Xylan degrading enzymes from Geobacillus thermoglucosidasius, an ethanologenic thermophile

Geobacillus thermoglucosidasius NCIMB 11955 is a thermophilic bacterium that ferments both C6 and C5 sugars.  It has been genetically engineered (to form strain TM242) to divert the fermentation pathways away from mixed acids to generate ethanol as the major product; however, hydrolysis of biomass feedstock with commercial enzymes remains a major economic consideration in the production of bioethanol.  The current project aims to improve biomass utilization through an investigation of the enzymes associated with hemicellulose hydrolysis.

Firstly, the Geobacillus thermoglucosidasius TM242 genome sequence revealed a number of genes encoding glycoside-hydrolases, and three, encoding two β-xylosidases and an α-L-arabinofuranosidase, have been cloned and expressed in Escherichia coli, and the recombinant enzymes characterised.  One of the two β-xylosidases belongs to the glycoside-hydrolase family 52 and a high-resolution (1.7Å) crystal structure has been determined.  This is the first reported structure of a GH52 family member, and it surprisingly shares structural similarities with other glycoside-hydrolases, despite sharing less than 13% sequence identity.  A lower resolution structure (2.6Å) of the enzyme-substrate complex shows the positioning of the xylobiose substrate to be consistent with the family’s proposed retaining mechanism; 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.

Secondly, as the bioethanol-producing strain TM242 lacks xylanase enzymes, four genes encoding xylanases from closely-related Geobacillus strains have been cloned and expressed in E. coli, and their incorporation into the genome of G. thermoglucosidasius is in progress.