Construction of a low-serine-type-carboxypeptidase-product mutant of Aspergillus oryzae by the expression of antisense RNA and its use as a host for heterologous protein secretion

Using an antisense control strategy, we isolated an Aspergillus oryzae mutant that produced low levels of carboxypeptidases (CPases). The mutant TFC-1 expressed the antisense RNA of the structural gene of CPase O and showed about 30% of the CPase activity in the parent strain. Gel filtration analysis indicated that this mutant decreased the CPase activities not only of CPase O but also of CPase O-1 and O-2. This result indicated that the antisense RNA was able to control the expression of the CPase genes as a group. Using the mutant as a heterologous protein expression host that produced the low levels of CPases, a stable and higher level of lysozyme expression could be obtained compared with the wild-type. In vitro proteolytic degradation assay also demonstrated that human lysozyme was degraded by purified CPase O.