Formate Dehydrogenase Activity Research Articles

Harnessing the potential of deep eutectic solvents in biocatalysis: design strategies using CO2 to formate reduction as a case study.

Deep eutectic solvents (DESs) have emerged as green solvents with versatile applications, demonstrating significant potential in biocatalysis. They often increase the solubility of poorly water-soluble substrates, serve as smart co-substrates, modulate enzyme stereoselectivity, and potentially improve enzyme activity and stability. Despite these advantages, screening for an optimal DES and determining the appropriate water content for a given biocatalytic reaction remains a complex and time-consuming process, posing a significant challenge. This paper discusses the rational design of DES tailored to a given biocatalytic system through a combination of experimental screening and computational tools, guided by performance targets defined by solvent properties and process constraints. The efficacy of this approach is demonstrated by the reduction of CO2 to formate catalyzed by NADH-dependent formate dehydrogenase (FDH). By systematically analyzing FDH activity and stability, NADH stability (both long-term and short-term stability after solvent saturation with CO2), and CO2 solubility in initially selected glycerol-based DESs, we were able to skillfully guide the DES screening process. Considering trade-offs between experimentally determined performance metrics of DESs, 20% solution of choline chloride:glycerol in phosphate buffer (ChCl:Gly80%B) was identified as the most promising solvent system for a given reaction. Using ChCl:Gly as a co-solvent resulted in an almost 15-fold increase in FDH half-life compared to the reference buffer and stabilized the coenzyme after the addition of CO2. Moreover, the 20% addition of ChCl:Gly to the buffer improved the volumetric productivity of FDH-catalyzed CO2 reduction in a batch system compared to the reference buffer. The exceptional stability of the enzyme in this co-solvent system shows great potential for application in continuous operation, which can significantly improve process productivity. Additionally, based on easily measurable physicochemical solvent properties and molecular descriptors derived from COSMO-RS, QSAR models were developed, which successfully predicted enzyme activity and stability, as well as coenzyme stability in selected solvent systems with DESs.

A growth-based screening strategy for engineering the catalytic activity of an oxygen-sensitive formate dehydrogenase.

Enzyme engineering is a powerful tool for improving or altering the properties of biocatalysts for industrial, research, and therapeutic applications. Fast and accurate screening of variant libraries is often the bottleneck of enzyme engineering and may be overcome by growth-based screening strategies with simple processes to enable high throughput. The currently available growth-based screening strategies have been widely employed for enzymes but not yet for catalytically potent and oxygen-sensitive metalloenzymes. Here, we present a screening system that couples the activity of an oxygen-sensitive formate dehydrogenase to the growth of Escherichia coli. This system relies on the complementation of the E. coli formate hydrogenlyase (FHL) complex by Mo-dependent formate dehydrogenase H (EcFDH-H). Using an EcFDH-H-deficient strain, we demonstrate that growth inhibition by acidic glucose fermentation products can be alleviated by FHL complementation. This allows the identification of catalytically active EcFDH-H variants at a readily measurable cell density readout, reduced handling efforts, and a low risk of oxygen contamination. Furthermore, a good correlation between cell density and formate oxidation activity was established using EcFDH-H variants with variable catalytic activities. As proof of concept, the growth assay was employed to screen a library of 1,032 EcFDH-H variants and reduced the library size to 96 clones. During the subsequent colorimetric screening of these clones, the variant A12G exhibiting an 82.4% enhanced formate oxidation rate was identified. Since many metal-dependent formate dehydrogenases and hydrogenases form functional complexes resembling E. coli FHL, the demonstrated growth-based screening strategy may be adapted to components of such electron-transferring complexes.IMPORTANCEOxygen-sensitive metalloenzymes are highly potent catalysts that allow the reduction of chemically inert substrates such as CO2 and N2 at ambient pressure and temperature and have, therefore, been considered for the sustainable production of biofuels and commodity chemicals such as ammonia, formic acid, and glycine. A proven method to optimize natural enzymes for such applications is enzyme engineering using high-throughput variant library screening. However, most screening methods are incompatible with the oxygen sensitivity of these metalloenzymes and thereby limit their relevance for the development of biosynthetic production processes. A microtiter plate-based assay was developed for the screening of metal-dependent formate dehydrogenase that links the activity of the tested enzyme variant to the growth of the anaerobically grown host cell. The presented work extends the application range of growth-based screening to metalloenzymes and is thereby expected to advance their adoption to biosynthesis applications.

