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A new approach to express transgenes in microalgae and its use to increase the flocculation ability of Chlamydomonas reinhardtii

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Abstract

The choice of strong efficient promoters is a critical step in the development of efficient transformation systems for microalgae; however, the physiological and genetic diversity among microalgae groups makes very difficult to develop standard universal plasmids for a wide number of microalgal species as has been achieved for higher plants. Here, we propose a new approach to express transgenes in microalgae: cotransformation with two naked promoterless genes, a selectable antibiotic-resistant gene and a gene of our interest. These genes are randomly inserted into the nuclear genome, where their transcription relies on their adequate insertion in a region adjacent to an endogenous genomic promoter or in frame with a native gene. In a high percentage of the transformants obtained, both genes are, not only adequately incorporated in the nuclear genome, but also efficiently transcribed and translated. This transformation method is validated in the model microalga Chlamydomonas reinhardtii with the bleomycin-resistant gene from Streptoalloteichus hindustanus (ShBLE) as gene of interest, and it is employed to express a flocculin gene from Saccharomyces bayanus (SbFLO5), which is responsible for the flocculation process in yeasts. Chlamydomonas reinhardtii transformants exhibited self-flocculation abilities between 2- and 3.5-fold higher than the control untransformed strain. The successful cotransformation of C. reinhardtii with two promoterless genes opens doors for the establishment of a universal transformation system based on endogenous promoters, applicable to any microalgal species.

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References

  • Alam MA, Wan C, Guo SL, Zhao XQ, Huang ZY, Yang YL, Chang JS, Bai FW (2014) Characterization of the flocculating agent from the spontaneously flocculating microalga Chlorella vulgaris JSC-7. J Biosci Bioeng 118:29–33

    Article  CAS  PubMed  Google Scholar 

  • Barney MC, Jansen GP, Helber GR (1990) Use of genetic transformation for the introduction of flocculence into yeast. J Am Soc Brew Chem 38:71–74

    Google Scholar 

  • Benemann J (2013) Microalgae for biofuels and animal feeds. Energies 6:5869–5886

    Article  Google Scholar 

  • Blanc G, Agarkova I, Grimwood J, Kuo A, Brueggeman A, Dunigan DD, Gurnon J, Ladunga I, Lindquist E, Lucas S, Pangilinan J, Pröschold T, Salamov A, Schmutz J, Weeks D, Yamada T, Lomsadze A, Borodovsky M, Claverie JM, Grigoriev IV, Van Etten JL (2012) The genome of the polar eukaryotic microalga Coccomyxa subellipsoidea reveals traits of cold adaptation. Genome Biol 13:R39

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Borowitzka MA (2013) High-value products from microalgae-their development and commercialization. J Appl Phycol 25:743–756

    Article  CAS  Google Scholar 

  • Christou P, Swain WF (1990) Cotransformation frequencies of foreign genes in soybean cell cultures. Theor Appl Genet 79:337–341

    Article  CAS  PubMed  Google Scholar 

  • Corellou F, Schwartz C, Motta JP, Djouani-Tahri B, Sanchez F, Bouget FY (2009) Clocks in the green lineage: comparative functional analysis of the circadian architecture of the picoeukaryote Ostreococcus. Plant Cell 21:3436–3449

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Derelle E, Ferraz C, Rombauts S, Rouzé P, Worden AZ, Robbens S, Partensky F, Degroeve S, Echeynié S, Cooke R, Saeys Y, Wuyts J, Jabbari K, Bowler C, Panaud O, Piégu B, Ball SG, Ral JP, Bouget FY, Piganeau G, De Baets B, Picard A, Delseny M, Demaille J, Van de Peer Y, Moreau H (2006) Genome analysis of the smallest free-living eukaryote Ostreococcus tauri unveils many unique features. Proc Natl Acad Sci U S A 103:11647–52

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Díaz-Santos E, De La Vega M, Vila M, Vigara J, León R (2013) Efficiency of different heterologous promoters in the unicellular microalga Chlamydomonas reinhardtii. Biotechnol Prog 29:319–28

    Article  PubMed  Google Scholar 

  • Díaz-Santos E, Vila M, De la Vega M, León R, Vigara J (2015) Study of bioflocculation induced by Saccharomyces bayanus var. uvarum and flocculating protein factors in microalgae. Algal Res 8:23–29

    Article  Google Scholar 

  • Eichler-Stahlberg A, Weisheit W, Ruecker O, Heitzer M (2009) Strategies to facilitate transgene expression in Chlamydomonas reinhardtii. Planta 229:873–883

