The facultatively-anaerobic, thermophilic bacterium Geobacillus thermoglucosidasius has been engineered and exploited for industrial bioethanol production. It is capable of fermenting a broad range of mono and short-chain oligosaccharides but is incapable of effective (hemi)cellulose utilisation. Here we report the construction of a system for the heterologous expression and secretion of glycosyl hydrolases (GHs) responsible for the degradation of (hemi)cellulose in G. thermoglucosidasius and provide an assessment of their activities on cellulosic substrates.
The GH expression cassette was assembled in the new E. coli - Geobacillus sp. shuttle vector pUCG3.8. It comprised a promoter inducible with cellobiose, a signal peptide (SP) from a Geobacillus sp.WCH70 β 1-4 xylanase, which targeted the GH for extracellular transport, followed by the GH of interest. Restriction enzyme recognition sites between the promoter, SP and GH modules facilitated facile substitution of these components.
GHs possessing cellulolytic or hemicellulolytic activities were sourced from both known cellulolytic thermophiles and from compost isolates grown on the bioenergy crop Miscanthus x giganteus. Activities in culture supernatants, assessed using the 3,5-dinitrosalicylic acid assay with xylan and amorphous/crystalline cellulose as substrates, confirmed the secretion of functional GHs and allowed for the construction of a G. thermoglucosidasius strain capable of utilising lignocellulosic biomass components.