Titanium carbide-based nanocomposite: A promising reinforcing material for enzymatic CO2 conversion

Enzymatic conversion of CO2 to formate is a highly significant pathway for CO2 utilization, but current level of conversion efficiency remains unsatisfactory due to low solubility of CO2 under mild conditions. CO2-philic materials can significantly augment the reaction by raising local concentration of substrate. Herein, the intercalated Ti3C2Tx, nano-silica (SiO2) and carbon nitride (CN) with abundant CO2 adsorption sites were introduced to fabricate Ti3C2Tx based nanocomposites, Ti3C2Tx/SiO2/CN (TSCN), for the first time. As the controls, binary composites including Ti3C2Tx/SiO2 (TS), Ti3C2Tx/CN (TCN) and SiO2/CN (SCN) were also studied. For each kind of material, the composition, structure, morphology, and application performance in the enzymatic CO2 conversion were characterized and influencing factors were investigated. Among these composites, TSCN exhibited the best application performance and the enzyme reaction was accelerated up to 23.9 times that of the blank system. The value could reach as high as 35.3 after polyethyleneimine (PEI) modification. Following that, PEI modified composites were used as carrier of formate dehydrogenase (FDH) and apparent specific activity was up to 2.8 times that of free enzyme, which could retain 88.2% of the initial value after 10 cycles associated with the structural integrity. This work provides new impetus to CO2 capture and conversion in a clean and mild way that shows bright application prospects.

Formate dehydrogenase activity by a Cu(II)-based molecular catalyst and deciphering the mechanism using DFT studies.

Due to the requirement to establish renewable energy sources, formic acid (FA), one of the most probable liquid organic hydrogen carriers (LOHCs), has received great attention. Catalytic formic acid dehydrogenation in an effective and environmentally friendly manner is still a challenge. The N3Q3 ligand (N3Q3 = N,N-bis(quinolin-8-ylmethyl)quinolin-8-amine) and the square pyramidal [Cu(N3Q3)Cl]Cl complex have been synthesised in this work and characterised using several techniques, such as NMR spectroscopy, mass spectrometry, EPR spectroscopy, cyclic voltammetry, X-ray diffraction and DFT calculations. This work investigates the dehydrogenation of formic acid using a molecular and homogeneous catalyst [Cu(N3Q3)Cl]Cl in the presence of HCOONa. The mononuclear copper complex exhibits catalytic activity towards the dehydrogenation of formic acid in H2O with the evolution of a 1 : 1 CO2 and H2 mixture. The activation energy of formic acid dehydrogenation was calculated to be Ea = 86 kJ mol-1, based on experiments carried out at various temperatures. The Gibbs free energy was found to be 82 kJ at 298 K for the decomposition of HCOOH. The DFT studies reveal that [Cu(N3Q3)(HCOO-)]+ undergoes an uphill process of rearrangement followed by decarboxylation to generate [Cu(N3Q3)(H-)]+. The initial uphill step for forming a transition state is the rate-determining step. The [Cu(N3Q3)(H-)]+ follows an activated state in the presence of HCOOH to liberate H2 and generate the [Cu(N3Q3)(OH2)]2+.

Identification of novel small molecule inhibitors of twin arginine translocation (Tat) pathway and their effect on the control of Campylobacter jejuni in chickens.

