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KMID : 0381120130350050631
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Genes and Genomics
2013 Volume.35 No. 5 p.631 ~ p.639
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Cloning, expression and characterization of squalene synthase from Inonotus obliquus
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Zheng Fangliang
Liu Nan
Che Yuchen
Zhang Li
Shao Lijie
Zhu Junfeng
Zhao Jian
Ai Haixin
Chang Alan K.
Liu Hongsheng
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Abstract
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The chaga mushroom Inonotus obliquus has been widely used as a folk medicine in Russia, Poland and most of the Baltic countries. The total triterpene saponins of I. obliquus have significant pharmacological activity. Though the triterpene component has been well characterized in terms of its pharmaceutical activity, there is little information on the genes responsible for the biosynthesis of these compounds in I. obliquus. Squalene synthase represents a potential branching point and the first committed step to diverge the carbon flux from the main isoprenoid pathway towards sterol biosynthesis. In this study, we cloned and characterized squalene synthase from I. obliquus. A 1476-bp full-length cDNA consisting of the entire coding region of squalene synthase (GenBank accession number is KC182754) was cloned by RT-PCR. The DNA sequence showed as much as 76 % similarity with the sequence of Fomitiporia mediterranea squalene synthase, and phylogenetic analysis indicated that it is most closely related to F. mediterranea squalene synthase at both DNA and protein levels. I. obliquus squalene synthase was actively expressed in the yeast Pichia pastoris as a secreted form and purified by gel filtration using Superdex G-75 column. The purified recombinant squalene synthase was able to convert farnesyl diphosphate (FPP) to squalene in an NADPH-dependent reaction. The result of this study could serve as an important step toward the manipulation of triterpenoids biosynthesis in I. obliquus at the level of squalene through engineering better SQS for reintroduction into the mushroom.
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KEYWORD
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Inonotus obliquus, Squalene synthase (SQS), Gene expression, Protein purification
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