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Evolution of a recombinant (gucoamylase‐producing) strain of Fusarium venenatum A3/5 in chemostat culture
Author(s) -
Wiebe Marilyn G.,
Robson Geoffrey D.,
Shuster Jeff,
Trinci Anthony P.J.
Publication year - 2001
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.1046
Subject(s) - chemostat , mutant , biology , population , gibberella fujikuroi , microbiology and biotechnology , strain (injury) , aspergillus niger , biochemistry , genetics , gene , fusarium , bacteria , demography , anatomy , sociology
Fusarium venenatum JeRS 325 is a transformant of strain A3/5 which produces Aspergillus niger glucoamylase (GAM) under the control of a Fusarium oxysporum trypsin‐like protease promoter. The evolution of JeRS 325 was studied in glucose‐limited chemostat cultures grown on NaNO 3 or (NH 4 ) 2 SO 4 as the nitrogen source. Thirteen mutants which were more highly branched and four mutants which were more sparsely branched than the parental strain were isolated from the NaNO 3 chemostat. The highly branched mutants detected in this chemostat did not displace the sparsely branched population. The mutants isolated from the NaNO 3 chemostat complemented representative strains previously isolated from glucose‐limited chemostat cultures of F. venenatum A3/5 grown on (NH 4 ) 2 SO 4 , but showed little complementation between themselves. By contrast, a highly branched mutant isolated from the (NH 4 ) 2 SO 4 chemostat culture displaced the sparsely branched mycelial population. None of the mutants isolated from the NaNO 3 or (NH 4 ) 2 SO 4 chemostats produced as much GAM as JeRS 325. Southern blot analysis showed that all except one mutant had lost copies of both the glucoamylase and the acetamidase (the selectable marker) genes. However, specific GAM production was not necessarily correlated with the extent of glaA gene loss observed. Further, 10 of the mutants had lost the ability to grow on acetamide as the sole nitrogen source, although they retained copies of the amdS gene. In competition studies, mutants which could not utilize acetamide displaced mutants which could. The presence of foreign DNA in JeRS 325 resulted in a reduced specific growth rate (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. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 73: 146–156, 2001.