Abstract
Objectives
To express a Δ6-desaturase gene and produce gamma-linolenic acid (GLA) and stearidonic acid (SDA) in prokaryotic expression system (Escherichia coli), and analyze its substrate specificity in the omega-3 fatty acid biosynthetic pathway.
Results
Full-length ORF (1448 bp) of Δ6Des-Iso was isolated from Isochrysis sp. and characterized using multiple sequence alignment, phylogenetic analysis, transmembrane domain, and protein tertiary structure. Δ6Des-Iso is a front-end desaturase consisting of three conserved histidine domains and a cytochrome b5 domain. Δ6Des-Iso was cloned and expressed in E. coli with the production of GLA and SDA. Recombinant E. coli utilized 27 and 8% of exogenously supplied alpha-linolenic acid (ALA) and linoleic acid (LA) to produce 6.3% of SDA and 2.3% of GLA, respectively, suggesting that isolated Δ6Des-Iso is specific to the omega-3 pathway.
Conclusion
For the first time production of GLA and SDA in a prokaryotic system was achieved.
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References
Amiri-Jami M, Abdelhamid AG, Hazaa M, Kakuda Y, Griffths MW (2015) Recombinant production of omega-3 fatty acids by probiotic Escherichia coli Nissle 1917. FEMS Micobiol Lett 362:fnv166
Cahoon EB, Mills LB, Shanklin J (1996) Modification of the fatty acid composition of Escherichia coli by coexpression of a plant acyl-acyl carrier protein desaturase and ferredoxin. J Bacteriol 178:936–939
Christie WW (1982) Lipid analysis, 2nd edn. Pergamon Press, New York, p 338, ISBN 0-08-017753-0
Eckert H, La Vallee B, Schweiger BJ, Kinney AJ, Cahoon EB, Clemente T (2006) Co-expression of the borage Delta 6 desaturase and the Arabidopsis delta 15 desaturase results in high accumulation of stearidonic acid in the seeds of transgenic soybean. Planta 224:1050–1057
Feng Y, Cronan JE (2009) Escherichia coli unsaturated fatty acid synthesis. Complex transcription of the fabA gene in vivo identification of the essential reaction catalyzed by FabB. J Biol Chem 284:29526–29535
Gill I, Valivety R (1997) Polyunsaturated fatty acids, part 1: occurrence, biological activities and applications. Trends Biotechnol 15:401–409
Iskandarov U, Khozin-Goldberg I, Cohen Z (2010) Identification and characterization of D12, D6, and D5 desaturases from the green microalga Parietochloris incise. Lipids 45:519–530
Jiang X, Liu H, Niu Y, Qi F, Zhang M, Huang J (2017) Functional identification and regulatory analysis of D6-fatty acid desaturase from the oleaginous fungus Mucor sp. EIM-10. Biotechnol Lett 39:453–461
Mishra AK, Muthamilarasan M, Khan Y, Parida SK, Prasad M (2014) Genome-wide investigation and expression analyses of WD40 protein family in the model plant Foxtail Millet (Setaria italica L.). PLoS ONE 9:e86852. https://doi.org/10.1371/journal.pone.0086852
Nykiforuk CL, Shewmaker C, Harry I, Yurchenko OP, Zhang M, Reed C, Oinam GS, Zaplachinski S, Fidantsef A, Boothe JG, Moloney MM (2012) High level accumulation of gamma linolenic acid (C18:3Δ6.9,12 cis) in transgenic safflower (Carthamus tinctorius) seeds. Transgenic Res 21:367–381
Pollak DW, Bostick MW, Yoon H, Wang J, Hollerbach DH, He H, Damude HG, Zhang H, Yadav NS, Hong S-P, Sharpe P, Xue Z, Zhu Q (2012) Isolation of a D5 desaturase gene from Euglena gracilis and functional dissection of its HPGG and HDASH motifs. Lipids 47:913–926
Qi B, Beaudoin F, Fraser T, Stobart AK, Napier JA, Lazarus CM (2002) Identification of a cDNA encoding a novel C18-v9 polyunsaturated fatty acid-specific elongating activity from the docosahexaenoic acid (DHA)-producing microalga, Isochrysis galbana. FEBS Lett 510:159–165
Sayanova O, Smith MA, Lapinskas P, Stobart AK, Dobson G, Christie WW, Shewry PR, Napier JA (1997) Expression of a borage desaturase cDNA containing an N-terminal cytochrome b5 domain results in the accumulation of high levels of D6-desaturated fatty acids in transgenic tobacco. Proc Natl Acad Sci USA 94:4211–4216
Walne PR (1970) Studies on the food value of nineteen genera of algae to juvenile bivalves of the genera Ostrea, Crassostrea, Mercenaria, and Mytilis. Fish Invest 26:1–62
Xue Z, Sharpe PL, Hong SP, Yadav NS, Xie D, Short DR, Damude HG, Rupert RA, Seip JE, Wang J, Pollak DW, Bostick MW, Bosak MD, Macool DJ, Hollerbach DH, Zhang H, Arcilla DM, Bledsoe SA, Croker K, McCord EF, Tyreus BD, Jackson EN, Zhu Q (2013) Production of omega-3 eicosapentaenoic acid by metabolic engineering of Yarrowia lipolytica. Nat Biotechnol 31:734–740
Yokoo EM (2003) Low level methylmercury exposure affects neruophysychological function in adults. Environ Health 2:1–11
Acknowledgement
Authors thank DBT-IPLS, NRCBS, CEGS, DST-PURSE and CAS-Phase III in School of Biological Sciences, MKU for the facilities and the supports provided for the study. This work was supported by a grant from Science and Engineering Research Board, Department of Science and Technology, India (SR/FT/LS-47/2011) and also supported by University Grants Commission, India (UGC.F.No.42–198/2013).
Supporting Information
Supplementary Table 1—List of primers used for isolation and cloning of Δ6-desaturase.
Supplementary Fig. 1—Isolation and cloning of Δ6Des-Iso.
Supplementary Fig. 2—Phylogenetic relationship of Δ6Des-Iso.
Supplementary Fig. 3—Transmembrane Topology model of Δ6Des-Iso.
Supplementary Fig. 4—3D homology model of Δ6Des-Iso.
Supplementary Fig. 5—Protein expression analysis of Δ6Des-Iso in E. coli.
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Thiyagarajan, S., Arumugam, M., Senthil, N. et al. Functional characterization and substrate specificity analysis of Δ6-desaturase from marine microalga Isochrysis sp.. Biotechnol Lett 40, 577–584 (2018). https://doi.org/10.1007/s10529-017-2501-4
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DOI: https://doi.org/10.1007/s10529-017-2501-4