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NAD-dependent Thermotoga maritima glycerol dehydrogenase (TmGlyDH) converts glycerol into dihydroxyacetone (DHA), a valuable synthetic precursor and sunless tanning agent. In this work, recombinant TmGlyDH was characterized to determine if it can be used to catalyze DHA production. The pH optima for glycerol oxidation and DHA reduction at 50 °C were 7.9 and 6.0, respectively. Under the conditions tested, TmGlyDH had a linear Arrhenius plot up to 80 °C. TmGlyDH was more thermostable than other glycerol dehydrogenases, remaining over 50 % active after 7 h at 50 °C. TmGlyDH was active on racemic 1,2-propanediol and produced (R)-1,2-propanediol from hydroxyacetone with an enantiomeric excess above 99 %, suggesting that TmGlyDH can also be used for chiral synthesis. (R)-1,2-propanediol production from hydroxyacetone was demonstrated for the first time in a one-enzyme cycling reaction using glycerol as the second substrate. Negative cooperativity was observed with glycerol and DHA, but not with the cofactor. Apparent kinetic parameters for glycerol, DHA, and NAD(H) were determined over a broad pH range. TmGlyDH showed little activity with N(6)-carboxymethyl-NAD(+) (N(6)-CM-NAD), an NAD(+) analog modified for easy immobilization to amino groups, but the double mutation V44A/K157G increased catalytic efficiency with N(6)-CM-NAD(+) ten-fold. Finally, we showed for the first time that a GlyDH is active with immobilized N(6)-CM-NAD(+), suggesting that N(6)-CM-NAD(+) can be immobilized on an electrode to allow TmGlyDH activity in a system that reoxidizes the cofactor electrocatalytically.

Mannitol is a fructose-derived, 6-carbon sugar alcohol that is widely found in bacteria, yeasts, fungi, and plants. Because of its desirable properties, mannitol has many applications in pharmaceutical products, in the food industry, and in medicine. The current mannitol chemical manufacturing process yields crystalline mannitol in yields below 20 mol% from 50% glucose/50% fructose syrups. Thus, microbial and enzymatic mannitol manufacturing methods have been actively investigated, in particular in the last 10 years. This review summarizes the most recent advances in biological mannitol production, including the development of bacterial-, yeast-, and enzyme-based transformations.

Thermotoga maritima TM0298 is annotated as an alcohol dehydrogenase, yet it shows high identity and similarity to mesophilic mannitol dehydrogenases. To investigate this enzyme further, its gene was cloned and expressed in Escherichia coli. The purified recombinant enzyme was most active on fructose and mannitol, making it the first known hyperthermophilic mannitol dehydrogenase. T. maritima mannitol dehydrogenase (TmMtDH) is optimally active between 90 and 100 degrees C and retains 63% of its activity at 120 degrees C but shows no detectable activity at room temperature. Its kinetic inactivation follows a first-order mechanism, with half-lives of 57 min at 80 degrees C and 6 min at 95 degrees C. Although TmMtDH has a higher V (max) with NADPH than with NADH, its catalytic efficiency is 2.2 times higher with NADH than with NADPH and 33 times higher with NAD(+) than with NADP(+). This cofactor specificity can be explained by the high density of negatively charged residues (Glu193, Asp195, and Glu196) downstream of the NAD(P) interaction site, the glycine motif. We demonstrate that TmMtDH contains a single catalytic zinc per subunit. Finally, we provide the first proof of concept that mannitol can be produced directly from glucose in a two-step enzymatic process, using a Thermotoga neapolitana xylose isomerase mutant and TmMtDH at 60 degrees C.

