Gene Targeting for Precision Glyco-Engineering: Production of Biopharmaceuticals Devoid of Plant-Typical Glycosylation in Moss Bioreactors

  • Eva L. Decker
  • Gertrud Wiedemann
  • Ralf Reski
Part of the Methods in Molecular Biology book series (MIMB, volume 1321)


One of the main challenges for the production of biopharmaceuticals in plant-based systems is the modulation of plant-specific glycosylation patterns towards a humanized form. Posttranslational modifications in plants are similar to those in humans, but several differences affect product quality and efficacy and can also cause immune responses in patients. In the moss Physcomitrella patens highly efficient gene targeting via homologous recombination enables glyco-engineering to obtain suitable platform lines for the production of recombinant proteins and biopharmaceuticals. Here we describe the methods which are effective for creating gene targeting constructs and transgenic moss lines as well as confirming successful homologous integration of the constructs and modification of target gene expression.

Key words

Physcomitrella patens Gene targeting Homologous recombination Knockout construct Protoplast transformation Glyco-engineering Biopharmaceutical production Plant-made pharmaceuticals Molecular farming 



This work was supported by contract research “Glykobiologie/Glykomik” of the Baden-Wuerttemberg Stiftung, by the Excellence Initiative of the German Federal and State Governments (EXC294 to R.R.), and EU-co-funded by INTERREG IV Project A17 “ITP-TIP” (ERDF). We thank Anne Katrin Prowse for proofreading of the manuscript.


