Abstract
BACKGROUND: Parageobacillus thermoglucosidasius is a thermophilic and ethanol-producing bacterium capable of utilising both hexose and pentose sugars for fermentation. The organism has been proposed to be a suitable organism for the production of bioethanol from lignocellulosic feedstocks. These feedstocks may be difficult to degrade, and a potential strategy to optimise this process is to engineer strains that secrete hydrolases that liberate increased amounts of sugars from those feedstocks. However, very little is known about protein transport in P. thermoglucosidasius and the limitations of that process, and as a first step we investigated whether there were bottlenecks in the secretion of a model protein. RESULTS: A secretory enzyme, xylanase (XynA1), was produced with and without its signal peptide. Cell cultures were fractionated into cytoplasm, membrane, cell wall, and extracellular milieu protein extracts, which were analysed using immunoblotting and enzyme activity assays. The main bottleneck identified was proteolytic degradation of XynA1 during or after its translocation. A combination of mass spectrometry and bioinformatics indicated the presence of several proteases that might be involved in this process. CONCLUSION: The creation of protease-deficient strains may be beneficial towards the development of P. thermoglucosidasius as a platform organism for industrial processes.