Molecular characterization of a glycerol-3-phosphate acyltransferase reveals key features essential for triacylglycerol production in Phaeodactylum tricornutum

Niu et al. Biotechnol Biofuels (2016) 9:60 DOI 10.1186/s13068-016-0478-1 Biotechnology for Biofuels Open Access RESEARCH Molecular characterization of a glycerol‑3‑phosphate acyltransferase reveals key features essential for triacylglycerol production in Phaeodactylum tricornutum Ying‑Fang Niu1,2†, Xiang Wang1†, Dong‑Xiong Hu1, Srinivasan Balamurugan1, Da‑Wei Li1, Wei‑Dong Yang1, Jie‑Sheng Liu1 and Hong‑Ye Li1* Abstract Background: The marine diatom, Phaeodactylum tricornutum, has become a model for studying lipid metabolism and its triacylglycerol (TAG) synthesis pathway makes it an ideal target for metabolic engineering to improve lipid productivity. However, the genetic background and metabolic networks of fatty acid biosynthesis in diatoms are not well understood. Glycerol-3-phosphate acyltransferase (GPAT) is the critical enzyme that catalyzes the first step of TAG formation. So far, characterization of GPAT in marine microalgae has not been reported, especially at the level of comprehensive sequence-structure and functional analysis. Results: A GPAT was cloned from P. tricornutum and overexpressed in P. tricornutum. Volumes of oil bodies were produced and the neutral lipid content was increased by twofold determined by Nile red fluorescence staining. Fatty acid composition was analyzed by GC–MS, which showed significantly higher proportion of unsaturated fatty acids compared to wild type. Conclusion: These results suggested that the identified GPAT could upregulate TAG biosynthesis in P. tricornutum. Moreover, this study offers insight into the lipid metabolism of diatoms and supports the role of microalgal strains for biofuels production. Keywords: GPAT, Lipid, Diatoms, Biofuels Background Renewable energy is one of the most effective solutions to address the carbon emission, energy security, and increased fuel consumption challenges that result in global warming and fossil fuel price concerns. These issues have prompted intensive interest in the capability of oleaginous microalgae to generate renewable oil sources which can be readily converted into biodiesel [1]. Microalgae can accumulate oil and are considered *Correspondence: † Ying-Fang Niu and Xiang Wang contributed equally to this work 1 Key Laboratory of Eutrophication and Red Tide Prevention of Guangdong Higher Education Institutes, College of Life Science, Jinan University, Guangzhou 510632, China Full list of author information is available at the end of the article to be a promising feedstock for renewable biofuel production. For example, microalgae demonstrate much higher biomass productivity compared to higher plants and algal growth facilities could potentially be located in aquatic environments, which will not increase arable land concerns. Although microalgae have reemerged as a potential 3rd generation feedstock for biofuel production, large-scale harvesting of microalgae is hampered by the lack of algal strains that can be selectively optimized for both high biomass generation capability and high TAG content [2]. One potential solution is to engineer robust oil-yielding microalgae by expressing the critical enzymes for TAG accumulation and to acquire a better overall understanding of lipid metabolic pathways in microalgae [3, 4]. © 2016 Niu et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/ zero/1.0/) applies to the data made available in this article, unless otherwise stated. Niu et al. Biotechnol Biofuels (2016) 9:60 In eukaryotes, TAGs are identified as neutral lipids that serve as the crucial storage form of energy. TAGs are the major feedstock for biodiesel production. TAG accumulation in microalgae is usually correlated to environmental stresses, such as high light intensity, high temperature, nitrogen limitation, and salinity [5, 6]. There are three major steps involved in TAG synthesis. Firstly, carboxylation of acetyl-CoA to form malonyl-CoA, which is the committing step of fatty acid biosynthesis in the plastid, secondly, acyl chain elongation in the plastid and cytosol, and finally, TAG formation in the endoplasmic reticulum (ER) [4]. The biosynthesis of fatty acids in chloroplast is catalyzed by two major, evolutionarily conserved enzymes, acetyl-CoA carboxylase (ACCase) and fatty acid synthase (FAS). The synthesized fatty acids are then esterified by glycerol-3-phosphate acyltransferase (GPAT) to glycerol 3-phosphate at the sn-1 position to form lysophosphatidic acid (LysoPA) [5, 7]. LysoPA is further catalyzed into phosphatidic acid (PA) by lysophosphatidic acid acyltransferase (LPAAT). The PA is then dephosphorylated by phosphatidic acid phosphatase (PAP) to form diacylglycerol (DAG). It has been reported that PA and DAG can also be formed in the chloroplast, where they serve as precursors for the synthesis of structural membrane lipids and neutral lipids [8, 9]. GPAT (EC 2.3.1.15) is considered as the initial enzyme for glycerolipid synthesis. In mammals, four GPAT isoforms have been identified [10–12]. In rats, hepatic glycerol-sn-3-phosphate acyltransferase 1 was overexpressed and caused hepatic insulin resistance, suggesting a role for lipid metabolites in the development of insulin resistance [7]. In humans, multiple isoforms of GPAT were expressed and differentially regulated in epidermis/ keratinocytes [13]. In the model dicot plant, Arabidopsis thaliana, ten GPAT isoforms have been reported recently [14, 15]. These ten genes can be divided into three clusters. The first cluster is plastid-localized GPAT, which uses acyl-ACP substrates, and exhibits sn-1 acyl transfer regiospecificity [16]. The second cluster is GPAT9, which is located in the endoplasmic reticulum, and is enable to synthesize non-plastid glycerolipid [17]. The remaining eight GPATs do not play a role in the Kennedy pathway [14, 17]. Daubossy et al. reported that disruption of the UDP-glucose pyrophosphorylase gene in Phaeodactylum tricornutum resulted in increased TAG accumulation [18]. In our previous report, we successfully developed transgenic P. tricornutum with increased lipid accumulation by overexpressing type 2 diacylglycerol acyl transferase (DGAT) [19]. Similarly, overexpression of DGAT in P. tricornutum resulted in increased proportion of polyunsaturated fatty acids [20, 21]. However, no GPAT has been characterized in microalgae. Report of research employing metabolic engineering to increase lipid Page 2 of 11 productivity in microalgae has been limited to date. In the present study, we first cloned a putative GPAT from the oleaginous marine dia (...truncated)