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Identification and Functional Characterization of Two Novel Fatty Acid Genes from Marine Microalgae for Eicosapentaenoic Acid Production

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Abstract

Marine microalgae such as Isochrysis sp. and Pavlova sp. are the predominant source of polyunsaturated fatty acids (PUFAs) such as eicosapentaenoic acid (EPA, 20:5n–3) and docosahexaenoic acid (DHA, 22:6n–3). EPA biosynthesis pathway is predominant in lower eukaryotes, and its biosynthetic gene expressions are not well established. Till date, the C18 elongation enzymes for EPA biosynthesis have not been identified from lower eukaryote. In the present study, we describe the identification of two microalgal genes Δ6-elongase and Δ5-desaturase involved for EPA biosynthesis. By PCR-based technique, a novel elongase gene (Δ6Elo-Iso) was isolated from Isochrysis sp., and 654 bp of full-length sequence was identified, which catalysed the conversion of SDA into ETr in E. coli. The identified gene displayed unique substrate specificity for both n-3 and n-6 C18-substrates for Δ6-elongation, with no activity towards Δ9-elongase. In addition, a novel Δ5-desaturase gene (Δ5Des-Pav) was isolated from Pavlova sp. and found an ORF of 1149 bp in length, which was capable of converting ETr into EPA in omega-3 pathway. For the first time, the heterologous expressions of two novel microalgal genes were successfully expressed in Escherichia coli. EPA production from E. coli is being considered as an alternative and economic source for industrial and pharmaceutical sectors.

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Acknowledgements

This work was supported by grants from Science and Engineering Research Board (SERB), Government of India (ECR/2017/002914, SB/EMEQ-219/2014). The authors thank DBT-IPLS, NRCBS, CEGS, DST-PURSE, UGC-CAS Phase III, School of Biological Sciences, and Madurai Kamaraj University, Tamil Nadu, India, for the instrumentation facilities and the supports provided for this study.

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Authors and Affiliations

  1. Department of Molecular Biology, School of Biological Sciences, Madurai Kamaraj University, Madurai, Tamil Nadu, 625021, India

    Thiyagarajan S &  Kathiresan S

  2. Microbial Processes and Technology Division, National Institute for Interdisciplinary Science and Technology (NIIST), Council of Scientific and Industrial Research (CSIR), Trivandrum, Kerala, 695 019, India

    Arumugam M

  3. Academy of Scientific and Innovative Research (AcSIR), New Delhi, 110001, India

    Arumugam M

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  1. Thiyagarajan S
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Correspondence to Kathiresan S.

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Fig. S1

Cloning and expression of Δ6-elongase gene from Isochrysis sp. and the PCR amplification were found at 0.65 kb in size (a), TA cloning of Δ6Elo-Iso in pXCM vector was confirmed by BamH1 restriction digestion (b), and colony PCR (c) showed gene of interest at 0.65 kb. For gene expression, Δ6Elo-Iso cloned into pGEX-4T2, and recombinant clone was confirmed by BamH1/EcoR1 restriction digestion (d) and colony PCR (e) respectively (JPG 50 kb)

Fig. S2

Phylogenetic tree analysis of Δ6-elongase and Δ5-desaturase gene from microalgae Isochrysis sp. and Pavlova sp. The tree was constructed by Mega 6.1 and neighbour-joining method with the other available elongases and desaturases sequences from different organisms. The identified genes of Δ6Elo-Iso from Isochrysis sp. (a) and Δ5Des-Pav from Pavlova sp. (b) are represented as * (JPG 62 kb)

Fig. S3

Protein expression analysis of Δ6Elo-Iso in E. coli and the overexpressed protein were found at 50 kDa in size (Lane 2, 3) than wild type pGEX-4T2 (Lane 1) (JPG 33 kb)

Fig. S4

Cloning and expression of Δ5-desaturase gene from Pavlova sp. and the PCR amplification were found at 1.2 kb in size (a), TA cloning in pGEMT-easy vector was confirmed by EcoR1 restriction digestion (b), and colony PCR (c) showed gene of interest at 1.2 kb. For gene expression, Δ5Des-Pav cloned into pGEX-4T2, and recombinant clone was confirmed by EcoR1/Sal1 restriction digestion (d) and colony PCR (e) respectively (JPG 72 kb)

Fig. S5

Protein 3D homology model for Δ5Des-Pav was developed by Phyre tool. (a) The 3D view for human strearoyl coA-desaturase2 has shared 99.9% confidence, and (b) 3D view for cytochrome b5 has shared 99.7% confidence to the newly identified Δ5Des-Pav (JPG 64 kb)

Fig. S6

Protein expression analysis of Δ5Des-Pav in E. coli and the overexpressed protein were found at 70 kDa in size (Lane 4–6) than wild type pGEX-4T2 (Lane 1) (JPG 31 kb)

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Thiyagarajan S, Arumugam M & Kathiresan S Identification and Functional Characterization of Two Novel Fatty Acid Genes from Marine Microalgae for Eicosapentaenoic Acid Production. Appl Biochem Biotechnol 190, 1371–1384 (2020). https://doi.org/10.1007/s12010-019-03176-x

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