  1. 1.
    Hohe A, Decker EL, Gorr G et al (2002) Tight control of growth and cell differentiation in photoautotrophically growing moss Physcomitrella patens bioreactor cultures. Plant Cell Rep 20:1135–1140CrossRefGoogle Scholar
  2. 2.
    Cerff M, Posten C (2012) Enhancing the growth of Physcomitrella patens by combination of monochromatic red and blue light—a kinetic study. Biotechnol J 7:527–536PubMedCrossRefGoogle Scholar
  3. 3.
    Hohe A, Schween G, Reski R (2001) Establishment of a semicontinuous bioreactor culture of Physcomitrella patens for mass production of protoplasts. Acta Hortic 560:425–428Google Scholar
  4. 4.
    Reutter K, Reski R (1996) Production of a heterologous protein in bioreactor cultures of fully differentiated moss plants. Plant Tissue Cult Biotechnol 2:142–147Google Scholar
  5. 5.
    Hohe A, Reski R (2002) Optimisation of a bioreactor culture of the moss Physcomitrella patens for mass production of protoplasts. Plant Sci 163:69–74CrossRefGoogle Scholar
  6. 6.
    Decker EL, Reski R (2004) The moss bioreactor. Curr Opin Plant Biol 7:166–170PubMedCrossRefGoogle Scholar
  7. 7.
    Hohe A, Reski R (2005) From axenic spore germination to molecular farming. Plant Cell Rep 23:513–521PubMedCrossRefGoogle Scholar
  8. 8.
    Lucumi A, Posten C (2006) Establishment of long-term perfusion cultures of recombinant moss in a pilot tubular photobioreactor. Proc Biochem 41:2180–2187CrossRefGoogle Scholar
  9. 9.
    Egener T, Granado M, Guitton M-C et al (2002) High frequency of phenotypic deviations in Physcomitrella patens plants transformed with a gene-disruption library. BMC Plant Biol 2:6PubMedCrossRefPubMedCentralGoogle Scholar
  10. 10.
    Decker EL, Reski R (2008) Current achievements in the production of complex biopharmaceuticals with moss bioreactor. Bioprocess Biosyst Eng 31:3–9PubMedCrossRefGoogle Scholar
  11. 11.
    Decker EL, Reski R (2007) Moss bioreactors producing improved biopharmaceuticals. Curr Opin Biotechnol 18:393–398PubMedCrossRefGoogle Scholar
  12. 12.
    Decker EL, Reski R (2012) Glycoprotein production in moss bioreactors. Plant Cell Rep 31:453–460PubMedCrossRefGoogle Scholar
  13. 13.
    Schaaf A, Tintelnot S, Baur A et al (2005) Use of endogenous signal sequences for transient production and efficient secretion by moss (Physcomitrella patens) cells. BMC Biotechnol 5:30PubMedCrossRefPubMedCentralGoogle Scholar
  14. 14.
    Weise A, Altmann F, Rodriguez-Franco M et al (2007) High-level expression of secreted complex glycosylated recombinant human erythropoietin in the Physcomitrella Delta-fuc-t Delta-xyl-t mutant. Plant Biotechnol J 5:389–401PubMedCrossRefGoogle Scholar
  15. 15.
    Rensing SA, Lang D, Zimmer A et al (2008) The Physcomitrella genome reveals insights into the conquest of land by plants. Science 319:64–69PubMedCrossRefGoogle Scholar
  16. 16.
    Zimmer AD, Lang D, Buchta K et al (2013) Reannotation and extended community resources for the genome of the non-seed plant Physcomitrella patens provide insights into the evolution of plant gene structures and functions. BMC Genomics 14:498PubMedCrossRefPubMedCentralGoogle Scholar
  17. 17.
    Hiss M, Laule O, Meskauskiene RM et al (2014) Large scale gene expression profiling data of the model moss Physcomitrella patens help to understand developmental progression, culture and stress conditions. Plant J. 79:530–539Google Scholar
  18. 18.
    Strepp R, Scholz S, Kruse S et al (1998) Plant nuclear gene knockout reveals a role in plastid division for the homolog of the bacterial cell division protein FtsZ, an ancestral tubulin. Proc Natl Acad Sci U S A 95:4368–4373PubMedCrossRefPubMedCentralGoogle Scholar
  19. 19.
    Lorenz S, Tintelnot S, Reski R, Decker EL (2003) Cyclin D-knockout uncouples developmental progression from sugar availability. Plant Mol Biol 53:227–236PubMedCrossRefGoogle Scholar
  20. 20.
    Wiedemann G, Koprivova A, Schneider M et al (2007) The role of the novel adenosine 5′-phosphosulfate reductase in regulation of sulfate assimilation of Physcomitrella patens. Plant Mol Biol 65:667–676PubMedCrossRefGoogle Scholar
  21. 21.
    Wiedemann G, Hermsen C, Melzer M et al (2010) Targeted knock-out of a gene encoding sulfite reductase in the moss Physcomitrella patens affects gametophytic and sporophytic development. FEBS Lett 584:2271–2278PubMedCrossRefGoogle Scholar
  22. 22.
    Khraiwesh B, Arif MA, Seumel GI et al (2010) Transcriptional control of gene expression by microRNAs. Cell 140:111–122PubMedCrossRefGoogle Scholar
  23. 23.
    Kamisugi Y, Cuming AC, Cove DJ (2005) Parameters determining the efficiency of gene targeting in the moss Physcomitrella patens. Nucleic Acids Res 33:6205–6216Google Scholar
  24. 24.
