Modular system for assessment of glycosyl hydrolase secretion in Geobacillus thermoglucosidasius

The facultatively-anaerobic, thermophilic bacterium Geobacillus thermoglucosidasius is being developed as an industrial micro-organism for cellulosic bioethanol production. Process improvement would be gained by enhanced secretion of glycosyl hydrolases. Here we report the construction of a modular system for combining promoters, signal peptide encoding regions and glycosyl hydrolase genes to facilitate selection of the optimal combination in G. thermoglucosidasius. Initially, a minimal 3-part E. coli - Geobacillus sp. shuttle vector pUCG3.8 was constructed using Gibson isothermal DNA assembly. The three PCR amplicons contained the pMB1 E. coli origin of replication and multiple cloning site of pUC18, the Geobacillus sp. origin of replication pBST1 and the thermostable kanamycin nucleotidyltransferase gene (knt), respectively. G. thermoglucosidasius could be transformed with pUCG3.8 at an increased efficiency (2.8 x 105c.f.u.μg-1 DNA) compared to a previously reported shuttle vector, pUCG18. A modular cassette for the inducible expression and secretion of proteins in G. thermoglucosidasius, designed to allow the simple interchange of parts, was demonstrated using the endoglucanase Cel5A from Thermotoga maritima as secretion target. In this example, expression of cel5A was placed under the control of a cellobiose-inducible promoter together with a signal peptide (SP) encoding sequence from a G.thermoglucosidasius C56-YS93 endo-β 1-4 xylanase. Cel5A activity was detected in culture supernatants indicating successful expression and secretion.