Mutation and screening to increase chymosin yield in a genetically-engineered strain ofAspergillus awamori

Abstract

Through the course of five rounds of mutagenesis of a genetically-engineered strain ofAspergillus awamori, the yield of a heterologous protein (the acid protease, calf chymosin) increased four-fold. This was accomplished through the use of an agar plate screen incorporating the colony restrictor 2,6-dichloro-4-nitroaniline (dichloran) and the acid protease inhibitor diazoacetyl-norleucine methyl ester (DAN) to reduce high background concentrations of the native acid protease. A miniaturized liquid culture growth method using 24-well culture plates was an intermediate screen between agar plate and shake flask cultures. Analysis of broth samples for active calf chymosin was accomplished with a highly specific, 96-well microtiter plate turbidimetric assay.