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Cloning and characterization of a GPAT-like gene from the microalga Lobosphaera incisa (Trebouxiophyceae): overexpression in Chlamydomonas reinhardtii enhances TAG production

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Abstract

The oleaginous green microalga Lobosphaera (formerly Parietochloris) incisa accumulates high amounts of arachidonic-acid-rich triacylglycerols (TAG), in particular under conditions of nitrogen starvation. This photosynthetic organism is of great interest for studying the mechanisms responsible for storage lipid biosynthesis and the deposition of long-chain polyunsaturated fatty acids (LC-PUFA) in TAG. In this work, we report on cloning a complementary DNA (cDNA) for the putative L. incisa glycerol-3-phosphate acyltransferase (GPAT), whose deduced amino acid sequence features distinctive motives found in those mammalian and Arabidopsis GPAT isoforms that have been implicated in TAG biosynthesis. Temporal analysis of LiGPAT expression in the course of nitrogen starvation showed a positive relationship between changes in the transcript level and patterns of fatty acid production. When expressed in Arabidopsis leaf mesophyll protoplasts, the green fluorescent protein (GFP) fused to the C-terminus of LiGPAT localized outside the chloroplasts in agreement with its predicted extraplastidial localization. Based on an in silico analysis of a deduced amino acid sequence and on similarity to other GPATs participating in TAG biosynthesis, LiGPAT was expressed in the green model microalga Chlamydomonas reinhardtii in order to confirm the predicted function in a heterologous microalgal system. Overexpression of LiGPAT resulted in an up to 50 % increase in the content of TAG on a cell dry weight basis as compared to the control in the stationary phase culture without negative impact on growth parameters. Total fatty acids and TAG of the transformant lines featured an elevated level of oleic acid (18:1 n-9) and a concurrent decrease in C18 PUFA. Additional studies on the acyl substrate preference of LiGPAT are required.

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Acknowledgments

This research was financially supported by the European Commission’s Seventh Framework Program for Research and Technology Development (FP7), project GIAVAP, grant no. 266401. The support of the Kreitman School of Advanced Graduate Studies and the Albert Katz International School for Desert Studies at the Ben-Gurion University of the Negev is gratefully acknowledged. The authors would like to thank Dr. Neeru Jain and Dr. Ilkhom Khalilov for their valuable contributions at the early stages of the research, and Prof. Gideon Grafi, Dr. Simon Barak, and Dr. Assif Khan for their helpful advice in the experiments with Arabidopsis protoplasts.

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  1. Umidjon Iskandarov

    Present address: Center for Plant Science Innovation and Department of Biochemistry, University of Nebraska—Lincoln, Lincoln, NE, 68588, USA

  2. Siarhei Sitnik

    Present address: Institute of Molecular Virology, Center for Molecular Biology of Inflammation (ZMBE), Westfälische Wilhelms-Universität Münster, Münster, 48149, Germany

Authors and Affiliations

  1. Microalgal Biotechnology Laboratory, French Associates Institute for Agriculture and Biotechnology of Drylands, J. Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, Midreshet Ben-Gurion, 84990, Israel

    Umidjon Iskandarov, Siarhei Sitnik, Nastassia Shtaida, Shoshana Didi-Cohen, Stefan Leu, Inna Khozin-Goldberg, Zvi Cohen & Sammy Boussiba

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  1. Umidjon Iskandarov
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Iskandarov, U., Sitnik, S., Shtaida, N. et al. Cloning and characterization of a GPAT-like gene from the microalga Lobosphaera incisa (Trebouxiophyceae): overexpression in Chlamydomonas reinhardtii enhances TAG production. J Appl Phycol 28, 907–919 (2016). https://doi.org/10.1007/s10811-015-0634-1

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