Transformation of an oleaginous zygomycete <Emphasis Type="Italic">Mortierella alpina</Emphasis> 1S-4 with the carboxin resistance gene conferred by mutation of the iron–sulfur subunit of succinate dehydrogenase |
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Authors: | Akinori Ando Eiji Sakuradani Kota Horinaka Jun Ogawa and Sakayu Shimizu |
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Institution: | (1) Laboratory of Fermentation Physiology and Applied Microbiology, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan;(2) Research Division of Microbial Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan |
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Abstract: | 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 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. |
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Keywords: | Mortierella alpina 1S-4 Transformation Carboxin Succinate dehydrogenase Polyunsaturated fatty acids β -Glucuronidase |
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