Plant Molecular Biology

, Volume 92, Issue 6, pp 629–641 | Cite as

UV-mediated Chlamydomonas mutants with enhanced nuclear transgene expression by disruption of DNA methylation-dependent and independent silencing systems

  • Sari Dewi Kurniasih
  • Tomohito Yamasaki
  • Fantao Kong
  • Sigeru Okada
  • Dwiyantari Widyaningrum
  • Takeshi OhamaEmail author


Key message

In this investigation, we succeeded to generate Chlamydomonas mutants that bear dramatically enhanced ability for transgene expression. To yield these mutants, we utilized DNA methyltransferase deficient strain. These mutants must be useful as a plant cell factory.


Chlamydomonas reinhardtii (hereafter Chlamydomonas) is a green freshwater microalga. It is a promising cell factory for the production of recombinant proteins because it rapidly grows in simple salt-based media. However, expression of transgenes integrated into the nuclear genome of Chlamydomonas is very poor, probably because of severe transcriptional silencing irrespective of the genomic position. In this study, we generated Chlamydomonas mutants by ultraviolet (UV)-mediated mutagenesis of maintenance-type DNA methyltransferase gene (MET1)-null mutants to overcome this disadvantage. We obtained several mutants with an enhanced ability to overexpress various transgenes irrespective of their integrated genomic positions. In addition, transformation efficiencies were significantly elevated. Our findings indicate that in addition to mechanisms involving MET1, transgene expression is regulated by a DNA methylation-independent transgene silencing system in Chlamydomonas. This is in agreement with the fact that DNA methylation occurs rarely in this organism. The generated mutants may be useful for the low-cost production of therapeutic proteins and eukaryotic enzymes based on their rapid growth in simple salt-based media.


Chlamydomonas UV-mediated mutation Heterologous gene expression Transcriptional gene silencing Maintenance-type DNA methyltransferase 



Special thanks to Ms. Miyuki Nakajima for her encouragement.


This research was partly supported by the Japan Science and Technology Agency (JST) and Core Research for Evolutional Science and Technology (CREST).

Author conrtibutions

T.Y. and T.O. designed the research, S.D.K., F.K., and D.W. performed experiments, T.O. wrote the manuscript with feedback from S.O.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11103_2016_529_MOESM1_ESM.docx (3 mb)
Supplementary material 1 (DOCX 3097 KB)