Control of Campylobacter from farm to fork is challenging due to the frequent emergence of antimicrobial-resistant isolates. Furthermore, poultry production systems are known reservoirs of Campylobacter. The twin-arginine translocation (Tat) pathway is a crucial bacterial secretion system that allows Campylobacter to colonize the host intestinal tract by using formate as the main source of energy. However, Tat pathway is also a major contributing factor for resistance to copper sulfate (CuSO4). Since mammals and chickens do not have proteins or receptors that are homologous to bacterial Tat proteins, identification of small molecule (SM) inhibitors targeting the Tat system would allow the development of safe and effective control methods to mitigate Campylobacter in infected or colonized hosts in both pre-harvest and post-harvest. In this study, we screened 11 commercial libraries (n = 50,917 SM) for increased susceptibility to CuSO4 (1 mM) in C. jejuni 81-176, a human isolate which is widely studied. Furthermore, we evaluated 177 SM hits (2.5 μg/mL and above) that increased the susceptibility to CuSO4 for the inhibition of formate dehydrogenase (Fdh) activity, a Tat-dependent substrate. Eight Tat-dependent inhibitors (T1-T8) were selected for further studies. These selected eight Tat inhibitors cleared all tested Campylobacter strains (n = 12) at >10 ng/mL in the presence of 0.5 mM CuSO4in vitro. These selected SMs were non-toxic to colon epithelial (Caco-2) cells when treated with 50 μg/mL for 24 h and completely cleared intracellular C. jejuni cells when treated with 0.63 μg/mL of SM for 24 h in the presence of 0.5 mM of CuSO4. Furthermore, 3 and 5-week-old chicks treated with SM candidates for 5 days had significantly decreased cecal colonization (up to 1.2 log; p < 0.01) with minimal disruption of microbiota. In silico analyses predicted that T7 has better drug-like properties than T2 inhibitor and might target a key amino acid residue (glutamine 165), which is located in the hydrophobic core of TatC protein. Thus, we have identified novel SM inhibitors of the Tat pathway, which represent a potential strategy to control C. jejuni spread on farms.

Leveraging liquid-liquid phase separation and volume modulation to regulate the enzymatic activity of formate dehydrogenase

Engineering of reaction media is an exciting alternative for modulating kinetic properties of biocatalytic reactions. We addressed the combined effect of an aqueous two-phase system (ATPS) and high hydrostatic pressure on the kinetics of the Candida boidinii formate dehydrogenase-catalyzed oxidation of formate to CO2. Pressurization was found to lead to an increase of the binding affinity (decrease of KM, respectively) and a decrease of the turnover number, kcat. The experimental approach was supported using thermodynamic modeling with the electrolyte Perturbed-Chain Statistical Associating Fluid Theory (ePC-SAFT) equation of state to predict the liquid-liquid phase separation and the molecular crowding effect of the ATPS on the kinetic properties. The ePC-SAFT was able to quantitatively predict the KM-values of the substrate in both phases at 1 bar as well as up to a pressure of 1000 bar. The framework presented enables significant advances in bioprocess engineering, including the design of processes with significantly fewer experiments and trial-and-error approaches.

Effect of an anti-methanogenic supplement on enteric methane emission, fermentation, and whole rumen metagenome in sheep.

