Abstract
Objectives
To enlarge the diversity of the desaturases associated with PUFA biosynthesis and to better understand the transcriptional regulation of desaturases, a Δ6-desaturase gene (Md6) from Mucor sp. and its 5′-upstream sequence was functionally identified in Saccharomyces cerevisiae.
Results
Expression of the Δ6-fatty acid desaturase (Md6) in S. cerevisiae showed that Md6 could convert linolenic acid to γ-linolenic acid. Computational analysis of the promoter of Md6 suggested it contains several eukaryotic fundamental transcription regulatory elements. In vivo functional analysis of the promoter showed the 5′-upstream sequence of Md6 could initiate expression of GFP and Md6 itself in S. cerevisiae. A series deletion analysis of the promoter suggested that sequence between −919 to −784 bp (relative to start site) named as eMd6 is the key factor for high activity of Δ6-desaturase. The activity of Δ6-desaturase was increased by 2.8-fold and 2.5-fold when the eMd6 sequence was placed upstream of −434 with forward or reverse orientations respectively.
Conclusion
To our best knowledge, the native promoter of Md6 from Mucor is the strongest promoter for Δ6-desaturase reported so far and the sequence between −919 to −784 bp is an enhancer for Δ6-desaturase activity.
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Acknowledgements
This work was supported by National Natural Science Foundation of China (Grant No. 30970047) and Natural Science Foundation of Fujian, China (Grant No. 2016J01147).
Supporting information
Supplementary Table 1—Primers used in the study.
Supplementary Table 2—Plasmid used in the study.
Supplementary Table 3—Conversion efficiency of typical Δ6 desaturases.
Supplementary Fig. 1—Multiple sequence alignment of the Δ6-desaturase using ClustalX.
Supplementary Fig. 2—Hydropathy analysis of Mucor sp. EIM-10 Δ6-desaturase.
Supplementary Fig. 3—Mass identification of the novel peak.
Supplementary Fig. 4—Phylogenetic analysis of promoter sequence of Δ6-desaturase gene.
Supplementary Fig. 5—Functional characterization of Δ6-desaturase promoter by GFP.
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The original version of this article was revised: the “Δ6-fatty acid desaturase” in the article title has been corrected.
An erratum to this article is available at http://dx.doi.org/10.1007/s10529-016-2284-z.
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Jiang, X., Liu, H., Niu, Y. et al. Functional identification and regulatory analysis of Δ6-fatty acid desaturase from the oleaginous fungus Mucor sp. EIM-10. Biotechnol Lett 39, 453–461 (2017). https://doi.org/10.1007/s10529-016-2268-z
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DOI: https://doi.org/10.1007/s10529-016-2268-z