    Schaefer DG (2001) Gene targeting in Physcomitrella patens. Curr Opin Plant Biol 4:143–150PubMedCrossRefGoogle Scholar
  25. 25.
    Hohe A, Reski R (2003) A tool for understanding homologous recombination in plants. Plant Cell Rep 21:1135–1142PubMedCrossRefGoogle Scholar
  26. 26.
    Koprivova A, Stemmer C, Altmann F et al (2004) Targeted knockouts of Physcomitrella lacking plant-specific immunogenic N-glycans. Plant Biotechnol J 2:517–523PubMedCrossRefGoogle Scholar
  27. 27.
    Huether CM, Lienhart O, Stemmer C et al (2005) Glyco-engineering of moss lacking plant-specific sugar residues. Plant Biol 7:292–299PubMedCrossRefGoogle Scholar
  28. 28.
    Parsons J, Altmann F, Arrenberg CK et al (2012) Moss-based production of asialo-erythropoietin devoid of Lewis A and other plant-typical carbohydrate determinants. Plant Biotechnol J 10:851–861PubMedCrossRefGoogle Scholar
  29. 29.
    Parsons J, Altmann F, Graf M et al (2013) A gene responsible for prolyl-hydroxylation of moss-produced recombinant human erythropoietin. Sci Rep 3:3019PubMedCrossRefPubMedCentralGoogle Scholar
  30. 30.
    Kubo M, Imai A, Nishiyama T et al (2013) System for stable β-estradiol-inducible gene expression in the moss Physcomitrella patens. PLoS One 8, e77356PubMedCrossRefPubMedCentralGoogle Scholar
  31. 31.
    Mosquna A, Katz A, Decker EL et al (2009) Regulation of stem cell maintenance by the Polycomb protein FIE has been conserved during land plant evolution. Development 136:2433–2444PubMedCrossRefGoogle Scholar
  32. 32.
    Mueller SJ, Lang D, Hoernstein SNW et al (2014) Quantitative analysis of the mitochondrial and plastid proteomes of the moss Physcomitrella patens reveals protein macrocompartmentation and microcompartmentation. Plant Physiol 164:2081–2095PubMedCrossRefPubMedCentralGoogle Scholar
  33. 33.
    Hohe A, Egener T, Lucht JM et al (2004) An improved and highly standardised transformation procedure allows efficient production of single and multiple targeted gene-knockouts in a moss, Physcomitrella patens. Curr Genet 44:339–347PubMedCrossRefGoogle Scholar
  34. 34.
    Maas C, Werr W (1989) Mechanism and optimized conditions for PEG mediated DNA transfection into plant protoplasts. Plant Cell Rep 8:148–151PubMedCrossRefGoogle Scholar
  35. 35.
    Reski R (1998) Development, genetics and molecular biology of mosses. Bot Acta 111:1–15CrossRefGoogle Scholar
  36. 36.
    Kamisugi Y, Cuming AC (2009) Gene targeting. In: Perroud P-F, Cove D, Knight C (eds) Annu. Plant Rev. Moss Physcomitrella patens. Blackwell Publishing, Chichester, pp 76–105Google Scholar
  37. 37.
    Strotbek C, Krinninger S, Frank W (2013) The moss Physcomitrella patens: methods and tools from cultivation to targeted analysis of gene function. Int J Dev Biol 57:553–564PubMedCrossRefGoogle Scholar
  38. 38.
    Schween G, Fleig S, Reski R (2002) High-throughput-PCR screen of 15,000 transgenic Physcomitrella plants. Plant Mol Biol Rep 20:43–47CrossRefGoogle Scholar
  39. 39.
    Büttner-Mainik A, Parsons J, Jérome H et al (2011) Production of biologically active recombinant human factor H in Physcomitrella. Plant Biotechnol J 9:373–383PubMedCrossRefGoogle Scholar
  40. 40.
    Decker EL, Parsons J, Reski R (2014) Glyco-engineering for biopharmaceutical production in moss bioreactors. Front Plant Sci 5:346PubMedCrossRefPubMedCentralGoogle Scholar
  41. 41.
    Schulte J, Reski R (2004) High throughput cryopreservation of 140,000 Physcomitrella patens mutants. Plant Biol 6:119–127PubMedCrossRefGoogle Scholar
  42. 42.
    Reski R, Abel WO (1985) Induction of budding on chloronemata and caulonemata of the moss, Physcomitrella patens, using isopentenyladenine. Planta 165:354–358PubMedCrossRefGoogle Scholar
  43. 43.
    Schween G, Hohe A, Koprivova A, Reski R (2003) Effects of nutrients, cell density and culture techniques on protoplast regeneration and early protonema development in a moss, Physcomitrella patens. J Plant Physiol 160:209–212PubMedCrossRefGoogle Scholar
  44. 44.
    Sambrook J, Russell DW, Cold Spring Harbor Laboratory (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NYGoogle Scholar
  45. 45.
    Gibson DG, Young L, Chuang R-Y et al (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6:343–345PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Eva L. Decker
    • 1
  • Gertrud Wiedemann
    • 1
    • 2
  • Ralf Reski
    • 1
    • 2
    • 3
    • 4
  1. 1.Plant Biotechnology, Faculty of BiologyUniversity of FreiburgFreiburgGermany
  2. 2.TIP Trinational Institute for Plant Research and Plant Biotechnology, Faculty of BiologyUniversity of FreiburgFreiburgGermany
  3. 3.BIOSS Centre for Biological Signalling StudiesFreiburgGermany
  4. 4.FRIAS Freiburg Institute for Advanced StudiesFreiburgGermany

Personalised recommendations