A study was conducted to investigate the impact of an anti-methanogenic product supplementation on enteric methane emissions, whole rumen metagenome and ruminal fermentation in sheep. Twelve adult male sheep were randomly divided into two groups of six animals each. Animals were fed ad libitum on a total mixed ration either without (CON) or with an anti-methanogenic supplement (Harit Dhara-HD). The anti-methanogenic supplement contained 22.1% tannic acid in a 3: 1 ratio of condensed and hydrolysable tannins. The supplementation of product revealed a significant reduction in daily enteric methane emission (21.9 vs. 17.2 g/d) and methane yield (23.2 vs. 18.2) without affecting the nutrient intake and digestibility. However, the propionate concentration in the HD treatment group was significantly higher than in the CON group. On the contrary, the ammonia nitrogen concentration was lower. The anti-methanogenic supplement significantly decreased the ruminal protozoa in the HD treatment group. Whole rumen metagenome analysis revealed that the core bacterial (Bacteroidetes and Firmicutes) and archaeal communities (Methanobrevibacter and Methanosarcina) were comparable between the CON and HD treatment groups. However, the supplementation of anti-methanogenic product led to a considerable reduction in the abundance of Proteobacteria, whereas the abundance of Lentisphaerae was greater. The supplementation significantly decreased the abundance of Methanocaldococcus, Methanococcoides, Methanocella, and Methanoregula methanogens. A total of 36 KO related to methanogenesis were identified in this study. The activities of formate dehydrogenase (EC 1.8.98.6) and tetrahydromethanopterin S-methyltransferase (EC 2.1.1.86) were significantly lowered by the anti-methanogenic product supplementation in sheep. In conclusion, the anti-methanogenic supplement has the potential to decrease enteric methane emission (~22%) at the recommended level (5% of DM) of supplementation. The contribution of minor methanogens vulnerable to supplementation to rumen methanogenesis is not known; hence, the culturing of these archaea should be taken on priority for determining the impact on overall rumen methanogenesis.

Influence of the Greater Protein Environment on the Electrostatic Potential in Metalloenzyme Active Sites: The Case of Formate Dehydrogenase.

The Mo/W-containing metalloenzyme formate dehydrogenase (FDH) is an efficient and selective natural catalyst that reversibly converts CO2 to formate under ambient conditions. In this study, we investigate the impact of the greater protein environment on the electrostatic potential (ESP) of the active site. To model the enzyme environment, we used a combination of classical molecular dynamics and multiscale quantum-mechanical (QM)/molecular-mechanical (MM) simulations. We leverage charge shift analysis to systematically construct QM regions and analyze the electronic environment of the active site by evaluating the degree of charge transfer between the core active site and the protein environment. The contribution of the terminal chalcogen ligand to the ESP of the metal center is substantial and dependent on the chalcogen identity, with similar, less negative ESPs for Se and S terminal chalcogens in comparison to O regardless of whether the metal is Mo or W. The orientation of the side chains and conformations of the cofactor also affect the ESP, highlighting the importance of sampling dynamic fluctuations in the protein. Overall, our observations suggest that the terminal chalcogen ligand identity plays an important role in the enzymatic activity of FDH, suggesting opportunities for a rational bioinspired catalyst design.

Use of Transposon Directed Insertion-Site Sequencing to Probe the Antibacterial Mechanism of a Model Honey on E. coli K-12.

Antimicrobial resistance is an ever-growing health concern worldwide that has created renewed interest in the use of traditional anti-microbial treatments, including honey. However, understanding the underlying mechanism of the anti-microbial action of honey has been hampered due to the complexity of its composition. High throughput genetic tools could assist in understanding this mechanism. In this study, the anti-bacterial mechanism of a model honey, made of sugars, hydrogen peroxide, and gluconic acid, was investigated using genome-wide transposon mutagenesis combined with high-throughput sequencing (TraDIS), with the strain Escherichia coli K-12 MG1655 as the target organism. We identified a number of genes which when mutated caused a severe loss of fitness when cells were exposed to the model honey. These genes encode membrane proteins including those involved in uptake of essential molecules, and components of the electron transport chain. They are enriched for pathways involved in intracellular homeostasis and redox activity. Genes involved in assembly and activity of formate dehydrogenase O (FDH-O) were of particular note. The phenotypes of mutants in a subset of the genes identified were confirmed by phenotypic screening of deletion strains. We also found some genes which when mutated led to enhanced resistance to treatment with the model honey. This study identifies potential synergies between the main honey stressors and provides insights into the global antibacterial mechanism of this natural product.