Actinobacillus succinogenes naturally produces high concentrations of succinate, a potential intermediary feedstock for bulk chemical productions. A. succinogenes responds to high CO(2) and H(2) concentrations by producing more succinate and by producing less formate, acetate, and ethanol. To determine how intermediary fluxes in A. succinogenes respond to CO(2) and H(2) perturbations, (13)C-metabolic flux analysis was performed in batch cultures at two different NaHCO(3) concentrations, with and without H(2), using a substrate mixture of [1-(13)C]glucose, [U-(13)C]glucose, and unlabeled NaHCO(3). The resulting amino acid, organic acid, and glycogen isotopomers were analyzed by gas chromatography-mass spectrometry and NMR. In all conditions, exchange flux was observed through malic enzyme and/or oxaloacetate decarboxylase. The presence of an exchange flux between oxaloacetate, malate, and pyruvate indicates that, in addition to phosphoenolpyruvate, oxaloacetate, and malate, pyruvate is a fourth node for flux distribution between succinate and alternative fermentation products. High NaHCO(3) concentrations decreased the amount of flux shunted by C(4)-decarboxylating activities from the succinate-producing C(4) pathway to the formate-, acetate-, and ethanol-producing C(3) pathway. In addition, pyruvate carboxylating flux increased in response to high NaHCO(3) concentrations. C(3)-pathway dehydrogenase fluxes increased or decreased appropriately in response to the different redox demands imposed by the different NaHCO(3) and H(2) concentrations. Overall, these metabolic flux changes allowed A. succinogenes to maintain a constant growth rate and biomass yield in all conditions. These results are discussed with respect to A. succinogenes' physiology and to metabolic engineering strategies to increase the flux to succinate.

Bio-based succinate is receiving increasing attention as a potential intermediary feedstock for replacing a large petrochemical-based bulk chemical market. The prospective economical and environmental benefits of a bio-based succinate industry have motivated research and development of succinate-producing organisms. Bio-based succinate is still faced with the challenge of becoming cost competitive against petrochemical-based alternatives. High succinate concentrations must be produced at high rates, with little or no by-products to most efficiently use substrates and to simplify purification procedures. Herein are described the current prospects for a bio-based succinate industry, with emphasis on specific bacteria that show the greatest promise for industrial succinate production. The succinate-producing characteristics and the metabolic pathway used by each bacterial species are described, and the advantages and disadvantages of each bacterial system are discussed.

Actinobacillus succinogenes is a promising candidate for industrial succinate production. However, in addition to producing succinate, it also produces formate and acetate. To understand carbon flux distribution to succinate and alternative products we fed A. succinogenes [1-(13)C]glucose and analyzed the resulting isotopomers of excreted organic acids, proteinaceous amino acids, and glycogen monomers by gas chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy. The isotopomer data, together with the glucose consumption and product formation rates and the A. succinogenes biomass composition, were supplied to a metabolic flux model. Oxidative pentose phosphate pathway flux supplied, at most, 20% of the estimated NADPH requirement for cell growth. The model indicated that NADPH was instead produced primarily by the conversion of NADH to NADPH by transhydrogenase and/or by NADP-dependent malic enzyme. Transhydrogenase activity was detected in A. succinogenes cell extracts, as were formate and pyruvate dehydrogenases, which the model suggested were contributing to NADH production. Malic enzyme activity was also detected in cell extracts, consistent with the flux analysis results. Labeling patterns in amino acids and organic acids showed that oxaloacetate and malate were being decarboxylated to pyruvate. These are the first in vivo experiments to show that the partitioning of flux between succinate and alternative fermentation products can occur at multiple nodes in A. succinogenes. The implications for designing effective metabolic engineering strategies to increase A. succinogenes succinate production are discussed.

