Search

Abstract Background Succinate is produced petrochemically from maleic anhydride to satisfy a small specialty chemical market. If succinate could be produced fermentatively at a price competitive with that of maleic anhydride, though, it could replace maleic anhydride as the precursor of many bulk chemicals, transforming a multi-billion dollar petrochemical market into one based on renewable resources. Actinobacillus succinogenes naturally converts sugars and CO2 into high concentrations of succinic acid as part of a mixed-acid fermentation. Efforts are ongoing to maximize carbon flux to succinate to achieve an industrial process. Results Described here is the 2.3 Mb A. succinogenes genome sequence with emphasis on A. succinogenes's potential for genetic engineering, its metabolic attributes and capabilities, and its lack of pathogenicity. The genome sequence contains 1,690 DNA uptake signal sequence repeats and a nearly complete set of natural competence proteins, suggesting that A. succinogenes is capable of natural transformation. A. succinogenes lacks a complete tricarboxylic acid cycle as well as a glyoxylate pathway, and it appears to be able to transport and degrade about twenty different carbohydrates. The genomes of A. succinogenes and its closest known relative, Mannheimia succiniciproducens, were compared for the presence of known Pasteurellaceae virulence factors. Both species appear to lack the virulence traits of toxin production, sialic acid and choline incorporation into lipopolysaccharide, and utilization of hemoglobin and transferrin as iron sources. Perspectives are also given on the conservation of A. succinogenes genomic features in other sequenced Pasteurellaceae. Conclusions Both A. succinogenes and M. succiniciproducens genome sequences lack many of the virulence genes used by their pathogenic Pasteurellaceae relatives. The lack of pathogenicity of these two succinogens is an exciting prospect, because comparisons with pathogenic Pasteurellaceae could lead to a better understanding of Pasteurellaceae virulence. The fact that the A. succinogenes genome encodes uptake and degradation pathways for a variety of carbohydrates reflects the variety of carbohydrate substrates available in the rumen, A. succinogenes's natural habitat. It also suggests that many different carbon sources can be used as feedstock for succinate production by A. succinogenes.

Succinate fermentation was investigated in Escherichia coli strains over expressing actinobacillus succinogenes phosphoenolpyruvate carboxykinase (PEPCK). Results showed that PEPCK overexpression increased succinate production 6.5- fold.

Research has been conducted on Pyrococcus furiosus alpha-amylase. The role of cysteine residues in alpha-amylase thermal stability and activity has been investigated and the details are reported.

This review discusses bacterial and archaeal enzymes isolated from terrestrial and marine environments that exhibit unique temperature stability. Structure-function relationships, molecular basis of thermostability, catalytic properties, genetic and evolutionary aspects, and mechanism of action of these thermozymes are discussed.

The cloning, sequencing and expression of Thermotoga neapolitana in Escherichia coli produced a recombinant XI (TNXI) with catalytic properties similar to those of native enzymes. Although tetrameric and dimeric forms were observed from the resulting TNXI, both displayed similar biochemical and physical properties. However, calorimetric results showed that the dimer is more catalytically viable and stable than the tetrameric form.

Microbiological research shows that the gene encoding the hyperthermophilic extracellular alpha-amylase from the microorganism Pyrococcus furiosus is a single 460-residue polypeptide chain with a 26-residue signal peptide. It has a half-life of 13 hours at 98 degrees centigrade and is more thermostable than the alpha-amylase enzyme from the microorganism Bacillus licheniformis.

Microbiological research shows that the gene encoding the anaerobic microorganism Pyrococcus furiosus hyperthermophilic enzyme amylopullulanase is a single 827-residue polypeptide with a 26-residue signal peptide. It is a mature protein which has a molecular weight of 89,000 and retains its three-dimensional shape at temperatures of up to 70 degrees centigrade. The substrate specificity, kinetic properties and hydrolysis product analysis are discussed.

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-amino acid polypeptide with a 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.

The xylA gene of Thermotoga neapolitana was cloned and expressed in Escherichia coli to study the structure and properties of the enzyme it codes for. Xylose isomerase is most active at 95 degrees centigrade, is stable over a wide range of pH and belongs to the xylose isomerase family II. The thermostability of this enzyme is probably because it has less glutamine and asparagine residues which are denatured at high temperatures. However, the properties of this enzyme are different from those of the enzyme produced by the closely related species Thermotoga maritima.

Combination of [super 15]N NMR relaxation measurements and NMR monitored hydrogen-deuterium exchange at 30 degree Celsius was used to study the backbone conformational dynamics of Thermotoga neapolitana adenylate kinase in the free form (TNAK) and inhibitor-bound form (TNAK*Ap5A). The results revealed that TNAK maintained high activity at 30 degree Celsius by localizing flexibility to the hinge regions that are key to facilitating conformational changes.

The enantiomers of a series of phenyl ring-containing secondary alcohols are produced by asymmetric reduction and enantioselective oxidation via kinetic resolution (KR) by using Thermoanaerobacter ethanolicus (W110A TESADH). W110A TESADH in Tris buffer/acetone is used to obtain (R)-alcohols, the anti-Prelog products, by enantiospecific oxidation of (S)-alcohols through oxidative kinetic resolution of the rac-alcohols.

A defined medium, AM3, is 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, NH4Cl as the main nitrogen source, and glutamate, cysteine, and methionine as required amino acids.