1887

Abstract

The chloroplast of and other microalgae represents an attractive new platform for the synthesis of recombinant therapeutics using synthetic biology (synbio) approaches. Transgenes can be designed , assembled from validated DNA parts and inserted at precise and predetermined locations within the chloroplast genome to give stable synthesis of a desired recombinant protein. Numerous recent examples of different therapeutic proteins produced successfully in the chloroplast highlight the potential of this green alga as a simple, low-cost and benign host. Furthermore, some of the features of the alga may offer additional advantages over more-established microbial, mammalian or plant-based systems. These include efficient folding and accumulation of the product in the chloroplast; a lack of contaminating toxins or infectious agents; reduced downstream processing requirements; the possibility to make complex therapeutics such as immunotoxins; and the opportunity to use the whole alga as a low-cost oral vaccine. In this paper we review the current status of algal chloroplast engineering with respect to therapeutic proteins. We also consider future advances in synbio tools, together with improvements to recipient strains, which will allow the design of bespoke strains with high levels of productivity.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.000599
2018-02-01
2024-03-29
Loading full text...

Full text loading...

/deliver/fulltext/micro/164/2/113.html?itemId=/content/journal/micro/10.1099/mic.0.000599&mimeType=html&fmt=ahah

References

  1. Murphy CD. The microbial cell factory. Org Biomol Chem 2012; 10:1949–1957 [View Article][PubMed]
    [Google Scholar]
  2. Wijffels RH, Kruse O, Hellingwerf KJ. Potential of industrial biotechnology with cyanobacteria and eukaryotic microalgae. Curr Opin Biotechnol 2013; 24:405–413 [View Article][PubMed]
    [Google Scholar]
  3. Guiry MD. How many species of algae are there?. J Phycol 2012; 48:1057–1063 [Crossref]
    [Google Scholar]
  4. Guarnieri MT, Pienkos PT. Algal omics: unlocking bioproduct diversity in algae cell factories. Photosynth Res 2015; 123:255–263 [View Article][PubMed]
    [Google Scholar]
  5. Gimpel JA, Specht EA, Georgianna DR, Mayfield SP. Advances in microalgae engineering and synthetic biology applications for biofuel production. Curr Opin Chem Biol 2013; 17:489–495 [View Article][PubMed]
    [Google Scholar]
  6. Scaife MA, Nguyen GT, Rico J, Lambert D, Helliwell KE et al. Establishing Chlamydomonas reinhardtii as an industrial biotechnology host. Plant J 2015; 82:532–546 [View Article][PubMed]
    [Google Scholar]
  7. Maul JE, Lilly JW, Cui L, Depamphilis CW, Miller W et al. The Chlamydomonas reinhardtii plastid chromosome: islands of genes in a sea of repeats. Plant Cell 2002; 14:2659–2679[PubMed] [Crossref]
    [Google Scholar]
  8. Purton S. Tools and techniques for chloroplast transformation of Chlamydomonas. Adv Exp Med Biol 2007; 616:34–45 [View Article][PubMed]
    [Google Scholar]
  9. Purton S, Szaub JB, Wannathong T, Young R, Economou CK. Genetic engineering of algal chloroplasts: progress and prospects. Rus J Plant Physiol 2013; 60:491–499 [View Article]
    [Google Scholar]
  10. Almaraz-Delgado AL, Flores-Uribe J, Pérez-España VH, Salgado-Manjarrez E, Badillo-Corona JA. Production of therapeutic proteins in the chloroplast of Chlamydomonas reinhardtii . AMB Express 2014; 4:57 [View Article][PubMed]
    [Google Scholar]
  11. Rasala BA, Mayfield SP. Photosynthetic biomanufacturing in green algae; production of recombinant proteins for industrial, nutritional, and medical uses. Photosynth Res 2015; 123:227–239 [View Article][PubMed]