    Article  CAS  PubMed  Google Scholar 

  • Enzing C, Sijtsma L, Parisi C, Vigani M, Barbosa M, Ploeg M, Rodriguez Cerezo E (2014) Microalgae-based products for the food and feed sector: an outlook for Europe. JRC Scientific and Policy Reports. Publications Office of the European Union. http://ipts.jrc.ec.europa.eu/publications/pub.cfm?id=7145

  • Garcia Sánchez R, Solodovnikova N, Wendland J (2012) Breeding of Lager yeast with Saccharomyces cerevisiae improves stress resistance and fermentation performance. Yeast 29:343–55

    Article  PubMed  Google Scholar 

  • Georgianna R, Mayfield SP (2012) Exploiting diversity and synthetic biology for the production of algal biofuels. Nature 488:329–335

    Article  CAS  PubMed  Google Scholar 

  • Goffeau A, Barrell BG, Bussey H, Davis RW, Dujon B, Feldmann H, Galibert F, Hoheisel JD, Jacq C, Johnston M, Louis EJ, Mewes HW, Murakami Y, Philippsen P, Tettelin H, Oliver SG (1996) Life with 6000 genes. Science 274:546–567

    Article  CAS  PubMed  Google Scholar 

  • Goossens K, Willaert R (2010) Flocculation protein structure and cell–cell adhesion mechanism in Saccharomyces cerevisiae. Biotechnol Lett 32:1571–1585

    Article  CAS  PubMed  Google Scholar 

  • Govender P, Domingo JL, Bester MC, Pretorius IS, Bauer FF (2008) Controlled expression of the dominant flocculation genes FLO1, FLO5, and FLO11 in Saccharomyces cerevisiae. Appl Environ Microbiol 74:6041–52

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Guo SL, Zhao XQ, Wan C, Huang ZY, Yang YL, Alam MA, Ho SH, Bai FW, Chang JS (2013) Characterization of flocculating agent from the self-flocculating microalga Scenedesmus obliquus AS-6-1 for efficient biomass harvest. Bioresour Technol 145:285–9

    Article  CAS  PubMed  Google Scholar 

  • Harris EH (2009) Chlamydomonas sourcebook: introduction to Chlamydomonas and its laboratory use. 2nd edn (Stern, D. and Witman, G., eds), San Diego, CA, Academic Press.

  • Hoffman CS, Winston F (1987) A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene 57:267–272

    Article  CAS  PubMed  Google Scholar 

  • Jinkerson RE, Jonikas MC (2015) Molecular techniques to interrogate and edit the Chlamydomonas nuclear genome. Plant J 82:393–412

    Article  CAS  PubMed  Google Scholar 

  • Kathirsesan S, Chandrashekar A, Ravishankar GA, Sarada R (2009) Agrobacterium-mediated transformation in the green alga Haematococcus pluvialis (Chlorophyceae, Volvocales). J Phycol 45:642–649

    Article  Google Scholar 

  • Kilian O, Benemann CSE, Niyogi KK, Vick B (2011) High-efficiency homologous recombination in the oil-producing alga Nannochloropsis sp. Proc Natl Acad Sci U S A 108:21265–21269

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kindle KL (1990) High-frequency nuclear transformation of Chlamydomonas reinhardtii. Proc Natl Acad Sci U S A 87:1228–1232

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lamesch P, Berardini TZ, Li D, Swarbreck D, Wilks C, Sasidharan R, Muller R, Dreher K, Alexander DL, Garcia-Hernandez M, Karthikeyan AS, Lee CH, Nelson WD, Ploetz L, Singh S, Wensel A, Huala E (2012) The Arabidopsis Information Resource (TAIR): improved gene annotation and new tools. Nucleic Acids Res 40:1202–1210

    Article  Google Scholar 

  • Larkum AW, Ross IL, Kruse O, Hankamer B (2012) Selection, breeding and engineering of microalgae for bioenergy and biofuel production. Trends Biotechnol 30:198–205

    Article  CAS  PubMed  Google Scholar 

  • Lee SK, Chou H, Ham TS, Lee TS, Keasling JD (2008) Metabolic engineering of microorganisms for biofuels production: from bugs to synthetic biology to fuels. Curr Opin Biotechnol 19:556–563

    Article  CAS  PubMed  Google Scholar 

  • León R, Fernández E (2007) Nuclear transformation of eukaryotic microalgae: historical overview, achievements and problems. Adv Exp Med Biol 616:1–11