Chemically defined media allow for a variety of metabolic studies that are not possible with undefined media. A defined medium, AM3, was created to expand the experimental opportunities for investigating the fermentative metabolism of succinate-producing Actinobacillus succinogenes . AM3 is a phosphate-buffered medium containing vitamins, minerals, NH 4 Cl as the main nitrogen source, and glutamate, cysteine, and methionine as required amino acids. A. succinogenes growth trends and end product distributions in AM3 and rich medium fermentations were compared. The effects of NaHCO 3 concentration in AM3 on end product distribution, growth rate, and metabolic rates were also examined. The A. succinogenes growth rate was 1.3 to 1.4 times higher at an NaHCO 3 concentration of 25 mM than at any other NaHCO 3 concentration, likely because both energy-producing metabolic branches (i.e., the succinate-producing branch and the formate-, acetate-, and ethanol-producing branch) were functioning at relatively high rates in the presence of 25 mM bicarbonate. To improve the accuracy of the A. succinogenes metabolic map, the reasons for A. succinogenes glutamate auxotrophy were examined by enzyme assays and by testing the ability of glutamate precursors to support growth. Enzyme activities were detected for glutamate synthesis that required glutamine or α-ketoglutarate. The inability to synthesize α-ketoglutarate from glucose indicates that at least two tricarboxylic acid cycle-associated enzyme activities are absent in A. succinogenes .

Pyrococcus furiosus amylopullulanase (PfAPU) belongs to glycosyl hydrolase family 57. Using sequence alignments of the known family 57 enzymes and site-directed mutagenesis, E291, D394, and E396 were identified as PfAPU putative catalytic residues. The apparent catalytic efficiencies (k(cat)/K(m)) of PfAPU mutants E291Q and D394N on pullulan were 123.0 and 24.4 times lower, respectively, than that of PfAPU. The activity of mutant E396Q on pullulan was too low to allow reliable determination of its catalytic efficiency. The apparent specific activities of these enzymes on starch also decreased 91.0 times (E291Q), 11.7 times (D394N), and 37.2 times (E396Q). The hydrolytic patterns for pullulan and starch were the same, while the hydrolysis rates differed as reported. Based on sequence alignment and a previous report, E291 is proposed as the catalytic nucleophile.

Succinate fermentation was investigated in Escherichia coli strains overexpressing Actinobacillus succinogenes phosphoenolpyruvate carboxykinase (PEPCK). In E. coli K-12, PEPCK overexpression had no effect on succinate fermentation. In contrast, in the phosphoenolpyruvate carboxylase mutant E. coli strain K-12 ppc :: kan , PEPCK overexpression increased succinate production 6.5-fold.

Fumarate reductase was used as a model oxidoreductase to demonstrate continuous electrical cofactor reduction-oxidation during the bioelectrochemical synthesis and detection of chemicals. The enzyme preparation was immobilized onto a graphite felt electrode that was modified with carboxymethylcellulose (CMC). Nicotinamide adenine dinucleotide (NAD), neutral red, and fumarate reductase (which contained menaquinone) were covalently linked by peptide bonds to the CMC. The electron mediator neutral red allowed NAD and menaquinone to be recycled electrically during enzymatic chemical synthesis. Succinate detection by the bioelectrocatalyst was linear from 5 microM to 10 mM succinate. Fumarate synthesis using this bioelectrode was dependent on succinate utilization and resulted in proportional production of electricity and fumarate. Succinate synthesis using this bioelectrocatalyst was dependent on current and fumarate concentration. This bioelectrocatalyst system may enhance the utility of menaquinone- and/or pyridine nucleotide-linked oxidoreductases in diverse enzymatic fuel cells and sensors. It may also enhance the utility of oxidoreductase-based chemical synthesis systems because it eliminates the problem of cofactor recycling.

Actinobacillus succinogenes 130Z naturally produces among the highest levels of succinate from a variety of inexpensive carbon substrates. A few studies have demonstrated that A. succinogenes can anaerobically metabolize glycerol, a waste product of biodiesel manufacture and an inexpensive feedstock, to produce high yields of succinate. However, all these studies were performed in the presence of yeast extract, which largely removes the redox constraints associated with fermenting glycerol, a highly reduced molecule. We demonstrated that A. succinogenes cannot ferment glycerol in minimal medium, but that it can metabolize glycerol by aerobic or anaerobic respiration. These results were expected based on the A. succinogenes genome, which encodes respiratory enzymes, but no pathway for 1,3-propanediol production. We investigated A. succinogenes’s glycerol metabolism in minimal medium in a variety of respiratory conditions by comparing growth, metabolite production, and in vitro activity of terminal oxidoreductases. Nitrate inhibited succinate production by inhibiting fumarate reductase expression. In contrast, growth in the presence of dimethylsulfoxide and in microaerobic conditions allowed high succinate yields. The highest succinate yield was 0.75 mol/mol glycerol (75 % of the maximum theoretical yield) in continuous microaerobic cultures. A. succinogenes could also grow and produce succinate on partially refined glycerols obtained directly from biodiesel manufacture. Finally, by expressing a heterologous 1,3-propanediol synthesis pathway in A. succinogenes, we provide the first proof of concept that A. succinogenes can be engineered to grow fermentatively on glycerol.

