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 184.108.40.206) 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 β-glucuronidase of Escherichia coli. Moreover, transformation efficiency was up to 40–50 transformants per 4.0 × 108 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.
KeywordsMortierella alpina 1S-4 Transformation Carboxin Succinate dehydrogenase Polyunsaturated fatty acids β-Glucuronidase
This work was partially supported by the Project for the Development of a Technological Infrastructure for Industrial Bioprocesses on R&D of New Industrial Science and Technology Frontiers (to S.S.), the Industrial Technology Research Grant Program (No. 05A07003d to E.S.) of the New Energy and Industrial Technology Development Organization, Japan, Grants-in-Aid for Scientific Research (No. 16688004 to J.O. and No. 19688006 to E.S.), and COE for Microbial-Process Development Pioneering Future Production Systems (to S.S.) from the Ministry of Education, Science, Sports, and Culture, Japan.
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