    Article  PubMed  Google Scholar 

  • León R, Couso I, Fernández E (2007) Metabolic engineering of ketocarotenoids biosynthesis in the unicellular microalga Chlamydomonas reinhardtii. J Biotechnol 130:143–152

    Article  PubMed  Google Scholar 

  • León-Bañares R, Gonzalez Ballester D, Galvan A, Fernandez E (2004) Transgenic microalgae as green cell-factories. Trends Biotechnol 22:45–52

    Article  PubMed  Google Scholar 

  • Loppes R, Radoux M, Ohresser MC, Matagne RF (1999) Transcriptional regulation of the Nia1 gene encoding nitrate reductase in Chlamydomonas reinhardtii: effects of various environmental factors on the expression of a reporter gene under the control of the Nia1 promoter. Plant Mol Biol 41:701–711

    Article  CAS  PubMed  Google Scholar 

  • Lumbreras V, Stevens DR, Purton S (1998) Efficient foreign gene expression in Chlamydomonas reinhardtii mediated by an endogenous intron. Plant J 14:441–447

    Article  CAS  Google Scholar 

  • Mamedov T, Yusibov V (2011) Green algae Chlamydomonas reinhardtii possess endogenous sialylated N-glycans. FEBS Open Bio 1:15–22

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Mathieu-Rivet E, Kiefer-Meyer MC, Vanier G, Ovide C, Burel C, Lerouge P, Bardor M (2014) Protein N-glycosylation in eukaryotic microalgae and its impact on the production of nuclear expressed biopharmaceuticals. Front Plant Sci 5:359

    Article  PubMed  PubMed Central  Google Scholar 

  • Merchant SS, Prochnik SE, Vallon O et al (2007) The Chlamydomonas genome reveals the evolution of key animal and plant Functions. Science 318:245–250

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Prochnik SE, Umen J, Nedelcu AM, Hallmann A, Miller SM, Nishii I, Ferris P, Kuo A, Mitros T, Fritz-Laylin LK, Hellsten U, Chapman J, Simakov O, Rensing SA, Terry A, Pangilinan J, Kapitonov V, Jurka J, Salamov A, Shapiro H, Schmutz J, Grimwood J, Lindquist E, Lucas S, Grigoriev IV, Schmitt R, Kirk D, Rokhsar DS (2010) Genomic analysis of organismal complexity in the multicellular green alga Volvox carteri. Science 329:223–6

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Pruess M, Fleischmann W, Kanapin A, Karavidopoulou Y, Kersey P, Kriventseva E, Mittard V, Mulder N, Phan I, Servant F, Apweiler R (2003) The Proteome Analysis database: a tool for the in silico analysis of whole proteomes. Nucleic Acids Res 31:414–417

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Radakovits R, Jinkerson RE, Darzins A, Posewitz MC (2010) Genetic engineering of algae for enhanced biofuel production. Eukaryot Cell 9:486–501

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Radakovits R, Jinkerson RE, Fuerstenberg SI, Tae H, Settlage RE, Boore JL, Posewitz MC (2012) Draft genome sequence and genetic transformation of the oleaginous alga Nannochloropis gaditana. Nat Commun 21:686

    Article  Google Scholar 

  • Rasala BA, Mayfield SP (2011) The microalga Chlamydomonas reinhardtii as a platform for the production of human protein therapeutics. Bioeng Bugs 2:50–54

    Article  PubMed  PubMed Central  Google Scholar 

  • Salim S, Vermuë MH, Wijffels RH (2012) Ratio between autoflocculating and target microalgae affects the energy-efficient harvesting by bio-flocculation. Bioresour Technol 118:49–55

    Article  CAS  PubMed  Google Scholar 

  • Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbour Laboratory Press, New York

    Google Scholar 

  • Scaife MA, Nguyen GTDT, Rico J, Lambert D, Helliwell KE, Smith AG (2015) Establishing Chlamydomonas reinhardtii as an industrial biotechnology host. Plant J 82:532–546

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Scranton MA, Ostrand JT, Fields FJ, Mayfield SP (2015) Chlamydomonas as a model for biofuels and bio-products production. Plant J 82:523–531

    Article  CAS  PubMed  Google Scholar 

  • Sizova I, Fuhrmann M, Hegemann P (2001) A Streptomyces rimosus aphVIII gene coding for a new type phosphotransferase provides stable antibiotic resistance to Chlamydomonas reinhardtii. Gene 277:221–229