  1. Babinger P, Kobl I, Mages W, Schmitt R (2001) A link between DNA methylation and epigenetic silencing in transgenic Volvox carteri. Nucleic Acids Res 29:1261–1271. doi: 10.1093/nar/29.6.1261 CrossRefPubMedPubMedCentralGoogle Scholar
  2. Babinger P, Völkl R, Cakstina I, Maftei A, Schmitt R (2007) Maintenance DNA methyltransferase (Met1) and silencing of CpG-methylated foreign DNA in Volvox carteri. Plant Mol Biol 63:325–336. doi: 10.1007/s11103-006-9091-1 CrossRefPubMedGoogle Scholar
  3. Barahimipour R, Neupert J, Bock R (2016) Efficient expression of nuclear transgenes in the green alga Chlamydomonas: synthesis of an HIV antigen and development of a new selectable marker. Plant Mol Biol. doi: 10.1007/s11103-015-0425-8 PubMedPubMedCentralGoogle Scholar
  4. Bernstein BE, Humphrey EL, Erlich RL, Schneider R, Bouman P, Liu JS, Kouzarides T, Schreiber SL (2002) Methylation of histone H3 Lys 4 in coding regions of active genes. Proc Natl Acad Sci USA 99:8695–8700. doi: 10.1073/pnas.082249499 CrossRefPubMedPubMedCentralGoogle Scholar
  5. Berthold P, Schmitt R, Mages W (2002) An engineered Streptomyces hygroscopicus aph 7″ gene mediates dominant resistance against hygromycin B in Chlamydomonas reinhardtii. Protist 153:401–412. doi: 10.1078/14344610260450136 CrossRefPubMedGoogle Scholar
  6. Bird A (2002) DNA methylation patterns and epigenetic memory DNA methylation patterns and epigenetic memory. Genes Dev 6–21. doi: 10.1101/gad.947102
  7. Casas-Mollano JA, van Dijk K, Eisenhart J, Cerutti H (2007) SET3p monomethylates histone H3 on lysine 9 and is required for the silencing of tandemly repeated transgenes in Chlamydomonas. Nucleic Acids Res 35:939–950. doi: 10.1093/nar/gkl1149 CrossRefPubMedPubMedCentralGoogle Scholar
  8. Casas-Mollano JA, Jeong B-R, Xu J, Moriyama H, Cerutti H (2008) The MUT9p kinase phosphorylates histone H3 threonine 3 and is necessary for heritable epigenetic silencing in Chlamydomonas. Proc Natl Acad Sci USA 105:6486–6491. doi: 10.1073/pnas.0711310105 CrossRefPubMedPubMedCentralGoogle Scholar
  9. Cerutti H, Johnson M, Gillham NW, Boynton JE (1997) Epigenetic silencing of a foreign gene in nuclear transformants of Chlamydomonas. Plant Cell 9:925–945. doi: 10.1105/tpc.9.6.925 CrossRefPubMedPubMedCentralGoogle Scholar
  10. Cerutti H, Ma X, Msanne J, Repas T (2011) RNA-mediated silencing in algae: biological roles and tools for analysis of gene function. Eukaryot Cell 10:1164–1172. doi: 10.1128/EC.05106-11 CrossRefPubMedPubMedCentralGoogle Scholar
  11. Chan SWL, Henderson IR, Jacobsen SE (2005) Gardening the genome: DNA methylation in Arabidopsis thaliana. Nat Rev Genet 6:351–360. doi: 10.1038/nrg1664 CrossRefPubMedGoogle Scholar
  12. Farkas G, Leibovitch B, Elgin SC (2000) Chromatin organization and transcriptional control of gene expression in Drosophila. Gene 253:117–136. doi:S0378-1119(00)00240-7CrossRefPubMedGoogle Scholar
  13. Feng S, Cokus SJ, Zhang X, Chen P-Y, Bostick M, Goll MG, Hetzel J, Jain J, Strauss SH, Halpern ME, Ukomadu C, Sadler KC, Pradhan S, Pellegrini M, Jacobsen SE (2010) Conservation and divergence of methylation patterning in plants and animals. Proc Natl Acad Sci USA 107:8689–8694. doi: 10.1073/pnas.1002720107 CrossRefPubMedPubMedCentralGoogle Scholar
  14. Fuhrmann M (2001) Grün fluorezierendes protein und gezielte genstillegung in Chlamydomonas reinhardtii. Dissertation. Universität Regensburg, RegensburgGoogle Scholar
  15. Fuhrmann M, Oertel W, Hegemann P (1999) A synthetic gene coding for the green fluorescent protein (GFP) is a versatile reporter in Chlamydomonas reinhardtii. Plant J 19:353–361CrossRefPubMedGoogle Scholar
  16. Fuks F, Hurd PJ, Wolf D, Nan X, Bird AP, Kouzarides T (2003) The methyl-CpG-binding protein MeCP2 links DNA methylation to histone methylation. J Biol Chem 278:4035–4040. doi: 10.1074/jbc.M210256200 CrossRefPubMedGoogle Scholar
  17. Jinkerson RE, Jonikas MC (2015) Molecular techniques to interrogate and edit the Chlamydomonas nuclear genome. Plant J. doi: 10.1111/tpj.12801 Google Scholar
  18. Kajikawa M, Kinohira S, Ando A, Shimoyama M, Kato M, Fukuzawa H (2015) Accumulation of squalene in a microalga Chlamydomonas reinhardtii by genetic modification of squalene synthase and squalene epoxidase genes. PLoS One 10:e0120446. doi: 10.1371/journal.pone.0120446 CrossRefPubMedPubMedCentralGoogle Scholar
