Evolution of a recombinant (gucoamylase-producing) strain of Fusarium venenatum A3/5 in chemostat culture

Fusarium venenatum JeRS 325 is a transformant of strain A3/5 which produces Aspergillus niger glucoamylase (GAM) under the control of a Fusarium oxysp orum trypsin-like protease promoter. The evolution of JeRS 325 was studied in glucose-limited chemostat cultures grown on NaNO3 or (NH4)(2)SO4 as the nitrogen source. Thirteen mutants which were more highly branched and four mutants which were more sparsely branched than the parental strain were iso lated from the NaNO3 chemostat, The highly branched mutants detected in thi s chemostat did not displace the sparsely branched population. The mutants isolated from the NaNO3 chemostat complemented representative strains previ ously isolated from glucose-limited chemostat cultures of F. venenatum A3/5 grown on (NH4)(2)SO4, but showed little complementation between themselves . By contrast, a highly branched mutant isolated from the (NH4)(2)SO4 chemo stat culture displaced the sparsely branched mycelial population. None of t he mutants isolated from the NaNO3 or (NH4)(2)SO4 chemostats produced as mu ch GAM as JeRS 325. Southern blot analysis showed that all except one mutan t had lost copies of both the glucoamylase and the acetamidase (the selecta ble marker) genes. However, specific GAM production was not necessarily cor related with the extent of glaA gene loss observed. Further, 10 of the muta nts had lost the ability to grow on acetamide as the sole nitrogen source, although they retained copies of the amdS gene. In competition studies, mut ants which could not utilize acetamide displaced mutants which could. The p resence of foreign DNA in JeRS 325 resulted in a reduced specific growth ra te (compared to A3/5), but the presence of the foreign DNA did not prevent the evolution of the strain or the isolation of mutants which had improved growth rates. (C) 2001 John Wiley & Sons, Inc.