Actinobacillus succinogenes is one of the best natural succinate-producing organisms, but it still needs engineering to further increase succinate yield and productivity. In this study, we developed a markerless knockout method for A. succinogenes using natural transformation or electroporation. The Escherichia coli isocitrate dehydrogenase gene with flanking flippase recognition target sites was used as the positive selection marker, making use of A. succinogenes 's auxotrophy for glutamate to select for growth on isocitrate. The Saccharomyces cerevisiae flippase recombinase (Flp) was used to remove the selection marker, allowing its reuse. Finally, the plasmid expressing flp was cured using acridine orange. We demonstrate that at least two consecutive deletions can be introduced into the same strain using this approach, that no more than a total of 1 kb of DNA is needed on each side of the selection cassette to protect from exonuclease activity during transformation, and that no more than 200 bp of homologous DNA is needed on each side for efficient recombination. We also demonstrate that electroporation can be used as an alternative transformation method to obtain knockout mutants and that an enriched defined medium can be used for direct selection of knockout mutants on agar plates with high efficiency. Single-knockout mutants of the fumarate reductase and of the pyruvate formate lyase-encoding genes were obtained using this knockout strategy. Double-knockout mutants were also obtained by deleting the citrate lyase-, β-galactosidase-, and aconitase-encoding genes in the pyruvate formate lyase knockout mutant strain.

The gene encoding the Pyrococcus furiosus hyperthermophilic amylopullulanase (APU) was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a single 827-residue polypeptide with a 26-residue signal peptide. The protein sequence had very low homology (17 to 21% identity) with other APUs and enzymes of the alpha-amylase family. In particular, none of the consensus regions present in the alpha-amylase family could be identified. P. furiosus APU showed similarity to three proteins, including the P. furiosus intracellular alpha-amylase and Dictyoglomus thermophilum alpha-amylase A. The mature protein had a molecular weight of 89,000. The recombinant P. furiosus APU remained folded after denaturation at temperatures of < or = 70 degrees C and showed an apparent molecular weight of 50,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Denaturating temperatures of above 100 degrees C were required for complete unfolding. The enzyme was extremely thermostable, with an optimal activity at 105 degrees C and pH 5.5. Ca2+ increased the enzyme activity, thermostability, and substrate affinity. The enzyme was highly resistant to chemical denaturing reagents, and its activity increased up to twofold in the presence of surfactants.

The xylA gene coding for xylose isomerase from the hyperthermophile Thermotoga neapolitana 5068 was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a polypeptide of 444 residues with a calculated molecular weight of 50,892. The native enzyme was a homotetramer with a molecular weight of 200,000. This xylose isomerase was a member of the family II enzymes (these differ from family I isomerases by the presence of approximately 50 additional residues at the amino terminus). The enzyme was extremely thermostable, with optimal activity above 95 degrees C. The xylose isomerase showed maximum activity at pH 7.1, but it had high relative activity over a broad pH range. The catalytic efficiency (kcat/Km) of the enzyme was essentially constant between 60 and 90 degrees C, and the catalytic efficiency decreased between 90 and 98 degrees C primarily because of a large increase in Km. The T. neapolitana xylose isomerase had a higher turnover number and a lower Km for glucose than other family II xylose isomerases. Comparisons with other xylose isomerases showed that the catalytic and cation binding regions were well conserved. Comparison of different xylose isomerase sequences showed that numbers of asparagine and glutamine residues decreased with increasing enzyme thermostability, presumably as a thermophilic strategy for diminishing the potential for chemical denaturation through deamidation at elevated temperatures.