  19. Kong F, Yamasaki T, Ohama T (2014) Expression levels of domestic cDNA cassettes integrated in the nuclear genomes of various Chlamydomonas reinhardtii strains. J Biosci Bioeng 117:613–616. doi: 10.1016/j.jbiosc.2013.10.025 CrossRefPubMedGoogle Scholar
  20. Kong F, Yamasaki T, Kurniasih SD, Hou L, Li X, Ivanova N, Okada S, Ohama T (2015) Robust expression of heterologous genes by selection marker fusion system in improved Chlamydomonas strains. J Biosci Bioeng 120x:239–245. doi: 10.1016/j.jbiosc.2015.01.005
  21. Kozminski KG, Diener DR, Rosenbaum JL (1993) High level expression of nonacetylatable α-tubulin in Chlamydomonas reinhardtii. Cell Motil Cytoskeleton 25:158–170. doi: 10.1002/cm.970250205 CrossRefPubMedGoogle Scholar
  22. Lauersen KJ, Berger H, Mussgnug JH, Kruse O (2013) Efficient recombinant protein production and secretion from nuclear transgenes in Chlamydomonas reinhardtii. J Biotechnol 167:101–110. doi: 10.1016/j.jbiotec.2012.10.010 CrossRefPubMedGoogle Scholar
  23. Lauersen KJ, Kruse O, Mussgnug JH (2015) Targeted expression of nuclear transgenes in Chlamydomonas reinhardtii with a versatile, modular vector toolkit. Appl Microbiol Biotechnol. doi: 10.1007/s00253-014-6354-7 PubMedGoogle Scholar
  24. Matzke M, Mette MF, Matzke J (2000) Transgene silencing by the host genome defense: implications for the evolution of epigenetic control mechanisms in plants and vertebrates. Plant Mol Biol 43:401–415. doi: 10.1023/A:1006484806925 CrossRefPubMedGoogle Scholar
  25. Meslet-Cladière L, Vallon O (2011) Novel shuttle markers for nuclear transformation of the green alga Chlamydomonas reinhardtii. Eukaryot Cell 10:1670–1678. doi: 10.1128/EC.05043-11 CrossRefPubMedPubMedCentralGoogle Scholar
  26. Mussgnug JH (2015) Genetic tools and techniques for Chlamydomonas reinhardtii. Appl Microbiol Biotechnol 5407–5418. doi: 10.1007/s00253-015-6698-7
  27. Neupert J, Karcher D, Bock R (2009) Generation of Chlamydomonas strains that efficiently express nuclear transgenes. Plant J 57:1140–1150. doi: 10.1111/j.1365-313X.2008.03746.x CrossRefPubMedGoogle Scholar
  28. Nguyen RL, Tam LW, Lefebvre PA (2005) The LF1 gene of Chlamydomonas reinhardtii encodes a novel protein required for flagellar length control. Genetics 169:1415–1424. doi: 10.1534/genetics.104.027615 CrossRefPubMedPubMedCentralGoogle Scholar
  29. Nishiyama R, Wada Y, Mibu M, Yamaguchi Y, Shimogawara K, Sano H (2004) Role of a nonselective de Novo DNA methyltransferase in maternal inheritance of chloroplast genes in the green alga, Chlamydomonas reinhardtii. Genetics 168:809–816. doi: 10.1534/genetics.104.030775 CrossRefPubMedPubMedCentralGoogle Scholar
  30. Plucinak, Horken KM, Jiang W, Fostvedt J, Nguyen ST, Weeks DP (2015) Improved and versatile viral 2A platforms for dependable and inducible high-level expression of dicistronic nuclear genes in Chlamydomonas reinhardtii. Plant J. doi: 10.1111/tpj.12844 PubMedGoogle Scholar
  31. Ponger L, Li W-H (2005) Evolutionary diversification of DNA methyltransferases in eukaryotic genomes. Mol Biol Evol 22:1119–1128. doi: 10.1093/molbev/msi098 CrossRefPubMedGoogle Scholar
  32. Quinn JM, Barraco P, Eriksson M, Merchant S (2000) Coordinate copper- and oxygen-responsive Cyc6 and Cpx1 expression in Chlamydomonas is mediated by the same element. J Biol Chem 275:6080–6089. doi: 10.1074/jbc.275.9.6080 CrossRefPubMedGoogle Scholar
  33. Rasala B, Lee P, Shen Z, Briggs SP, Mendez M, Mayfield SP (2012) Robust expression and secretion of Xylanase1 in Chlamydomonas reinhardtii by fusion to a selection gene and processing with the FMDV 2A peptide. PLoS One 7:e43349. doi: 10.1371/journal.pone.0043349 CrossRefPubMedPubMedCentralGoogle Scholar
  34. Rasala B, Barrera DJ, Ng J, Plucinak, Rosenberg JN, Weeks DP, Oyler G, Peterson TC, Haerizadeh F, Mayfield SP (2013) Expanding the spectral palette of fluorescent proteins for the green microalga Chlamydomonas reinhardtii. Plant J 74:545–556. doi: 10.1111/tpj.12165 CrossRefPubMedGoogle Scholar
  35. Ruecker O, Zillner K, Groebner-Ferreira R, Heitzer M (2008) Gaussia-luciferase as a sensitive reporter gene for monitoring promoter activity in the nucleus of the green alga Chlamydomonas reinhardtii. Mol Genet Genomics 280:153–162. doi: 10.1007/s00438-008-0352-3 CrossRefPubMedGoogle Scholar
