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Two novel Physcomitrella patens fatty acid elongases (ELOs): identification and functional characterization

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Abstract

The lower plant Physcomitrella patens synthesizes several long-chain polyunsaturated fatty acids (LC-PUFAs) by a series of desaturation and elongation reactions. In the present study, the full-length cDNAs for two novel fatty acid elongases designated PpELO1 and PpELO2 were isolated from P. patens using a PCR-based cloning strategy. These cDNAs encoding proteins of 335 and 280 amino acids with predicted molecular masses of 38.7 and 32.9 kDa, respectively, are predicted to contain seven transmembrane domains with a possible localization in the subcellular endoplasmic reticulum. Sequence comparisons and phylogenetic analysis revealed that they are closely related to other LC-PUFA elongases of the lower eukaryotes such as the Δ5- and Δ6-elongases of Marchantia polymorpha as well as the Δ6-elongase of P. patens. Heterologous expression of the PpELO1 in Saccharomyces cerevisiae led to the elongation of Δ9-, Δ6-C18, and Δ5-C20 LC-PUFAs, whereas only Δ9- and Δ6-C18 LC-PUFA substrates were used by PpELO2. Chimeric proteins were constructed to identify the amino acid regions most likely to be involved in the determination of the fatty acid substrate specificity. The expression of eight chimeric proteins in yeast revealed that substitution of the C-terminal 50 amino acids from PpELO1 into PpELO2 resulted in a high specificity for C20 fatty acid substrates. As a result, we suggest that the C-terminal region of PpELO1 is sufficient for C20 substrate elongation. Overall, these results provide important insights into the structural basis for substrate specificity of PUFA-generating ELO enzymes.

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Acknowledgments

This work was granted by the Office of the Higher Education Commission, Thailand, to Ms. Pradinunt Eiamsa-ard under the program Strategic Scholarships for Frontier Research Network for the Ph.D. Program Thai Doctoral degree, Grant No. 80/2551 and the Graduated School, Prince of Songkla University (PSU), Thailand. The research fund was also supported by PSU (PHA530058S) and the Higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission (PHA5405395) and the Marine Natural Products Research Unit (MNP) at the Faculty of Pharmaceutical Sciences, PSU. We gratefully acknowledge the Faculty of Agro-Industry and Faculty of Pharmaceutical Sciences at PSU for partial funding and providing the laboratory facilities. Also thanks to Ms. Rebecca E. Cahoon at the University of Nebraska and Dr. Brian Hodgson of PSU for assistance with the English.

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Authors and Affiliations

  1. Department of Industrial Biotechnology, Faculty of Agro-Industry, Prince of Songkla University, Songkhla, 90112, Thailand

    Pradinunt Eiamsa-ard & Akkharawit Kanjana-Opas

  2. Center for Plant Science Innovation and Department of Biochemistry, University of Nebraska-Lincoln, Lincoln, NE, 68588, USA

    Edgar B. Cahoon

  3. Marine Natural Products Research Unit (MNP), Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Songkhla, 90112, Thailand

    Pichit Chodok & Sireewan Kaewsuwan

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  1. Pradinunt Eiamsa-ard
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  2. Akkharawit Kanjana-Opas
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Correspondence to Sireewan Kaewsuwan.

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Eiamsa-ard, P., Kanjana-Opas, A., Cahoon, E.B. et al. Two novel Physcomitrella patens fatty acid elongases (ELOs): identification and functional characterization. Appl Microbiol Biotechnol 97, 3485–3497 (2013). https://doi.org/10.1007/s00253-012-4556-4

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