A novel ß-xylosidase structure from Geobacillus thermoglucosidasius: the first crystal structure of a glycoside hydrolase family GH52 enzyme reveals unpredicted similarity to other glycoside hydrolase folds.

Geobacillus thermoglucosidasius is a thermophilic bacterium that is able to ferment both C6 and C5 sugars to produce ethanol. During growth on hemicellulose biomass, an intracellular ß-xylosidase catalyses the hydrolysis of xylo-oligosaccharides to the monosaccharide xylose, which can then enter the pathways of central metabolism. The gene encoding a G. thermoglucosidasius ß-xylosidase belonging to CAZy glycoside hydrolase family GH52 has been cloned and expressed in Escherichia coli. The recombinant enzyme has been characterized and a high-resolution (1.7 Å) crystal structure has been determined, resulting in the first reported structure of a GH52 family member. A lower resolution (2.6 Å) structure of the enzyme-substrate complex shows the positioning of the xylobiose substrate to be consistent with the proposed retaining mechanism of the family; additionally, the deep cleft of the active-site pocket, plus the proximity of the neighbouring subunit, afford an explanation for the lack of catalytic activity towards the polymer xylan. Whilst the fold of the G. thermoglucosidasius ß-xylosidase is completely different from xylosidases in other CAZy families, the enzyme surprisingly shares structural similarities with other glycoside hydrolases, despite having no more than 13% sequence identity.

Structure of a bifunctional alcohol dehydrogenase involved in bioethanol generation in Geobacillus thermoglucosidasius.

Bifunctional alcohol/aldehyde dehydrogenase (ADHE) enzymes are found within many fermentative microorganisms. They catalyse the conversion of an acyl-coenzyme A to an alcohol via an aldehyde intermediate; this is coupled to the oxidation of two NADH molecules to maintain the NAD(+) pool during fermentative metabolism. The structure of the alcohol dehydrogenase (ADH) domain of an ADHE protein from the ethanol-producing thermophile Geobacillus thermoglucosidasius has been determined to 2.5 Šresolution. This is the first structure to be reported for such a domain. In silico modelling has been carried out to generate a homology model of the aldehyde dehydrogenase domain, and this was subsequently docked with the ADH-domain structure to model the structure of the complete ADHE protein. This model suggests, for the first time, a structural mechanism for the formation of the large multimeric assemblies or `spirosomes' that are observed for this ADHE protein and which have previously been reported for ADHEs from other organisms.

Application of pheB as a reporter gene for Geobacillus spp., enabling qualitative colony screening and quantitative analysis of promoter strength.

The pheB gene from Geobacillus stearothermophilus DSM6285 has been exploited as a reporter gene for Geobacillus spp. The gene product, catechol 2,3-dioxygenase (C23O), catalyzes the formation of 2-hydroxymuconic semialdehyde, which can be readily assayed. The reporter was used to examine expression from the ldh promoter associated with fermentative metabolism.

Microbial responses to solvent and alcohol stress.

Increasing fuel prices and doubts over the long-term availability of oil are currently major global concerns. Such concerns have led to national policies and objectives to develop microbially produced alcohols as fuel additives or substitutes. However, in South Africa this solution poses the further dilemma of sourcing a suitable fermentative carbohydrate that will not impact negatively on the availability of staple foods. The solution lies in the use of lignocellulosic materials, currently a waste product of the food and agriculture industries, which could be used in conjunction with a catabolically suitable production strain. In the pursuit of lignocellulosic biofuel production, conventional fermentation strains have been shown to have limited catabolic versatility. However, catabolically versatile engineered strains and novel isolates engineered with ethanologenic pathways have subsequently been shown to exhibit limitations in solvent tolerance, hindering their full potential as economically viable production strains. A considerable volume of research has been reported on the general cellular mechanisms and physiological responses to solvent shock as well as adaptive changes responsible for solvent tolerant phenotypes in mutant progeny. Here we review a number of the more common cell responses to solvents with particular focus on alcohol tolerance.

Biosynthesis of the 2-pyridone tenellin in the insect pathogenic fungus Beauveria bassiana.

Genomic DNA from the insect pathogenic fungus Beauveria bassiana was used as a template in a PCR with degenerate primers designed to amplify a fragment of a C-methyl transferase (CMeT) domain from a highly reduced fungal polyketide synthase (PKS). The resulting 270-bp PCR product was homologous to other fungal PKS CMeT domains and was used as a probe to isolate a 7.3-kb fragment of genomic DNA from a BamH1 library. Further library probing and TAIL-PCR then gave a 21.9-kb contig that encoded a 12.9-kb fused type I PKS-NRPS ORF together with ORFs encoding other oxidative and reductive enzymes. A directed knockout experiment with a BaR cassette, reported for the first time in B. bassiana, identified the PKS-NRPS as being involved in the biosynthesis of the 2-pyridone tenellin. Other fungal PKS-NRPS genes are known to be involved in the formation of tetramic acids in fungi, and it thus appears likely that related compounds are precursors of 2-pyridones in fungi. B. bassiana tenellin KO and WT strains proved to be equally pathogenic towards insect larvae; this indicated that tenellin is not involved in insect pathogenesis.

Directed evolution of a non-heme-iron-dependent extradiol catechol dioxygenase: identification of mutants with intradiol oxidative cleavage activity.

The non-heme-iron(II)-dependent extradiol catechol dioxygenases catalyse the oxidative cleavage of substituted catechols found on bacterial aromatic degradation pathways. The reaction mechanism of the extradiol dioxygenases is believed to proceed through the same proximal hydroperoxide intermediate as the iron(III)-dependent intradiol catechol dioxygenases. Directed evolution was carried out on members of the class III extradiol catechol dioxygenases, by using 1) error-prone polymerase chain reaction, 2) a primer-based cross-over method; the mutant dioxygenases were then screened for their ability to process a range of substituted catechols. Several mutant enzymes were found to show higher activity towards certain substituted catechols, including 4-chlorocatechol, and higher affinity for the iron(II) cofactor. Two mutants isolated from error-prone PCR of Escherichia coli MhpB (mutants R215W and K273R) were found to produce a mixture of extradiol and intradiol cleavage products, as detected by GC-MS and 1H NMR spectroscopy. The residue corresponding to K273 in protocatechuate 4,5-dioxygenase (LigAB), Val244, is located approximately 12 A from the iron(II) centre, but close to the putative dioxygen channel; R215 is found on a sequence loop not present in LigB.