a promising approach for cost-efficient in vivo immobilization of a tetrameric organophosphorus degrading enzyme. The immobilization process expands the utility of the enzyme, and may inspire further commercial developments of PHA nano-biocatalysts. As revealed by our results, combination of covalent and non-covalent binding is recommended for display of enzymes on PHA granules.

Complementing urea hydrolysis and nitrate reduction for improved microbially induced calcium carbonate precipitation.

Microbial-induced CaCO3 precipitation has been widely applied in bacterial-based self-healing concrete. However, the limited biogenetic CaCO3 production by bacteria after they were introduced into the incompatible concrete matrix is a major challenge of this technology. In the present study, the potential of combining two metabolic pathways, urea hydrolysis and nitrate reduction, simultaneously in one bacteria strain for improving the bacterial CaCO3 yield has been investigated. One bacterial strain, Ralstonia eutropha H16, which has the highest Ca2+ tolerance and is capable of performing both urea hydrolysis and nitrate reduction in combined media was selected among three bacterial candidates based on the enzymatic examinations. Results showed that H16 does not need oxygen for urea hydrolysis and urease activity was determined primarily by cell concentration. However, the additional urea in the combined medium slowed down the nitrate reduction rate to 7 days until full NO3- decomposition. Moreover, the nitrate reduction of H16 was significantly restricted by an increased Ca2+ ion concentration in the media. Nevertheless, the overall CaCO3 precipitation yield can be improved by 20 to 30% after optimization through the combination of two metabolic pathways. The highest total CaCO3 precipitation yield achieved in an orthogonal experiment was 14 g/L. It can be concluded that Ralstonia eutropha H16 is a suitable bacterium for simultaneous activation of urea hydrolysis and nitrate reduction for improving the CaCO3 precipitation and it can be studied later, on activation of multiple metabolic pathways in bacteria-based self-healing concrete.

Deconvolution of reduction potentials of formate dehydrogenase from Cupriavidus necator.

The formate dehydrogenase enzyme from Cupriavidus necator (FdsABG) carries out the two-electron oxidation of formate to CO2, but is also capable of reducing CO2 back to formate, a potential biofuel. FdsABG is a heterotrimeric enzyme that performs this transformation using nine redox-active cofactors: a bis(molybdopterin guanine dinucleotide) (bis-MGD) at the active site coupled to seven iron-sulfur clusters, and one equivalent of flavin mononucleotide (FMN). To better understand the pathway of electron flow in FdsABG, the reduction potentials of the various cofactors were examined through direct electrochemistry. Given the redundancy of cofactors, a truncated form of the FdsA subunit was developed that possesses only the bis-MGD active site and a singular [4Fe-4S] cluster. Electrochemical characterization of FdsABG compared to truncated FdsA shows that the measured reduction potentials are remarkably similar despite the truncation with two observable features at - 265 mV and - 455 mV vs SHE, indicating that the voltammetry of the truncated enzyme is representative of the reduction potentials of the intact heterotrimer. By producing truncated FdsA without the necessary maturation factors required for bis-MGD insertion, a form of the truncated FdsA that possesses only the [4Fe-4S] was produced, which gives a single voltammetric feature at - 525 mV, allowing the contributions of the molybdenum cofactor to be associated with the observed feature at - 265 mV. This method allowed for the deconvolution of reduction potentials for an enzyme with highly complex cofactor content to know more about the thermodynamic landscape of catalysis.

In vivo and in vitro characterization of hydrophilic protein tag-fused Ralstonia eutropha polyhydroxyalkanoate synthase.

Polyhydroxyalkanoates (PHAs) are synthesized by bacteria as an intracellular storage polyester, where PHA synthase (PhaC) catalyzes the polymerization of its substrate hydroxyacyl-coenzyme A (HA-CoA) to form PHA. When PhaC is overexpressed in Escherichia coli, most PhaC protein is produced as insoluble inclusion bodies due to its low aqueous solubility. This study aimed to improve the solubility of Ralstonia eutropha PHA synthase (PhaCRe) by fusing a hydrophilic tag, glutathione S-transferase (GST), to the protein's N-terminus. In in vivo assays, the GST tag had no obvious effect on solubility and enzymatic activity of PhaCRe. However, an in vitro assay revealed that the surface of GST-fused PhaCRe (GST-PhaCRe) had increased hydrophilicity, and tended to form correct PhaCRe dimers when added to the (R)-3-hydroxybutyryl-CoA substrate. Although GST-PhaCRe displayed a long lag phase at the start of a polymerization reaction, granule-associated GST-PhaCRe showed higher catalytic activity than PhaCRe in kinetic analysis. The results are discussed in light of the dimerization mechanisms of PhaCRe.

2-Ketogluconate Kinase from Cupriavidus necator H16: Purification, Characterization, and Exploration of Its Substrate Specificity.

We have cloned, overexpressed, purified, and characterized a 2-ketogluconate kinase (2-dehydrogluconokinase, EC 2.7.1.13) from Cupriavidus necator (Ralstonia eutropha) H16. Exploration of its substrate specificity revealed that three ketoacids (2-keto-3-deoxy-d-gluconate, 2-keto-d-gulonate, and 2-keto-3-deoxy-d-gulonate) with structures close to the natural substrate (2-keto-d-gluconate) were successfully phosphorylated at an efficiency lower than or comparable to 2-ketogluconate, as depicted by the measured kinetic constant values. Eleven aldo and keto monosaccharides of different chain lengths and stereochemistries were also assayed but not found to be substrates. 2-ketogluconate-6-phosphate was synthesized at a preparative scale and was fully characterized for the first time.