Emiliania huxleyi is the most prominent modern coccolithophore, a group of marine unicellular eukaryotes that play a critical role in ocean biogeochemistry. Coccolithoviruses are large double stranded DNA viruses, which is responsible for the demise of large oceanic blooms formed by E. huxleyi. E. huxleyi virus (EhVs) acquired a series of enzyme-coding genes predicted to be involved in the sphingolipid biosynthesis by horizontal gene transfer between virus-host. Currently, there is limited experimental validation identifying the functions of these genes in EhV. Genetic transformation of eukaryotic cells is a powerful tool to get an insight into gene functions of the studied organisms. Serine palmitoyltransferase (SPT) catalyzes the first committed step in de novo sphingolipid biosynthetic pathway. Here, a novel vector system for the transformation of E. huxleyi was designed. It contained fragments of promoter and terminator sequences of E. huxleyi endogenic fucoxanthin chlorophyll a/c-binding protein gene “fcp” and harbored EhV-99B1 spt gene. The resultant recombinant transformation vectors pEhux-I-spt and pEhux-II were co-transferred into E. huxleyi BOF92 by electroporation. Transformants were obtained upon glufosinate-ammonium selection, and confirmed by Southern hybridization, genome PCR, qRT-PCR and Western blot screening of spt gene, which indicated that spt gene was integrated into the nuclear genome and was expressed at the mRNA and protein levels. The expression of the viral spt gene led to differences in lipid compositions analyzed using thin-layer chromatography (TLC). The results present the genetic transformation system for E. huxleyi, providing additional genetic resource with potential for exploring basic biological questions such as the virus-host interactions.
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Ausubel F M, Kingston R E, Seidman J G, Struhl K, Brent R, Moore D D, Smith J A. 1999. Short protocols in molecular biology, 4th edn. Wiley, NY. https://doi.org/10.1038/206645a0.
Bratbak G, Egge J K, Heldal M. 1993. Viral mortality of the marine alga Emiliania huxleyi (Haptophyceae) and termination of algal blooms. Marine Ecology Progress Series, 93(1–2): 39–48, https://doi.org/10.3354/meps093039.
Brown L E, Sprecher S L, Keller L R. 1991. Introduction of exogenous DNA into Chlamydomonas reinhardtii by electroporation. Molecular and Cellular Biology, 11(4): 2 328–2 332, https://doi.org/10.1128/MCB.1L4.2328.
Brussaard C P D. 2004. Viral control of phytoplankton populations—a review. Journal of Eukaryotic Microbiology, 51(2): 125–138, https://doi.org/10.1111/j.1550-7408.2004.tb00537.x.
Čgovnik U, Novaković S. 2004. Setting optimal parameters for in vitro electrotransfection of B16F1, SA1, LPB, SCK, L929 and CHO cells using predefined exponentially decaying electric pulses. Bioelectrochemistry, 62(1): 73–82, https://doi.org/10.1016/j.bioelechem.2003.10.009.
Coll J M. 2006. Methodologies for transferring DNA into eukaryotic microalgae: a review. Spanish Journal of Agricultural Research, 4(4): 316–330, https://doi.org/10.5424/sjar/2006044-209.
Dymond J, Lyle M. 1985. Flux comparisons between sediments and sediment traps in the eastern tropical Pacific: implications for atmospheric CO2 variations during the Pleistocene. Limnology and Oceanography, 30(4): 699–712, https://doi.org/10.4319/lo.1985.30.4.0699.
Endo H, Yoshida M, Uji T, Saga N, Inoue K, Nagasawa H. 2016. Stable nuclear transformation system for the coccolithophorid alga Pleurochrysis carterae. Scientific Reports, 6(1): 22–252, https://doi.org/10.1038/srep22252.
Falciatore A, Casotti R, Leblanc C, Abrescia C, Bowler C. 1999. Transformation of nonselectable reporter genes in marine diatoms. Marine Biotechnology, 1(3): 239–251, https://doi.org/10.1007/PL00011773.