The conversion of glucose to fructose at elevated temperatures, as catalyzed by soluble and immobilized xylose (glucose) isomerases from the hyperthermophiles Thermotoga maritima (TMGI) and Thermotoga neapolitana 5068 (TNGI) and from the mesophile Streptomyces murinus (SMGI), was examined. At pH 7.0 in the presence of Mg(2+), the temperature optima for the three soluble enzymes were 85 degrees C (SMGI), 95 degrees to 100 degrees C (TNGI), and >100 degrees C (TMGI). Under certain conditions, soluble forms of the three enzymes exhibited an unusual, multiphasic inactivation behavior in which the decay rate slowed considerably after an initial rapid decline. However, the inactivation of the enzymes covalently immobilized to glass beads, monophasic in most cases, was characterized by a first-order decay rate intermediate between those of the initial rapid and slower phases for the soluble enzymes. Enzyme productivities for the three immobilized GIs were determined experimentally in the presence of Mg(2+). The highest productivities measured were 750 and 760 kg fructose per kilogram SMGI at 60 degrees C and 70 degrees C, respectively. The highest productivity for both TMGI and TNGI in the presence of Mg(2+) occurred at 70 degrees C, pH 7.0, with approximately 230 and 200 kg fructose per kilogram enzyme for TNGI and TMGI, respectively. At 80 degrees C and in the presence of Mg(2+), productivities for the three enzymes ranged from 31 to 273. A simple mathematical model, which accounted for thermal effects on kinetics, glucose-fructose equilibrium, and enzyme inactivation, was used to examine the potential for high-fructose corn syrup (HFCS) production at 80 degrees C and above using TNGI and SMGI under optimal conditions, which included the presence of both Co(2+) and Mg(2+). In the presence of both cations, these enzymes showed the potential to catalyze glucose-to-fructose conversion at 80 degrees C with estimated lifetime productivities on the order of 2000 kg fructose per kilogram enzyme, a value competitive with enzymes currently used at 55 degrees to 65 degrees C, but with the additional advantage of higher fructose concentrations. At 90 degrees C, the estimated productivity for SMGI dropped to 200, whereas, for TNGI, lifetime productivities on the order of 1000 were estimated. Assuming that the most favorable biocatalytic and thermostability features of these enzymes can be captured in immobilized form and the chemical lability of substrates and products can be minimized, HFCS production at high temperatures could be used to achieve higher fructose concentrations as well as create alternative processing strategies.

The xylA gene from Thermotoga neapolitana 5068 was expressed in Escherichia coli . Gel filtration chromatography showed that the recombinant enzyme was both a homodimer and a homotetramer, with the dimer being the more abundant form. The purified native enzyme, however, has been shown to be exclusively tetrameric. The two enzyme forms had comparable stabilities when they were thermoinactivated at 95°C. Differential scanning calorimetry revealed thermal transitions at 99 and 109.5°C for both forms, with an additional shoulder at 91°C for the tetramer. These results suggest that the association of the subunits into the tetrameric form may have little impact on the stability and biocatalytic properties of the enzyme.

The phosphoenolpyruvate (PEP) carboxykinase-encoding gene from the anaerobic, CO2-fixing, succinate-producing bacterium Anaerobiospirillum succiniciproducens was cloned, sequenced, and expressed in Escherichia coli. The gene encoded a 532-residue polypeptide with a calculated molecular mass of 58.7 kDa. The sequence of the A. succiniciproducens PEP carboxykinase was similar to those of all known ATP/ADP-dependent PEP carboxykinases. In particular, the A. succiniciproducens enzyme was 67.3% identical and 79.2% similar to the E. coli enzyme. The A. succiniciproducens pckA transcription start site was determined, and putative promoter regions were identified. The recombinant enzyme was overexpressed in E. coli. The purified enzyme was indiscernible from the native enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had the same activity as the native enzyme.