Recombinant Expression of a Medium-temperature Amylase Production in Bacillus amyloliquefaciens LT

Bacillus amyloliquefaciens is one of best host cells for production of a wide spectrum of enzymes applied in food industry. However,studies for strain optimization are always difficult due to the low efficiency in transforming foreign DNA into those industrial strains. A novel type IV DNA methylation/restriction system was identified in B. amyloliquefaciens and other close species that was not found in model bacillus strain through genomic data mining,such as B. subtilis 164 or 168. It was confirmed that transformation efficiency of non-methylated DNA was much higher than DNAs purified from E. coli strain with methylation activity. With the optimized vector preparation and transformation protocols,two recombinant B. amyloliquefaciens LT transformants expressing its native medium-temperature amylase were obtained,namely LT-J1 and LT-J2,they produced the enzyme up to 145.9 U/mL and 153.6 U/mL,respectively,more than two times higher than the original strain that does not harbor the expression vector pMK4E-dx.