Guillard R R L. 1975. Culture of phytoplankton for feeding marine invertebrates. In: Smith W L, Chanley M H eds. Culture of Marine Invertebrates Animals, Plenum Publishing, New York, p.29–60, https://doi.org/10.1007/978-1-4615-8714-9_3.
Hanada K, Hara T, Nishijima M. 2000. Purification of the serine palmitoyltransferase complex responsible for sphingoid base synthesis by using affinity peptide chromatography techniques. Journal of Biological Chemistry, 275(12): 8 409–8 415, https://doi.org/10.1074/jbc.275.12.8409.
Hernández A S R, Flores J A, Sierro F J, Fuertes M A, Cros L, Trull T W. 2018. Coccolithophore populations and their contribution to carbonate export during an annual cycle in the Australian sector of the Antarctic zone. Biogeosciences, 15(6): 1 843–1 862, https://doi.org/10.5194/bg-15-1843-2018.
Hlavova M, Turoczy Z, Bisova K. 2015. Improving microalgae for biotechnology — from genetics to synthetic biology. Biotechnology Advances, 33(6): 1 194–1 203, https://doi.org/10.1016/j.biotechadv.2015.01.009.
Kammerer W, Cove D J. 1996. Genetic analysis of the effects of re-transformation of transgenic lines of the moss Physcomitrella patens. Molecular and General Genetics MGG, 250(3): 380–382, https://doi.org/10.1007/BF02174397.
Kilian O, Benemann C S E, Niyogi K K, Vick B. 2011. High-efficiency homologous recombination in the oil-producing alga Nannochloropsis sp. Proceedings of the National Academy of Science of the United State of America, 108(52): 21 265–21 269, https://doi.org/10.1073/pnas.1105861108.
Laguna R, Romo J, Read B A, Wahlund T M. 2001. Induction of phase variation events in the life cycle of the marine coccolithophorid Emiliania huxleyi. Applied and Environmental Microbiology, 67(9): 3 824–3 831, https://doi.org/10.1128/AEM.67.9.3824-3831.2001.
Li F C, Qin S, Jiang P, Wu Y, Zhang W. 2009. The integrative expression of GUS gene driven by FCP promoter in the seaweed Laminaria japonica (Phaeophyta). Journal of Applied Phycology, 21(3): 287–293, https://doi.org/10.1007/s10811-008-9366-9.
Liu J W, Cai W C, Fang X, Wang X T, Li G L. 2018. Virus-induced apoptosis and phosphorylation form of metacaspase in the marine coccolithophorid Emiliania huxleyi. Archives of Microbiology, 200(3): 413–422, https://doi.org/10.1007/s00203-017-1460-4.
Liu X H, Zheng T L, Cai Y Q, Liu J W. 2012. Cloning, expression and characterization of serine palmitoyltransferase (SPT)-like gene subunit (LCB2) from marine Emiliania huxleyi virus (Coccolithovirus). Acta Oceanologica Sinica, 31(6): 127–138, https://doi.org/10.1007/s13131-012-0259-z.
Michaelson L V, Dunn T M, Napier J A. 2010. Viral trans-dominant manipulation of algal sphingolipids. Trends in Plant Science, 15(12): 651–655, https://doi.org/10.1016/j.tplants.2010.09.004.
Miyagawa A, Okami T, Kira N, Yamaguchi H, Ohnishi K, Adachi M. 2009. Research note: high efficiency transformation of the diatom Phaeodactylum tricornutum with a promoter from the diatom Cylindrotheca fusiformis. Phycological Research, 57(2): 142–146, https://doi.org/10.1111/j.1440-1835.2009.00531.x.
Miyagawa-Yamaguchi A, Okami T, Kira N, Yamaguchi H, Ohnishi K, Adachi M. 2011. Stable nuclear transformation of the diatom Chaetoceros sp. Phycological Research, 59(2): 113–119, https://doi.org/10.1111/j.1440-1835.2011.00607.x.
Monier A, Pagarete A, de Vargas C, Allen M J, Read B, Claverie J M, Ogata H. 2009. Horizontal gene transfer of an entire metabolic pathway between a eukaryotic alga and its DNA virus. Genome Research, 19(8): 1 441–1 449, https://doi.org/10.1101/gr.091686.109.
