Effect of pH on hen egg white lysozyme production and evolution of a recombinant strain of Aspergillus niger

An Aspergillus niger strain (B1) transformed to produce mature hen egg whit e lysozyme (HEWL) from a glucoamylase fusion protein under control of the A . niger glucoamylase promoter was grown in glucose-limited chemostat cultur e at a dilution rate of 0.07 h(-1) at various pH values. Maximum HEWL produ ction (9.3 mg g(-1); specific production rate = 0.65 mg g(-1) per h) was ob tained at pH 4.5. However, in chemostat culture, HEWL production was not st able at any pH tested. After 240 h in steady state, specific production dec reased to only 0.03 +/- 0.01 and 0.24 +/- 0.02 mg g(-1) per h at pH 6.5 and 4.5, respectively. Some isolates removed from the chemostat cultures had l ost copies of the HEWL gene and when grown in shake flask cultures all of t he isolates produced less HEWL than the parental strain. Morphological muta nts with similar phenotypes were isolated at all pHs, but their rate of inc rease in the population was pH dependent, with cultures at low pH (< 4.5) b eing more morphologically stable than cultures at high (> 4.5) pH. The sele ctive advantage of these mutants was also generally dependent on pH. Both y ellow pigment producing mutants and brown sporulation mutants had higher se lective advantages over the parental strain at high than at low pH, regardl ess of the pH at which they were isolated. However, the selective advantage of densely sporulating mutants was independent of pH. (C) 1999 Elsevier Sc ience B.V. All rights reserved.