Efficient 2,3-Butanediol/Acetoin Production Using Whole-Cell Biocatalyst with a New Nadh/Nad(+) Regeneration System

An auto-inducing expression system was developed that could express target genes in S. marcescens MG1. Using this system, MG1 was constructed as a whole-cell biocatalyst to produce 2,3-butanediol/acetoin. Formate dehydrogenase (FDH) and 2,3-butanediol dehydrogenase were expressed together to build an NADH regeneration system to transform diacetyl to 2,3-butanediol. After fermentation, the extract of recombinant S. marcescens MG1ABC (pETDuet-bdhA-fdh) showed 2,3-BDH activity of 57.8 U/mg and FDH activity of 0.5 U/mg. And 27.95 g/L of 2,3-BD was achieved with a productivity of 4.66 g/Lh using engineered S. marcescens MG1(Pswnb+pETDuet-bdhA-fdh) after 6 h incubation. Next, to produce 2,3-butanediol from acetoin, NADH oxidase and 2,3-butanediol dehydrogenase from Bacillus subtilis were co-expressed to obtain a NAD+ regeneration system. After fermentation, the recombinant strain S. marcescens MG1ABC (pSWNB+pETDuet-bdhA-yodC) showed AR activity of 212.4 U/mg and NOX activity of 150.1 U/mg. We obtained 44.9 g/L of acetoin with a productivity of 3.74 g/Lh using S. marcescens MG1ABC (pSWNB+pETDuet-bdhA-yodC). This work confirmed that S. marcescens could be designed as a whole-cell biocatalyst for 2,3-butanediol and acetoin production.

Identification and characterization of a noncanonical menaquinone-linked formate dehydrogenase

The molybdenum/tungsten—bis-pyranopterin guanine dinucleotide family of formate dehydrogenases (FDHs) plays roles in several metabolic pathways ranging from carbon fixation to energy harvesting because of their reaction with a wide variety of redox partners. Indeed, this metabolic plasticity results from the diverse structures, cofactor content, and substrates used by partner subunits interacting with the catalytic hub. Here, we unveiled two noncanonical FDHs in Bacillus subtilis, which are organized into two-subunit complexes with unique features, ForCE1 and ForCE2. We show that the formate oxidoreductase catalytic subunit interacts with an unprecedented partner subunit, formate oxidoreductase essential subunit, and that its amino acid sequence within the active site deviates from the consensus residues typically associated with FDH activity, as a histidine residue is naturally substituted with a glutamine. The formate oxidoreductase essential subunit mediates the utilization of menaquinone as an electron acceptor as shown by the formate:menadione oxidoreductase activity of both enzymes, their copurification with menaquinone, and the distinctive detection of a protein-bound neutral menasemiquinone radical by multifrequency electron paramagnetic resonance (EPR) experiments on the purified enzymes. Moreover, EPR characterization of both FDHs reveals the presence of several [Fe-S] clusters with distinct relaxation properties and a weakly anisotropic Mo(V) EPR signature, consistent with the characteristic molybdenum/bis-pyranopterin guanine dinucleotide cofactor of this enzyme family. Altogether, this work enlarges our knowledge of the FDH family by identifying a noncanonical FDH, which differs in terms of architecture, amino acid conservation around the molybdenum cofactor, and reactivity.

Initial Step of Selenite Reduction via Thioredoxin for Bacterial Selenoprotein Biosynthesis.

Many organisms reductively assimilate selenite to synthesize selenoprotein. Although the thioredoxin system, consisting of thioredoxin 1 (TrxA) and thioredoxin reductase with NADPH, can reduce selenite and is considered to facilitate selenite assimilation, the detailed mechanism remains obscure. Here, we show that selenite was reduced by the thioredoxin system from Pseudomonas stutzeri only in the presence of the TrxA (PsTrxA), and this system was specific to selenite among the oxyanions examined. Mutational analysis revealed that Cys33 and Cys36 residues in PsTrxA are important for selenite reduction. Free thiol-labeling assays suggested that Cys33 is more reactive than Cys36. Mass spectrometry analysis suggested that PsTrxA reduces selenite via PsTrxA-SeO intermediate formation. Furthermore, an in vivo formate dehydrogenase activity assay in Escherichia coli with a gene disruption suggested that TrxA is important for selenoprotein biosynthesis. The introduction of PsTrxA complemented the effects of TrxA disruption in E. coli cells, only when PsTrxA contained Cys33 and Cys36. Based on these results, we proposed the early steps of the link between selenite and selenoprotein biosynthesis via the formation of TrxA–selenium complexes.

