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
- 326 Downloads
The sdhB gene encoding an iron–sulfur (Ip) subunit of succinate dehydrogenase (SDH, EC 22.214.171.124) 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.
- Ohta S, Mita S, Hattori T, Nakamura K (1990) Construction and expression in tobacco of a β-glucuronidase (GUS) reporter gene containing an intron within the coding sequence. Plant Cell Physiol 31:805–813Google Scholar
- Sakuradani E, Takeno S, Abe T, Shimizu S (2005) Arachidonic acid-producing Mortierella alpina: creation of mutants and molecular breeding. In: Cohen Z, Ratledge C (eds) Single cell oils. AOCS Press, Champaign, pp 21–35Google Scholar
- Takeno S, Sakuradani E, Murata S, Inohara-Ochiai M, Kawashima H, Ashikari T, Shimizu S (2004a) Cloning and sequencing of the ura3 and ura5 genes, and isolation and characterization of uracil auxotrophs of the fungus Mortierella alpina 1S-4. Biosci Biotechnol Biochem 68:277–285PubMedCrossRefGoogle Scholar