    [Google Scholar]
  12. Hempel F, Maier UG. Microalgae as solar-powered protein factories. Adv Exp Med Biol 2016; 896:241–262 [View Article][PubMed]
    [Google Scholar]
  13. Green BR. Chloroplast genomes of photosynthetic eukaryotes. Plant J 2011; 66:34–44 [View Article][PubMed]
    [Google Scholar]
  14. Barkan A. Expression of plastid genes: organelle-specific elaborations on a prokaryotic scaffold. Plant Physiol 2011; 155:1520–1532 [View Article][PubMed]
    [Google Scholar]
  15. Boynton JE, Gillham NW, Harris EH, Hosler JP, Johnson AM et al. Chloroplast transformation in Chlamydomonas with high velocity microprojectiles. Science 1988; 240:1534–1538 [View Article][PubMed]
    [Google Scholar]
  16. Doron L, Segal N, Shapira M. Transgene expression in microalgae-from tools to applications. Front Plant Sci 2016; 7:505 [View Article][PubMed]
    [Google Scholar]
  17. Gutiérrez CL, Gimpel J, Escobar C, Marshall SH, Henríquez V. Chloroplast genetic tool for the green microalgae Haematococcus pluvialis (chlorophyceae, volvocales). J Phycol 2012; 48:976–983 [View Article][PubMed]
    [Google Scholar]
  18. Georgianna DR, Hannon MJ, Marcuschi M, Wu S, Botsch K et al. Production of recombinant enzymes in the marine alga Dunaliella tertiolecta . Algal Res 2013; 2:2–9 [View Article]
    [Google Scholar]
  19. Zienkiewicz M, Krupnik T, Drożak A, Golke A, Romanowska E. Transformation of the Cyanidioschyzon merolae chloroplast genome: prospects for understanding chloroplast function in extreme environments. Plant Mol Biol 2017; 93:171–183 [View Article][PubMed]
    [Google Scholar]
  20. Xie WH, Zhu CC, Zhang NS, Li DW, Yang WD et al. Construction of novel chloroplast expression vector and development of an efficient transformation system for the diatom Phaeodactylum tricornutum . Mar Biotechnol 2014; 16:538–546 [View Article][PubMed]
    [Google Scholar]
  21. Bock R. Engineering plastid genomes: methods, tools, and applications in basic research and biotechnology. Annu Rev Plant Biol 2015; 66:211–241 [View Article][PubMed]
    [Google Scholar]
  22. Zhang B, Shanmugaraj B, Daniell H. Expression and functional evaluation of biopharmaceuticals made in plant chloroplasts. Curr Opin Chem Biol 2017; 38:17–23 [View Article][PubMed]
    [Google Scholar]
  23. Chen Q, Davis KR. The potential of plants as a system for the development and production of human biologics. F1000Res 2016; 5:912 [View Article][PubMed]
    [Google Scholar]
  24. Yan N, Fan C, Chen Y, Hu Z. The potential for microalgae as bioreactors to produce pharmaceuticals. Int J Mol Sci 2016; 17:962 [View Article][PubMed]
    [Google Scholar]
  25. Zhao Y, Shi X, Zhang Z. High-frequency electroporation and expression of human interleukin 4 gene in Chlamydomonas reinhardtii chloroplast. Joumal of Huazhong Agricultural University 2006; 25:110–116
    [Google Scholar]
  26. Economou C, Wannathong T, Szaub J, Purton S. A simple, low-cost method for chloroplast transformation of the green alga Chlamydomonas reinhardtii . Methods Mol Biol 2014; 1132:401–411 [View Article][PubMed]
    [Google Scholar]
  27. Bertalan I, Munder MC, Weiß C, Kopf J, Fischer D et al. A rapid, modular and marker-free chloroplast expression system for the green alga Chlamydomonas reinhardtii . J Biotechnol 2015; 195:60–66 [View Article][PubMed]
    [Google Scholar]
  28. Wannathong T, Waterhouse JC, Young RE, Economou CK, Purton S. New tools for chloroplast genetic engineering allow the synthesis of human growth hormone in the green alga Chlamydomonas reinhardtii . Appl Microbiol Biotechnol 2016; 100:5467–5477 [View Article][PubMed]
