Enhanced triacylglycerol production in the diatom Phaeodactylum tricornutum by inactivation of a Hotdog-fold thioesterase gene using TALEN-based targeted mutagenesis
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 l...
| Main Authors: | , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2018-11-01
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| Series: | Biotechnology for Biofuels |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s13068-018-1309-3 |
| _version_ | 1818264281532071936 |
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| author | Xiahui Hao Ling Luo Juliette Jouhet Fabrice Rébeillé Eric Maréchal Hanhua Hu Yufang Pan Xiaoming Tan Zhuo Chen Lingjie You Hong Chen Fang Wei Yangmin Gong |
| author_facet | Xiahui Hao Ling Luo Juliette Jouhet Fabrice Rébeillé Eric Maréchal Hanhua Hu Yufang Pan Xiaoming Tan Zhuo Chen Lingjie You Hong Chen Fang Wei Yangmin Gong |
| author_sort | Xiahui Hao |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-12T19:32:25Z |
| format | Article |
| id | doaj.art-edbb143f8f3d41d6a46388dad5c9649a |
| institution | Directory Open Access Journal |
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| language | English |
| last_indexed | 2024-12-12T19:32:25Z |
| publishDate | 2018-11-01 |
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| series | Biotechnology for Biofuels |
| spelling | doaj.art-edbb143f8f3d41d6a46388dad5c9649a2022-12-22T00:14:23ZengBMCBiotechnology for Biofuels1754-68342018-11-0111111810.1186/s13068-018-1309-3Enhanced triacylglycerol production in the diatom Phaeodactylum tricornutum by inactivation of a Hotdog-fold thioesterase gene using TALEN-based targeted mutagenesisXiahui Hao0Ling Luo1Juliette Jouhet2Fabrice Rébeillé3Eric Maréchal4Hanhua Hu5Yufang Pan6Xiaoming Tan7Zhuo Chen8Lingjie You9Hong Chen10Fang Wei11Yangmin Gong12Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Oil Crops Research Institute of Chinese Academy of Agricultural SciencesKey Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Oil Crops Research Institute of Chinese Academy of Agricultural SciencesLaboratoire de Physiologie Cellulaire et Végétale, Centre National de la Recherche Scientifique, Commissariat à l’Energie Atomique et aux Energies Alternatives, Institut National de la Recherche Agronomique, Université Grenoble Alpes, UMR 5168Laboratoire de Physiologie Cellulaire et Végétale, Centre National de la Recherche Scientifique, Commissariat à l’Energie Atomique et aux Energies Alternatives, Institut National de la Recherche Agronomique, Université Grenoble Alpes, UMR 5168Laboratoire de Physiologie Cellulaire et Végétale, Centre National de la Recherche Scientifique, Commissariat à l’Energie Atomique et aux Energies Alternatives, Institut National de la Recherche Agronomique, Université Grenoble Alpes, UMR 5168Key Laboratory of Algal Biology, Institute of Hydrobiology, Chinese Academy of SciencesKey Laboratory of Algal Biology, Institute of Hydrobiology, Chinese Academy of SciencesHubei Collaborative Innovation Center for Green Transformation of Bioresources, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei UniversityShandong Provincial Key Laboratory of Plant Stress, College of Life Science, Shandong Normal UniversityKey Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Oil Crops Research Institute of Chinese Academy of Agricultural SciencesKey Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Oil Crops Research Institute of Chinese Academy of Agricultural SciencesKey Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Oil Crops Research Institute of Chinese Academy of Agricultural SciencesKey Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Oil Crops Research Institute of Chinese Academy of Agricultural SciencesAbstract 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.http://link.springer.com/article/10.1186/s13068-018-1309-3Acyl-CoA thioesteraseAcyl-CoAFatty acidsPhaeodactylum tricornutumTALENTriacylglycerols |
| spellingShingle | Xiahui Hao Ling Luo Juliette Jouhet Fabrice Rébeillé Eric Maréchal Hanhua Hu Yufang Pan Xiaoming Tan Zhuo Chen Lingjie You Hong Chen Fang Wei Yangmin Gong Enhanced triacylglycerol production in the diatom Phaeodactylum tricornutum by inactivation of a Hotdog-fold thioesterase gene using TALEN-based targeted mutagenesis Biotechnology for Biofuels Acyl-CoA thioesterase Acyl-CoA Fatty acids Phaeodactylum tricornutum TALEN Triacylglycerols |
| title | Enhanced triacylglycerol production in the diatom Phaeodactylum tricornutum by inactivation of a Hotdog-fold thioesterase gene using TALEN-based targeted mutagenesis |
| title_full | Enhanced triacylglycerol production in the diatom Phaeodactylum tricornutum by inactivation of a Hotdog-fold thioesterase gene using TALEN-based targeted mutagenesis |
| title_fullStr | Enhanced triacylglycerol production in the diatom Phaeodactylum tricornutum by inactivation of a Hotdog-fold thioesterase gene using TALEN-based targeted mutagenesis |
| title_full_unstemmed | Enhanced triacylglycerol production in the diatom Phaeodactylum tricornutum by inactivation of a Hotdog-fold thioesterase gene using TALEN-based targeted mutagenesis |
| title_short | Enhanced triacylglycerol production in the diatom Phaeodactylum tricornutum by inactivation of a Hotdog-fold thioesterase gene using TALEN-based targeted mutagenesis |
| title_sort | enhanced triacylglycerol production in the diatom phaeodactylum tricornutum by inactivation of a hotdog fold thioesterase gene using talen based targeted mutagenesis |
| topic | Acyl-CoA thioesterase Acyl-CoA Fatty acids Phaeodactylum tricornutum TALEN Triacylglycerols |
| url | http://link.springer.com/article/10.1186/s13068-018-1309-3 |
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