Secreted expression of a hyperthermophilic α-amylase gene from Thermococcus sp. HJ21 in Bacillus subtilis

The hyperthermophilic α-amylase from Thermococcus sp. HJ21 possesses unique traits (Ca(2+)-independent thermostability and optimal temperature of 95°C) that make it a great potential candidate for use in the food industry. However, this Archaea isolated from a deep-sea thermal vent requires strict control of culture conditions and produces only small amounts of α-amylase. To solve these problems, the α-amylase gene was cloned and expressed in Bacillus subtilis, which is an ideal food-grade host for heterologous protein expression. To express high levels of this α-amylase in B. subtilis, the promoters Pgrac, PxylA, P43, and Phag were used to construct four different expression vectors for testing. The vector containing the PxylA promoter was found to have the highest transcriptional activity and produce the highest amylase activity (19.6 U/ml). To test the secretion efficiency of signal peptides in B. subtilis, three signal peptides were cloned and fused to the α-amylase gene (lacking its native signal peptide). The optimal signal peptide was SamyQ, with a secretion efficiency of approximately 90%. These results indicate that the promoter PxylA and signal peptide SamyQ tested in this study may be useful for the expression and secretion of archaeal proteins in B. subtilis.