Enhanced triacylglycerol production in the diatom Phaeodactylum tricornutum by inactivation of a Hotdog-fold thioesterase gene using TALEN-based targeted mutagenesis

(2018) 11:312 Hao et al. Biotechnol Biofuels https://doi.org/10.1186/s13068-018-1309-3 Biotechnology for Biofuels Open Access RESEARCH Enhanced triacylglycerol production in the diatom Phaeodactylum tricornutum by inactivation of a Hotdog‑fold thioesterase gene using TALEN‑based targeted mutagenesis Xiahui Hao1,2, Ling Luo1,2, Juliette Jouhet3, Fabrice Rébeillé3, Eric Maréchal3, Hanhua Hu4, Yufang Pan4, Xiaoming Tan5, Zhuo Chen6, Lingjie You1,2, Hong Chen1,2, Fang Wei1,2 and Yangmin Gong1,2* Abstract Background: In photosynthetic oleaginous microalgae, acyl-CoA molecules are used as substrates for the biosynthesis of membrane glycerolipids, triacylglycerol (TAG) and other acylated molecules. Acyl-CoA can also be directed to beta-oxidative catabolism. They can be utilized by a number of lipid metabolic enzymes including endogenous thioesterases, which catalyze their hydrolysis to release free fatty acids. Acyl-CoA availability thus plays fundamental roles in determining the quantity and composition of membrane lipids and storage lipids. Results: Here, we have engineered the model diatom Phaeodactylum tricornutum to produce significantly increased TAGs by disruption of the gene encoding a Hotdog-fold thioesterase involved in acyl-CoA hydrolysis (ptTES1). This plastidial thioesterase can hydrolyze both medium- and long-chain fatty acyl-CoAs, but has the highest activity toward long-chain saturated and monounsaturated fatty acyl-CoAs. The maximum rate was found with oleoyl-CoA, which is hydrolyzed at 50 nmol/min/mg protein. The stable and targeted interruption of acyl-CoA thioesterase gene was achieved using a genome editing technique, transcription activator-like effector nucleases (TALENs). Disruption of native ptTES1 gene resulted in a 1.7-fold increase in TAG content when algal strains were grown in nitrogen-replete media for 8 days, whereas the content of other lipid classes, including phosphoglycerolipids and galactoglycerolipids, remained almost unchanged. The engineered algal strain also exhibited a marked change in fatty acid profile, including a remarkable increase in 16:0 and 16:1 and a decrease in 20:5. Nitrogen deprivation for 72 h further increased TAG content and titer of the engineered strain, reaching 478 μg/109 cells and 4.8 mg/L, respectively. Quantitative determination of in vivo acyl-CoAs showed that the total acyl-CoA pool size was significantly higher in the engineered algal strain than that in the wild type. Conclusions: This study supports the role of ptTES1 in free fatty acid homeostasis in the plastid of Phaeodactylum and demonstrates the potential of TALEN-based genome editing technique to generate an enhanced lipid-producing algal strain through blocking acyl-CoA catabolism. Keywords: Acyl-CoA thioesterase, Acyl-CoA, Fatty acids, Phaeodactylum tricornutum, TALEN, Triacylglycerols *Correspondence: 1 Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Oil Crops Research Institute of Chinese Academy of Agricultural Sciences, No. 2 Xudong Second Road, Wuhan 430062, People’s Republic of China Full list of author information is available at the end of the article © The Author(s) 2018. 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. Hao et al. Biotechnol Biofuels (2018) 11:312 Background Some photosynthetic microalgae synthesize and accumulate large amounts of triacylglycerols (TAGs) and deposit them into lipid droplets (LDs) under stress conditions, especially nutrient deprivation. As one of energy dense compounds, these biologically synthesized TAG molecules have gained great attention owing to the potential to be used as liquid biofuel feedstock [1, 2]. Up to now, despite its technological feasibility, microalgae-to-biofuel technology remains significantly challenging and economic viability is hampered by energetic costs and sustainability issues. One major hurdle is the low biomass and oil productivities of the selected algal strains. Engineering and optimization of algal strains and metabolic pathways provide a powerful tool and efficient strategy to increase oil productivity, with the promise of advancing the research and development of economically feasible algae-to-biofuel technology [3]. Over the past 10 years, the oleaginous diatom Phaeodactylum tricornutum has been used as a microalgal model to engineer lipid metabolism. Some important advances include increased accumulation of shorter chain length fatty acids by heterologous expression of plant-derived fatty acyl-ACP thioesterases [4], and enhanced accumulation of high-value omega-3 longchain polyunsaturated fatty acids by expression of heterologous desaturase and elongase [5]. In addition, enhanced lipid production was achieved via heterologous co-expression of a yeast diacylglycerol acyltransferase and a plant oleosin [6], overexpression of malic enzyme [7], glycerol-3-phosphate acyltransferase [8, 9], targeted knockdown of phosphoenolpyruvate carboxykinase [10], disruption of the UDP-glucose pyrophosphorylase gene [11], overexpression of a gene involved in nitric oxide emission via the by-production of fumaric acid [12] and inhibition of acetyl-CoA utilization for sterol metabolism [13, 14]. These studies for enhancing lipid production in P. tricornutum can be classified into two different approaches: (1) overexpressing enzymes, especially acyltransferases, to increase acyl flux being channeled to TAG synthesis pathway or enhance reductant (NADPH) supply for lipogenic enzymes; (2) inhibition of or blocking off competing pathway to decrease lipid catabolism or increase substrate supply. In eukaryotic microalgae, there are two primary metabolic pathways for TAG biosynthesis: acyl-CoA-dependent pathway and acyl-CoA-independent pathway. In the first pathway, which is also known as the Kennedy pathway, three molecules of acyl-CoAs are used as substrates for successive acylation of glycerol backbone at sn-1, -2 and -3 positions by three distinct acyltransferases, leading to the formation of one molecule of TAG. The final and committed step of TAG biosynthesis is catalyzed by Page 2 of 18 acyl-CoA:diacylglycerol acyltransferase (DGAT). In acylCoA-independent pathway, phospholipid:diacylglycerol acyltransferase (PDAT) catalyzes the transfer of a fatty acyl moiety from the sn-2 position of phosphatidylcholine (PC) to the sn-3 position of DAG to form TAG and sn-1 lyso-PC. DGAT is considered a key enzyme for biotechnological purpo (...truncated)