  36. Sahdev S, Khattar SK, Saini KS (2008) Production of active eukaryotic proteins through bacterial expression systems: a review of the existing biotechnology strategies. Mol Cell Biochem 307:249–264. doi: 10.1007/s11010-007-9603-6 CrossRefPubMedGoogle Scholar
  37. Schroda M, Blöcker D, Beck CF (2000) The HSP70A promoter as a tool for the improved expression of transgenes in Chlamydomonas. Plant J 21:121–131CrossRefPubMedGoogle Scholar
  38. Shaver S, Casas-Mollano JA, Cerny RL, Cerutti H (2010) Origin of the polycomb repressive complex 2 and gene silencing by an E(z) homolog in the unicellular alga Chlamydomonas. Epigenetics 5:301–312.CrossRefPubMedGoogle Scholar
  39. Spolaore P, Joannis-Cassan C, Duran E, Isambert A (2006) Commercial applications of microalgae. J Biosci Bioeng 101:87–96. doi: 10.1263/jbb.101.87 CrossRefPubMedGoogle Scholar
  40. Strenkert D, Schmollinger S, Sommer F, Schulz-Raffelt M, Schroda M (2011) Transcription factor-dependent chromatin remodeling at heat shock and copper-responsive promoters in Chlamydomonas reinhardtii. Plant Cell 23:2285–2301. doi: 10.1105/tpc.111.085266 CrossRefPubMedPubMedCentralGoogle Scholar
  41. Strenkert D, Schmollinger S, Schroda M (2013) Heat shock factor 1 counteracts epigenetic silencing of nuclear transgenes in Chlamydomonas reinhardtii. Nucleic Acids Res 41:5273–5289. doi: 10.1093/nar/gkt224 CrossRefPubMedPubMedCentralGoogle Scholar
  42. Terpe K (2006) Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol 72:211–222. doi: 10.1007/s00253-006-0465-8 CrossRefPubMedGoogle Scholar
  43. Vaissière T, Sawan C, Herceg Z (2008) Epigenetic interplay between histone modifications and DNA methylation in gene silencing. Mutat Res Rev 659:40–48CrossRefGoogle Scholar
  44. Wu J, Grunstein M (2000) 25 years after the nucleosome model: chromatin modifications. Trends Biochem Sci 25:619–623. doi: 10.1016/S0968-0004(00)01718-7 CrossRefPubMedGoogle Scholar
  45. Yamano T, Iguchi H, Fukuzawa H (2013) Rapid transformation of Chlamydomonas reinhardtii without cell-wall removal. J Biosci Bioeng 115:691–694. doi: 10.1016/j.jbiosc.2012.12.020 CrossRefPubMedGoogle Scholar
  46. Yamasaki T, Ohama T (2011) Involvement of Elongin C in the spread of repressive histone modifications. Plant J 65:51–61. doi: 10.1111/j.1365-313X.2010.04400.x CrossRefPubMedGoogle Scholar
  47. Yamasaki T, Miyasaka H, Ohama T (2008) Unstable RNAi effects through epigenetic silencing of an inverted repeat transgene in Chlamydomonas reinhardtii. Genetics 180:1927–1944. doi: 10.1534/genetics.108.092395 CrossRefPubMedPubMedCentralGoogle Scholar
  48. Yamasaki T, Kim EJ, Cerutti H, Ohama T (2015) Argonaute3 is a key player in miRNA-mediated target cleavage and translational repression in Chlamydomonas. Plant J 258–268. doi: 10.1111/tpj.13107
  49. Yamasaki Y, Onishi M, Kim E-J, Cerutti H, Ohama T (2016) The RNA-binding protein DUS16 plays an essential role in primary miRNA processing in the unicellular alga Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 113:10720–10725. doi: 10.1073/pnas.1523230113
  50. Zhang C, Wu-Scharf D, Jeong B, Cerutti H (2002) A WD40-repeat containing protein, similar to a fungal co-repressor, is required for transcriptional gene silencing in Chlamydomonas. Plant J 31:25–36CrossRefPubMedGoogle Scholar
  51. Zhang R, Patena W, Armbruster U, Gang SS, Blum SR, Jonikas MC (2014) High-throughput genotyping of green algal mutants reveals random distribution of mutagenic insertion sites and endonucleolytic cleavage of transforming DNA. Plant Cell 1–13. doi: 10.1105/tpc.114.124099

Copyright information

© Springer Science+Business Media Dordrecht 2016

Authors and Affiliations

  • Sari Dewi Kurniasih
    • 1
  • Tomohito Yamasaki
    • 1
  • Fantao Kong
    • 1
  • Sigeru Okada
    • 2
  • Dwiyantari Widyaningrum
    • 1
  • Takeshi Ohama
    • 1
    Email author
  1. 1.School of Environmental Science and EngineeringKochi University of TechnologyKochiJapan
  2. 2.Laboratory of Aquatic Natural Products Chemistry, Graduate School of Agricultural & Life SciencesThe University of TokyoTokyoJapan

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