Metabolic engineering of the oleaginous alga Nannochloropsis for enriching eicosapentaenoic acid in triacylglycerol by combined pulling and pushing strategies |
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| Affiliation: | 1. School of Life Science and Technology, Tokyo Institute of Technology, 4259-B-65 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan;2. Graduate School of Integrated Sciences for Life, Hiroshima University, 1-4-3, Kagamiyama, Higashi-Hiroshima 739-8526, Japan;3. Technical Research Center, Mazda Motor Corporation, 3-1, Shinchi, Fuchu-cho, Aki-gun, Hiroshima, 730-8670, Japan;1. Single-Cell Center, CAS Key Laboratory of Biofuels and Shandong Key Laboratory of Energy Genetics, Qingdao Institute of BioEnergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong 266101, China;2. University of Chinese Academy of Sciences, Beijing 100049, China;3. Laboratory for Algae Research and Biotechnology, Department of Applied Biological Sciences, AZ State University, Mesa, AZ85212, USA;4. Institute of Marine and Environmental Technology, University of Maryland, Baltimore, MD 21202;5. Center for Microalgal Biotechnology and Biofuels, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei 430072, China |
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| Abstract: | The marine alga Nannochloropsis oceanica has been considered as a promising photosynthetic cell factory for synthesizing eicosapentaenoic acid (EPA), yet the accumulation of EPA in triacylglycerol (TAG) is restricted to an extreme low level. Poor channeling of EPA to TAG was observed in N. oceanica under TAG induction conditions, likely due to the weak activity of endogenous diacylglycerol acyltransferases (DGATs) on EPA-CoA. Screening over thirty algal DGATs revealed potent enzymes acting on EPA-CoA. Whilst overexpressing endogenous DGATs had no or slight effect on EPA abundance in TAG, introducing selected DGATs with strong activity on EPA-CoA, particularly the Chlamydomonas-derived CrDGTT1, which resided at the outermost membrane of the chloroplast and provided a strong pulling power to divert EPA to TAG for storage and protection, led to drastic increases in EPA abundance in TAG and TAG-derived EPA level in N. oceanica. They were further promoted by additional overexpression of an elongase gene involved in EPA biosynthesis, reaching 5.9- and 12.3-fold greater than the control strain, respectively. Our results together demonstrate the concept of applying combined pulling and pushing strategies to enrich EPA in algal TAG and provide clues for the enrichment of other desired fatty acids in TAG as well. |
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| Keywords: | Eicosapentaenoic acid Nannochloropsis Metabolic engineering Pulling Pushing Triacylglycerol |
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