    [Google Scholar]
  29. Young RE, Purton S. Codon reassignment to facilitate genetic engineering and biocontainment in the chloroplast of Chlamydomonas reinhardtii . Plant Biotechnol J 2016; 14:1251–1260 [View Article][PubMed]
    [Google Scholar]
  30. Ramundo S, Rochaix JD. Controlling expression of genes in the unicellular alga Chlamydomonas reinhardtii with a vitamin-repressible riboswitch. Methods Enzymol 2015; 550:267–281 [View Article][PubMed]
    [Google Scholar]
  31. Stoffels L, Taunt HN, Charalambous B, Purton S. Synthesis of bacteriophage lytic proteins against Streptococcus pneumoniae in the chloroplast of Chlamydomonas reinhardtii . Plant Biotechnol J 2017; 15:1130–1140 [View Article][PubMed]
    [Google Scholar]
  32. Su ZL, Qian KX, Tan CP, Meng CX, Qin S. Recombination and heterologous expression of allophycocyanin gene in the chloroplast of Chlamydomonas reinhardtii . Acta Biochim Biophys Sin 2005; 37:709–712[PubMed] [Crossref]
    [Google Scholar]
  33. Tran M, Zhou B, Pettersson PL, Gonzalez MJ, Mayfield SP. Synthesis and assembly of a full-length human monoclonal antibody in algal chloroplasts. Biotechnol Bioeng 2009; 104:n/a–673 [View Article][PubMed]
    [Google Scholar]
  34. Zedler JAZ, Mullineaux CW, Robinson C. Efficient targeting of recombinant proteins to the thylakoid lumen in Chlamydomonas reinhardtii using a bacterial Tat signal peptide. Algal Res 2016; 19:57–62 [View Article]
    [Google Scholar]
  35. Gangl D, Zedler JA, Włodarczyk A, Jensen PE, Purton S et al. Expression and membrane-targeting of an active plant cytochrome P450 in the chloroplast of the green alga Chlamydomonas reinhardtii . Phytochemistry 2015; 110:22–28 [View Article][PubMed]
    [Google Scholar]
  36. Jones CS, Mayfield SP. Steps toward a globally available malaria vaccine: harnessing the potential of algae for future low cost vaccines. Bioengineered 2013; 4:164–167 [View Article][PubMed]
    [Google Scholar]
  37. Dreesen IA, Charpin-El Hamri G, Fussenegger M. Heat-stable oral alga-based vaccine protects mice from Staphylococcus aureus infection. J Biotechnol 2010; 145:273–280 [View Article][PubMed]
    [Google Scholar]
  38. Gregory JA, Topol AB, Doerner DZ, Mayfield S. Alga-produced cholera toxin-Pfs25 fusion proteins as oral vaccines. Appl Environ Microbiol 2013; 79:3917–3925 [View Article][PubMed]
    [Google Scholar]
  39. Rosales-Mendoza S. Algae-made vaccines targeting human diseases. In Biopharmaceuticals Algae-Based Springer, Cham: 2016 [Crossref]
    [Google Scholar]
  40. Gupta SK, Shukla P. Advanced technologies for improved expression of recombinant proteins in bacteria: perspectives and applications. Crit Rev Biotechnol 2016; 36:1089–1098 [View Article][PubMed]
    [Google Scholar]
  41. Matthews CB, Wright C, Kuo A, Colant N, Westoby M et al. Reexamining opportunities for therapeutic protein production in eukaryotic microorganisms. Biotechnol Bioeng 2017; 114:2432–2444 [View Article][PubMed]
    [Google Scholar]
  42. Mayfield SP, Franklin SE, Lerner RA. Expression and assembly of a fully active antibody in algae. Proc Natl Acad Sci USA 2003; 100:438–442 [View Article][PubMed]
    [Google Scholar]
  43. Tran M, Henry RE, Siefker D, van C, Newkirk G et al. Production of anti-cancer immunotoxins in algae: ribosome inactivating proteins as fusion partners. Biotechnol Bioeng 2013; 110:2826–2835 [View Article][PubMed]
    [Google Scholar]
  44. Tran M, van C, Barrera DJ, Pettersson PL, Peinado CD et al. Production of unique immunotoxin cancer therapeutics in algal chloroplasts. Proc Natl Acad Sci USA 2013; 110:E15E22 [View Article][PubMed]
    [Google Scholar]
