(620bt) Screening Signal Peptides for Heterologous Protein Secretion in Geobacillus Thermoglucosidans

Zhou, J., University of Illinois at Urbana-Champaign
Rivera, A., Centers for Disease Control and Prevention
Rao, C. V., University of Illinois at Urbana-Champaign

Bacillus is widely used in industry for enzymes production because they are capable of secreting proteins at high concentration. We identified a related thermophile Geobacillus thermoglucosidans capable of secreting proteins at high titers and then engineered it to secrete heterologous protein. We examined the native capacity of three strains (G. thermoglucosidasius BGSC 95A1, BGSC 95A2, and C56-YS93) to secrete protein on different carbohydrate substrates. They were able to secrete grams per liter of protein on C5 sugars, which was much higher than on glucose and cellobiose. The highest yields are observed during growth on xylan with strain C56-YS93. We have also performed shotgun proteomics to determine and compare which proteins are secreted by strain C56-YS93 during growth on different substrates (xylan, xylose and cellobiose). Based on this analysis, the signal peptides (SPs) for highly secreted proteins were identified. Additionally, SPs were analyzed by prediction algorithms (SignalP 4.1) for strong signals and cleavage sequences. By using a thermostable amylase from G. stearothermophilis as a reporter, the top 25 SPs from prediction result were characterized for their potential for heterologous protein secretion.