Development of a fermentation strategy to enhance the catalytic efficiency of recombinant Escherichia coli for l-2-aminobutyric acid production.

Microbial fermentation for enzyme production and then whole-cell catalysis for l-2-aminobutyric acid (l-ABA) production have huge potential for industrial application, but the catalytic capacities of cells are directly related to the fermentation process. Using a 50L fermenter, the effects of initial glycerol concentration in the medium and rotating speed on cell catalytic capacity were investigated. Fermentation cells showed the best catalytic activity when the initial glycerol concentration was 12g/L and the rotating speed was 250rpm. Furthermore, we studied the difference between glycerol and glycerol mixtures as fed-batch media in pH-stat fed-batch fermentation. Results showed that glycerol had better catalytic activity than the glycerol mixture, and the effect of fed-batch fermentation was better than batch fermentation. Meanwhile, the enzyme activities of leucine dehydrogenase and formate dehydrogenase reached 129.87 U/g DCW and 437.02 U/g DCW, respectively, and the intracellular NAD(H) concentration reached 14.94μmol/g DCW. Using the optimized fermentation parameters, amplified fermentation was then carried out in a 5000L fermenter to demonstrate the industrial production of l-ABA by Escherichia coli BL21.

Cross-linking of carbonic anhydrase and formate dehydrogenase based on amino acid specific recognition: Conversion of carbon dioxide to formic acid

Inspired by the cascades performed in vivo, the assembly of multiple enzymes in vitro has strongly moved into the focus of researchers in the field of biocatalysis. In this study, a new, mild and accurate enzyme cross-linking method is revealed. Microbial transglutaminase (MTG) acts as a "cross-linking medium" by identifying the amide group of the glutamine and the primary amine group of lysine in the artificial peptide tags specifically to form an iso-peptide bond. Here, carbonic anhydrase (CA) and formate dehydrogenase (FDH) with different peptide tags that can be recognized by MTG were linked together to obtain different proportions of cross-linked enzymes for efficient conversion of greenhouse gas carbon dioxide to formic acid. After cross-linking, we obtained "one-to-one" and "one-to-more" cross-linked enzyme aggregates. There is a minor residual loss of the two enzymes, the remaining enzyme activity of CA is more than 93%, and the remaining enzyme activity of FDH is more than 84%. In particular, the overall catalytic efficiency of the cross-linked enzyme is increased by 5.8 times compared with free enzymes and the thermal stability of FDH at different temperatures is improved. The applied strategy demonstrates the potential application of MTG in multi-enzyme assembly and synthetic biology.

Interdependence of Escherichia coli formate dehydrogenase and hydrogen-producing hydrogenases during mixed carbon sources fermentation at different pHs

The impact of the four membrane-bound [NiFe]-hydrogenases (Hyd) of Escherichia coli on total H2-oxidizing activity during fermentation of a mixture of glucose, glycerol and formate at different pHs was examined. It was shown that Hyd-2 had a major contribution to total Hyd activity at pH 7.5 in early-stationary phase (24 h) cells, while the main contribution was made by Hyd-3 in late-stationary phase (72 h). Hyd-4-dependent Hyd activity could be demonstrated at pH 6.5 in cells lacking Hyd-1, Hyd-2 and Hyd-3. at pH 7.5 Hyd-4-dependent formate dehydrogenase (FDH-H) activity was demonstrated. Growth properties and fermentation end product patterns during 72 h demonstrated that the cells retained viability deep into stationary phase. Our findings emphasize the importance of formate in modulating H2 metabolism, presumably by contributing to maintain redox, pH and pmf balance. This is important for regulating and enhancing H2 production when a mixture of carbon sources is applied.