Mussgnug J H. 2015. Genetic tools and techniques for Chlamydomonas reinhardtii. Applied Microbiology and Biotechnology, 99(13): 5 407–5 418, https://doi.org/10.1007/s00253-015-6698-7.
Muto M, Fukuda Y, Nemoto M, Yoshino T, Matsunaga T, Tanaka T. 2013. Establishment of a genetic transformation system for the marine pennate diatom Fistulifera sp. strain JPCC DA0580—a high triglyceride producer. Marine Biotechnology, 15(1): 48–55, https://doi.org/10.1007/s10126-012-9457-0.
Niu Y F, Yang Z K, Zhang M H, Zhu C C, Yang W D, Liu J S, Li H Y. 2012. Transformation of diatom Phaeodactylum tricornutum by electroporation and establishment of inducible selection marker. BioTechniques, 52(6): 1–3, https://doi.org/10.2144/000113881.
Niu Y F, Zhang M H, Li D W, Yang W D, Liu J S, Bai W B, Li H Y. 2013. Improvement of neutral lipid and polyunsaturated fatty acid biosynthesis by overexpressing a type 2 diacylglycerol acyltransferase in marine diatom Phaeodactylum tricornutum. Marine Drugs, 11(11): 4 558–4 569, https://doi.org/10.3390/md11114558.
Oeltjen A, Marquardt J, Rhiel E. 2004. Differential circadian expression of genes fcp2 and fcp6 in Cyclotella cryptica. International Microbiology, 7(2): 127–131.
Qin S, Lin H Z, Jiang P. 2012. Advances in genetic engineering of marine algae. Biotechnology Advances, 30(6): 1 602–1 613, https://doi.org/10.1016/j.biotechadv.2012.05.004.
Radakovits R, Jinkerson R E, Darzins A, Posewitz M C. 2010. Genetic engineering of algae for enhanced biofuel production. Eukaryotic Cell, 9(4): 486–501, https://doi.org/10.1128/EC.00364-09.
Read B A, Kegel J, Klute M J, Kuo A, Lefebvre S C, Maumus F, Mayer C, Miller J, Monier A, Salamov A, Young J, Aguilar M, Claverie J M, Frickenhaus S, Gonzalez K, Herman E K, Lin Y C, Napier J, Ogata H, Sarno A F, Shmutz J, Schroeder D, de Vargas C, Verret F, von Dassow P, Valentin K, Van de Peer Y, Wheeler G, Dacks J B, Delwiche C F, Dyhrman S T, Glöckner G, John U, Richards T, Worden A Z, Zhang X Y, Grigoriev I V. 2013. Pan genome of the phytoplankton Emiliania underpins its global distribution. Nature, 499(7457): 209–213, https://doi.org/10.1038/nature12221.
Rose S L, Fulton J M, Brown C M, Natale F, Van Mooy B A S, Bidle K D. 2014. Isolation and characterization of lipid rafts in Emiliania huxleyi: a role for membrane microdomains in host-virus interactions. Environmental Microbiology, 16(4): 1 150–1 166, https://doi.org/10.1111/1462-2920.12357.
Rosenwasser S, Mausz M A, Schatz D, Sheyn U, Malitsky S, Aharoni A, Weinstock E, Tzfadia O, Ben-Dor S, Feldmesser E, Pohnert G, Vardi A. 2014. Rewiring host lipid metabolism by large viruses determines the fate of Emiliania huxleyi, a bloom-forming alga in the Ocean. Plant Cell, 26(6): 2 689–2 707, https://doi.org/10.1105/tpc.114.125641.
Schneider-Schaulies J, Schneider-Schaulies S. 2015. Sphingolipids in viral infection. Biological Chemistry, 396(6–7): 585–595, https://doi.org/10.1515/hsz-2014-0273.
Sekino K, Shiraiwa Y. 1996. Evidence for the involvement of mitochondrial respiration in calcification in a marine coccolithophorid, Emiliania huxleyi. Plant and Cell Physiology, 37(7): 1 030–1 033, https://doi.org/10.1093/oxfordjournals.pcp.a029034.
