Effect of in vitro DNA Rearrangement in the NH2-terminal Region of the Penicillinase Gene from Bacillus licheniformis on the Mode of Expression in Bacillus subtilis

Abstract

Secretion vector plasmids were constructed that have unique BglII sites within or near the signal sequence of B. licheniformis penicillinase, and penicillinase cartridges were also constructed that lack either 1, 2 or 3 of the processing sites for the membrane-bound, exo-large and exo-small enzymes. Each of these penicillinase cartridges was cloned on secretion vectors in B. subtilis and enzyme production was examined. The presence of both the signal sequence and the 3 host-specific processing sites on the secretion vector was required for an effective expression of the enzyme in B. subtilis. The presence of processing sites on the cartridge reduced the accumulation of penicillinase in the culture medium. When a vector plasmid lacking part of the hydrophobic region of the signal sequence and lacking the 3 processing sites was used, total penicillinase production decreased and enzyme accumulation in the medium was extremely low, despite the complete or incomplete presence of the processing sites on the cartridge. MW determination of these extracellular penicillinases suggested the existence of a new cleavage site for the enzyme.