Effect of Metal Ions on the Activity of Ten NAD-Dependent Formate Dehydrogenases.

NAD-dependent formate dehydrogenase (FDH) enzymes are frequently used in industrial and scientific applications. FDH is a reversible enzyme that reduces the NAD molecule to NADH and produces CO2 by oxidation of the formate ion, whereas it causes CO2 reduction in the reverse reaction. Some transition metal elements - Fe3+, Mo6+ and W6 + - can be found in the FDH structure of anaerobic and archaeal microorganisms, and these enzymes require cations and other redox-active cofactors for their FDH activity. While NAD-dependent FDHs do not necessarily require any metal cations, the presence of various metal cations can still affect FDH activities. To study the effect of 11 different metal ions, NAD-dependent FDH enzymes from ten different microorganisms were tested: Ancylobacter aquaticus (AaFDH), Candida boidinii (CboFDH), Candida methylica (CmFDH), Ceriporiopsis subvermispora (CsFDH), Chaetomium thermophilum (CtFDH), Moraxella sp. (MsFDH), Myceliophthora thermophila (MtFDH), Paracoccus sp. (PsFDH), Saccharomyces cerevisiae (ScFDH) and Thiobacillus sp. (TsFDH). It was found that metal ions (mainly Cu2+ and Zn2+) could have quite strong inhibition effects on several enzymes in the forward reaction, whereas several cations (Li+, Mg2+, Mn2+, Fe3+ and W6+) could increase the forward reaction of two FDHs. The highest activity increase (1.97 fold) was caused by Fe3+ in AaFDH. The effect on the reverse reaction was minimal. The modelled structures of ten FDHs showed that the active site is formed by 15 highly conserved amino acid residues spatially settling around the formate binding site in a conserved way. However, the residue differences at some of the sites close to the substrate do not explain the activity differences. The active site space is very tight, excluding water molecules, as observed in earlier studies. Structural examination indicated that smaller metal ions might be spaced close to the active site to affect the reaction. Metal ion size showed partial correlation to the effect on inhibition or activation. Affinity of the substrate may also affect the sensitivity to the metal's effect. In addition, amino acid differences on the protein surface may also be important for the metal ion effect.

Degradation of Methanol Catabolism Enzymes of Formaldehyde Dehydrogenase and Formate Dehydrogenase in Methylotrophic Yeast Komagataella phaffii

Abstract—The investigation of the mechanisms of cytosolic protein degradation is of great fundamental and applied importance. The decrease in the specific activity of formaldehyde dehydrogenase (Fldh1) and formate dehydrogenase (Fdh1) in the wild type strain GS200, the strain with the deletion of the GSS1 hexose sensor gene, and the strain that is defective in autophagy pathway SMD1163 of K. phaffii during short-term and long-term induction with methanol, with or without the addition of the MG132 (proteasome degradation inhibitor), was investigated. It was shown that the duration of cell incubation on methanol had no particular effect on the inactivation of enzymes. The effect of the proteasome inhibitor MG132 was insignificant. Catabolic inactivation of cytosolic and peroxisomal enzymes was damaged in the gss1Δ mutant since glucose signaling was impaired. Fldh1 and Fdh1 are probably degraded via the vacuolar pathway regardless of the duration of methanol induction.

Regulation of lanthanide-dependent methanol oxidation pathway in the legume symbiotic nitrogen-fixing bacterium Bradyrhizobium sp. strain Ce-3

