Supplementary Material for: 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 (Ca²⁺-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 P_grac, P_xylA, P43, and P_hag were used to construct four different expression vectors for testing. The vector containing the P_xylA 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 S_amyQ, with a secretion efficiency of approximately 90%. These results indicate that the promoter P_xylA and signal peptide S_amyQ tested in this study may be useful for the expression and secretion of archaeal proteins in B. subtilis.