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Cloning and Functional Identification of Delta5 Fatty Acid Desaturase Gene and Its 5′-Upstream Region from Marine Fungus Thraustochytrium sp. FJN-10

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Abstract

A gene encoding delta5 fatty acid desaturase (fad5) was cloned from marine fungus Thraustochytrium sp. FJN-10, a species capable of producing docosahexaenoic acid. The open reading frame of fad5 was 1,320 bp and encoded a protein comprising 439 amino acids. Expression of the fad5 in Saccharomyces cerevisiae INVSC1 revealed that FAD5 is able to introduce a double bond at position 5 of the dihomo-γ-linolenic acid (20:3 Δ8,11,14), resulting in arachidonic acid (20:4 Δ5,8,11,14) with a conversion rate of 56.40% which is the highest among engineering yeasts reported so far. The 5′-upstream region of fad5 was cloned by LA-PCR and analyzed. Phylogenetic analysis of this sequence with the 5′-upstream region of other delta5 desaturases showed that the 5′-upstream region of fad5 from Thraustochytrium share the smallest evolution distance with human and rhesus. Computational analysis of the nucleotide sequence of the 5′-upstream region of fad5 has revealed several basic transcriptional elements including five TATA boxes, three CCAAT boxes, 12 GC boxes, and several putative target-binding sites for transcription factors such as HSF, CAP, and ADR1. Preliminary functional analysis of this promoter in S. cerevisiae shows that the 5′-upstream region of fad5 could drive the expression of green fluorescent protein.

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Acknowledgments

This work is supported by National Natural Science Foundation of China, No. 30370028 and 30970047, the Natural Science Foundation of Fujian, China (General and major Program), No. 2003F005, 2008F3036, and The Project of Fujian Development and Reform Commission, No. [2005]847, 2005Q007.

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Authors and Affiliations

  1. Engineering Research Center of Industrial Microbiology, Ministry of Education, Fujian Normal University, Fuzhou, Fujian, 350108, People’s Republic of China

    Jian-Zhong Huang, Xian-Zhang Jiang, Xiao-Feng Xia, Ai-Qun Yu, Ruo-Yu Mao, Xiao-Feng Chen & Bao-Yu Tian

  2. Engineering Research Center of Fujian Modern Fermentation Technology, Fujian Normal University, Fuzhou, Fujian, 350108, People’s Republic of China

    Jian-Zhong Huang, Xian-Zhang Jiang, Xiao-Feng Xia, Ai-Qun Yu, Ruo-Yu Mao, Xiao-Feng Chen & Bao-Yu Tian

  3. College of Life Sciences, Fujian Normal University (New Campus), Shangjie Town, Minhou District, Fuzhou, Fujian, 350108, People’s Republic of China

    Jian-Zhong Huang, Xian-Zhang Jiang, Xiao-Feng Xia, Ai-Qun Yu, Ruo-Yu Mao, Xiao-Feng Chen & Bao-Yu Tian

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  1. Jian-Zhong Huang
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  2. Xian-Zhang Jiang
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Correspondence to Jian-Zhong Huang.

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Jian-Zhong Huang, Xian-Zhang Jiang, and Xiao-Feng Xia contributed equally to this work.

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Huang, JZ., Jiang, XZ., Xia, XF. et al. Cloning and Functional Identification of Delta5 Fatty Acid Desaturase Gene and Its 5′-Upstream Region from Marine Fungus Thraustochytrium sp. FJN-10. Mar Biotechnol 13, 12–21 (2011). https://doi.org/10.1007/s10126-010-9262-6

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