Lanthanide (Ln)-dependent XoxF-type methanol dehydrogenase (MDH) genes can be found in bacteria that are not believed to be methylotrophs, and studies on their methylotrophic pathways and their use of Ln are now emerging. Ln-dependent methanol utilization in Bradyrhizobium sp. strain Ce-3, which belongs to the Bradyrhizobium elkanii superclade (clade II), was investigated in this study. Strain Ce-3 was able to grow in a media containing methanol as a sole carbon source and light Ln (L-Ln, i.e., La3+, Ce3+, Pr3+, and Nd3+), whereas the strain did not show any growth with Ca2+ or the heavy Ln, Sm3+. We found that the uptake of L-Ln is enhanced mainly by methanol and L-Ln species, and the strain incorporates each L-Ln species evenly into the cell. The genome of strain Ce-3 encodes the xox cluster for Ln-dependent methanol dehydrogenase (xoxF) and the enzymes participating in the methanol oxidation pathway (xoxG, fldA, and gfaA) and regulation (xoxR), but the gene encoding formate dehydrogenase (FDH) was not found in the cluster. MDH, formaldehyde dehydrogenase, and FDH activities were induced by methanol/Ln. Moreover, expression of the genes on the xox cluster was upregulated by methanol/Ln. Based on these results, we concluded that strain Ce-3 possesses a complete L-Ln-dependent methanol oxidation pathway, which is dissimilar to plant phyllospheric bacteria, Methylobacterium species, with a transport system for L-Ln species.

Influence of C4 -Dcu transporters on hydrogenase and formate dehydrogenase activities in stationary phase-grown fermenting Escherichia coli.

During mixed-acid fermentation, Escherichia coli transports succinate mainly via transporters of the Dcu family. Here, we analyze the influence of Dcu transporters on hydrogenase (Hyd) and fermentative formate dehydrogenase (FDH-H) activities and how this is affected by external pH and carbon source. Using selected dcu mutations, it was shown that Dcu carriers mainly affect Hyd and FDH-H activities during glycerol but not glucose fermentation at acidic pH. During glycerol fermentation at pH 5.5, inactivation of either one or all Dcu carriers increased total Hyd activity by 60% compared with wild type. Under the same growth conditions, a dcuACBD mutant had a twofold higher FDH-H activity. When glucose was fermented in dcuD single mutant at pH 5.5, the FDH-H activity was also increased twofold compared with wild type. Interestingly, in dcuD or dcuACBD mutants at pH 7.5, Hyd activity was lowered by 20%. Taken together, it can be concluded that during glucose fermentation at pH 7.5, lack of DcuD affects Hyd enzyme activity, but at pH 5.5, it has a stronger effect on FDH-H activity. During glycerol fermentation, lack of Dcu carriers increased Hyd and FDH-H activities as revealed at pH 5.5. The results suggest that impairing Dcu transport function increases intracellular formate levels and thus affects H2 cycling and proton-motive force generation.

Modeling a co-culture of Clostridium autoethanogenum and Clostridium kluyveri to increase syngas conversion to medium-chain fatty-acids

Microbial fermentation of synthesis gas (syngas) is becoming more attractive for sustainable production of commodity chemicals. To date, syngas fermentation focuses mainly on the use of Clostridium species for the production of small organic molecules such as ethanol and acetate. The co-cultivation of syngas-fermenting microorganisms with chain-elongating bacteria can expand the range of possible products, allowing, for instance, the production of medium-chain fatty acids (MCFA) and alcohols from syngas. To explore these possibilities, we report herein a genome-scale, constraint-based metabolic model to describe growth of a co-culture of Clostridium autoethanogenum and Clostridium kluyveri on syngas for the production of valuable compounds. Community flux balance analysis was used to gain insight into the metabolism of the two strains and their interactions, and to reveal potential strategies enabling production of butyrate and hexanoate.The model suggests that one strategy to optimize the production of medium-chain fatty-acids from syngas would be the addition of succinate. According to the prediction, addition of succinate would increase the pool of crotonyl-CoA and the ethanol/acetate uptake ratio in C. kluyveri, resulting in a flux of up to 60% of electrons into hexanoate. Another potential way to further optimize butyrate and hexanoate production would be an increase of C. autoethanogenum ethanol production. Blocking either acetaldehyde dehydrogenase or formate dehydrogenase (ferredoxin) activity or formate transport, in the C. autoethanogenum metabolic model could potentially lead to an up to 150% increase in ethanol production.