Transformation of an oleaginous zygomycete Mortierella alpina 1S-4 with the carboxin resistance gene conferred by mutation of the iron-sulfur subunit of succinate dehydrogenase.
The sdhB gene encoding an iron-sulfur (Ip) subunit of succinate dehydrogenase (SDH, EC 1.3.99.1) complex was cloned from Mortierella alpina 1S-4. The deduced amino acid sequence of SdhB from M. alpina 1S-4 showed high similarity to those of SdhB from other organisms. The mutated sdhB (CBXB) gene encodes a modified SdhB with an amino-acid substitution (a highly conserved histidine residue within the third cysteine-rich cluster of SdhB replaced by a leucine residue) and is known to confer carboxin resistance. We succeeded in transforming M. alpina 1S-4 by using the CBXB gene as a selectable marker gene and expressing the heterologous uidA gene encoding beta-glucuronidase of Escherichia coli. Moreover, transformation efficiency was up to 40-50 transformants per 4.0 x 10(8) spores. This carboxin-transformation system, characterized by marginal background growth and mitotic stability in M. alpina 1S-4, is considered to be widely useful for the wild strain, M. alpina 1S-4, and various derivative mutants without laborious preparation of auxotrophic mutants as a host strain.
Ando
Akinori
A
Kyoto University, Sakyo-ku, Japan. aando@kais.kyoto-u.ac.jp
Sakuradani
Eiji
E
Horinaka
Kota
K
Ogawa
Jun
J
Shimizu
Sakayu
S
D000595:Amino Acid Sequence; D015139:Blotting, Southern; D002261:Carboxin / Q000494:pharmacology*; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D025141:Drug Resistance, Fungal / Q000235:genetics; D005659:Fungicides, Industrial / Q000494:pharmacology; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D020094:Mortierella / Q000201:enzymology / Q000235:genetics*; D009154:Mutation; D021122:Protein Subunits / Q000235:genetics; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013385:Succinate Dehydrogenase / Q000235:genetics*; D014170:Transformation, Genetic
D004271:DNA, Fungal; D005659:Fungicides, Industrial; D021122:Protein Subunits; D002261:Carboxin; D013385:Succinate Dehydrogenase; D005966:Glucuronidase