    Article  CAS  PubMed  Google Scholar 

  • Steinbrenner J, Sandmann G (2006) Transformation of the green alga Haematococcus pluvialis with a phytoene desaturase for accelerated astaxanthin biosynthesis. Appl Environ Microbiol 72:7477–748

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Stevens DR, Rochaix JD, Purton S (1996) The bacterial phleomycin resistance gene ble as a dominant selectable marker in Chlamydomonas. Mol Gen Genet 251:23–30

    CAS  PubMed  Google Scholar 

  • Talebi AF, Tohidfar M, Tabatabaei M, Bagheri A, Mohsenpor M, Mohtashami SK (2013) Genetici manipulation, as feasible tool to enhance unique characteristic of Chlorella vulgaris as a feedstock for biodiesel production. Mol Biol Rep 40:4421–4428

    Article  CAS  PubMed  Google Scholar 

  • Tan C, Qin S, Zhang Q, Jiang P, Zhao F (2005) Establishment of a micro-particle bombardment transformation system for Dunaliella salina. J Microbiol 43:361–365

    CAS  PubMed  Google Scholar 

  • Úbeda-Mínguez P, Chileh T, Dautor Y, García-Maroto F, Alonso DL (2015) Tools for microalgal biotechnology: development of an optimized transformation method for an industrially promising microalga—Tetraselmis chuii. J Appl Phycol 27:223–232

    Article  Google Scholar 

  • Vanthoor-Koopmans M, Wijffels RH, Barbosa MJ, Eppink MH (2013) Biorefinery of microalgae for food and fuel. Bioresour Technol 135:142–149

    Article  CAS  PubMed  Google Scholar 

  • Vila M, Couso I, León R (2008) Carotenoids content in mutants of the chlorophyte Chlamydomonas reinhardtii with low expression levels of phytoene desaturase. Process Biochem 43:1147–1152

    Article  CAS  Google Scholar 

  • Vila M, Díaz-Santos E, De la Vega M, Rodríguez H, Vargas A, León R (2012) Promoter trapping in microalgae using the antibiotic paromomycin as selective agent. Mar Drugs 10:2749–2765

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Watari J, Kudo M, Nishikawa N, Kamimura M (1990) Construction of flocculent yeast cells (Saccharomyces cerevisiae) by mating or protoplast fusion using a yeast cell containing the flocculation gene FLO5. Agric Biol Chem 54:1677–1681

    CAS  Google Scholar 

  • Wijffels RH, Barbosa MJ (2010) An outlook on microalgal biofuels. Science 329:796–799

    Article  CAS  PubMed  Google Scholar 

  • Yamamoto QQ, Tsuhara Y, Gohda K, Suzuki K, Matsui M (2003) Gene trapping of the Arabidopsis genome with a firefly luciferase reporter. Plant J 35:273–283

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

We gratefully acknowledge the financial support from the Andalusian Government (P09-CVI-5053 and BIO214) and the CEICAMBIO. Dra. M. Vila thanks the University of Huelva for financial support. We also want to thank Dr. Fernández from the University of Córdoba for critical discussion about the manuscript and Dr. Pérez-Catañeira from the University of Seville for providing protocols and advice for isolation of yeast genomic DNA. We also acknowledge Canvax Biotech (Córdoba, Spain) for technical support on FLO5 cloning. This is 106 publication from CEIMAR Publication Series.

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  1. Laboratory of Biochemistry and Molecular Biology, Faculty of Experimental Sciences, Marine International Campus of Excellence, CEIMAR and CEICAMBIO, University of Huelva, Avda Fuerzas Armadas s/n, 21007, Huelva, Spain

    Encarnación Díaz-Santos, Marta Vila, Javier Vigara & Rosa León

  2. PhycoGenetics SL, Joan Miró Street, 21110, Aljaraque, Huelva, Spain

    Marta Vila & Rosa León

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  1. Encarnación Díaz-Santos
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  2. Marta Vila
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  3. Javier Vigara
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Correspondence to Rosa León.

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Díaz-Santos, E., Vila, M., Vigara, J. et al. A new approach to express transgenes in microalgae and its use to increase the flocculation ability of Chlamydomonas reinhardtii . J Appl Phycol 28, 1611–1621 (2016). https://doi.org/10.1007/s10811-015-0706-2

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  • DOI: https://doi.org/10.1007/s10811-015-0706-2

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