Adenine auxotrophic mutants of Aspergillus oryzae: development of a novel transformation system with triple auxotrophic hosts.
adeA and adeB genes homologous to Saccharomyces cerevisiae ADE1 and ADE2, respectively, were cloned from Aspergillus oryzae. AdeA and AdeB share 62.8% and 52.5% identities with S. cerevisiae Ade1 and Ade2, respectively. In order to obtain triple auxotrophic mutants from A. oryzae, 12 red-colored mutant colonies were isolated by UV mutagenesis of a double auxotrophic host, NS4 (niaD(-), sC(-)), as a parent strain. All the mutants exhibited adenine auxotrophy and showed fluorescence in the vacuoles due to accumulation of a purine biosynthetic pathway precursor. Adenine auxotrophy of all the mutants was restored by introduction of either A. oryzae adeA or adeB genes. Sequence analysis demonstrated that substitutions or deletions of a single base pair occurred, inducing substitutions or frame shifts of amino acid sequences in both ade genes complementing the mutants. This study provides a novel host-vector system with triple auxotrophy in A. oryzae.
Jin
Feng Jie
FJ
Department of Biotechnology, The University of Tokyo, Japan.
Maruyama
Jun-ichi
J
Juvvadi
Praveen Rao
PR
Arioka
Manabu
M
Kitamoto
Katsuhiko
K
D000225:Adenine / Q000096:biosynthesis*; D000595:Amino Acid Sequence; D001236:Aspergillus oryzae / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D020868:Gene Silencing; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D014170:Transformation, Genetic; D014466:Ultraviolet Rays; D014617:Vacuoles / Q000648:ultrastructure
D000225:Adenine