Suttle C A. 2005. Viruses in the sea. Nature, 437(7057): 356–361, https://doi.org/10.1038/nature04160.
Vardi A, Van Mooy B A S, Fredricks H F, Popendorf K J, Ossolinski J E, Haramaty L, Bidle K D. 2009. Viral glycosphingolipids induce lytic infection and cell death in marine phytoplankton. Science, 326(5954): 861–865, https://doi.org/10.1126/science.1177322.
Velmurugan N, Deka D. 2018. Transformation techniques for metabolic engineering of diatoms and haptophytes: current state and prospects. Applied Microbiology and Biotechnology, 102(10): 4 255–4 267, https://doi.org/10.1007/s00253-018-8925-5.
Watanabe S, Ohnuma M, Sato J, Yoshikawa H, Tanaka K. 2011. Utility of a GFP reporter system in the red alga Cyanidioschyzon merolae. The Journal of General and Applied Microbiology, 57(1): 69–72, https://doi.org/10.2323/jgam.57.69.
Westbroek P, Brown C W, Van Bleijswijk J, Brownlee C, Brummer G J, Conte M, Egge J, Fernández E, Jordan R, Knappertsbusch M, Stefels J, Veldhuis M, van der Wal P, Young J. 1993. A model system approach to biological climate forcing. The example of Emiliania huxleyi. Global and Planetary Change, 8(1–2): 27–46, https://doi.org/10.1016/0921-8181(93)90061-R.
Wilson W H, Schroeder D C, Allen M J, Holden M T G, Parkhill J, Barrell B G, Churcher C, Hamlin N, Mungall K, Norbertczak H, Quail M A, Price C, Rabbinowitsch E, Walker D, Craigon M, Roy D, Ghazal P. 2005. Complete genome sequence and lytic phase transcription profile of a Coccolithovirus. Science, 309(5737): 1 090–1 092, https://doi.org/10.1126/science.1113109.
Xue J, Niu Y F, Huang T, Yang W D, Liu J S, Li H Y. 2015. Genetic improvement of the microalga Phaeodactylum tricornutum for boosting neutral lipid accumulation. Metabolic Engineering, 27: 1–9, https://doi.org/10.1016/j.ymben.2014.10.002.
Zeng J, Liu S S Y, Cai W C, Jiang H R, Lu X, Li G L, Li J, Liu J W. 2019. Emerging lipidome patterns associated with marine Emiliania huxleyi-virus model system. Science of the Total Environment, 688: 521–528, https://doi.org/10.1016/j.scitotenv.2019.06.284.
Ziv C, Malitsky S, Othman A, Ben-Dor S, Wei Y, Zheng S N, Aharoni A, Hornemann T, Vardi A. 2016. Viral serine palmitoyltransferase induces metabolic switch in sphingolipid biosynthesis and is required for infection of a marine alga. Proceedings of the National Academy of Science of the United State of America, 113(13): E1 907–E1 916, https://doi.org/10.1073/pnas.1523168113.
Our deepest thanks go to Prof. Gunnar BRATBAK (Department of Biology, University of Bergen) for providing the E. huxleyi BOF92 strain and the E. huxleyi virus 99B1 strain friendly. We also would appreciate Prof. Kehou PAN and Baohua ZHU (Ocean University of China) for providing plasmid pSP73.
Supported by the National Natural Science Foundation of China (Nos. 41576166, 21707042, 31771972) and Fujian Province Natural Science Foundation of China (Nos. 2019J01696, 2017J01447, 2017J01636)
Data Availability Statement
The datasets generated during and/or analyzed in this study are available from the corresponding author upon reasonable request.
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Cai, W., Wang, X., Su, J. et al. Transformation of coccolithophorid Emiliania huxleyi harboring a marine virus (Coccolithoviruses) serine palmitoyltransferase (SPT) gene by electroporation. J. Ocean. Limnol. (2020). https://doi.org/10.1007/s00343-020-9325-0
- Emiliania huxleyi
- genetic transformation
- serine palmitoyltransferase (SPT)
- total lipid