  45. Ochoa-Méndez CE, Lara-Hernández I, González LM, Aguirre-Bañuelos P, Ibarra-Barajas M et al. Bioactivity of an antihypertensive peptide expressed in Chlamydomonas reinhardtii . J Biotechnol 2016; 240:76–84 [View Article][PubMed]
    [Google Scholar]
  46. Yang Z, Li Yinü, Chen F, Li D, Zhang Z et al. Expression of human soluble TRAIL in Chlamydomonas reinhardtii chloroplast. Chin Sci Bull 2006; 51:1703–1709 [View Article]
    [Google Scholar]
  47. Rasala BA, Muto M, Lee PA, Jager M, Cardoso RM et al. Production of therapeutic proteins in algae, analysis of expression of seven human proteins in the chloroplast of Chlamydomonas reinhardtii . Plant Biotechnol J 2010; 8:719–733 [View Article][PubMed]
    [Google Scholar]
  48. Wang X, Brandsma M, Tremblay R, Maxwell D, Jevnikar AM et al. A novel expression platform for the production of diabetes-associated autoantigen human glutamic acid decarboxylase (hGAD65). BMC Biotechnol 2008; 8:87 [View Article][PubMed]
    [Google Scholar]
  49. Ferreira F, Wolf M, Wallner M. Molecular approach to allergy diagnosis and therapy. Yonsei Med J 2014; 55:839–852 [View Article][PubMed]
    [Google Scholar]
  50. Gregory JA, Shepley-Mctaggart A, Umpierrez M, Hurlburt BK, Maleki SJ et al. Immunotherapy using algal-produced Ara h 1 core domain suppresses peanut allergy in mice. Plant Biotechnol J 2016; 14:1541–1550 [View Article][PubMed]
    [Google Scholar]
  51. Larson MA, Wei SH, Weber A, Mack DR, McDonald TL. Human serum amyloid A3 peptide enhances intestinal MUC3 expression and inhibits EPEC adherence. Biochem Biophys Res Commun 2003; 300:531–540 [View Article][PubMed]
    [Google Scholar]
  52. Manuell AL, Beligni MV, Elder JH, Siefker DT, Tran M et al. Robust expression of a bioactive mammalian protein in Chlamydomonas chloroplast. Plant Biotechnol J 2007; 5:402–412 [View Article][PubMed]
    [Google Scholar]
  53. Humer E, Schwarz C, Schedle K. Phytate in pig and poultry nutrition. J Anim Physiol Anim Nutr 2015; 99:605–625 [View Article][PubMed]
    [Google Scholar]
  54. Yoon SM, Kim SY, Li KF, Yoon BH, Choe S et al. Transgenic microalgae expressing Escherichia coli AppA phytase as feed additive to reduce phytate excretion in the manure of young broiler chicks. Appl Microbiol Biotechnol 2011; 91:553–563 [View Article][PubMed]
    [Google Scholar]
  55. Erpel F, Restovic F, Arce-Johnson P. Development of phytase-expressing chlamydomonas reinhardtii for monogastric animal nutrition. BMC Biotechnol 2016; 16:29 [View Article][PubMed]
    [Google Scholar]
  56. Noor-Mohammadi S, Pourmir A, Johannes TW. Method to assemble and integrate biochemical pathways into the chloroplast genome of Chlamydomonas reinhardtii . Biotechnol Bioeng 2012; 109:2896–2903 [View Article][PubMed]
    [Google Scholar]
  57. Oey M, Ross IL, Hankamer B. Gateway-assisted vector construction to facilitate expression of foreign proteins in the chloroplast of single celled algae. PLoS One 2014; 9:e86841 [View Article][PubMed]
    [Google Scholar]
  58. Scharff LB, Bock R. Synthetic biology in plastids. Plant J 2014; 78:783–798 [View Article][PubMed]
    [Google Scholar]
  59. Gimpel JA, Nour-Eldin HH, Scranton MA, Li D, Mayfield SP. Refactoring the six-gene Photosystem II core in the chloroplast of the green algae Chlamydomonas reinhardtii . ACS Synth Biol 2016; 5:589–596 [View Article][PubMed]
    [Google Scholar]
  60. O'Neill BM, Mikkelson KL, Gutierrez NM, Cunningham JL, Wolff KL et al. An exogenous chloroplast genome for complex sequence manipulation in algae. Nucleic Acids Res 2012; 40:2782–2792 [View Article][PubMed]
    [Google Scholar]
  61. Rasala BA, Muto M, Sullivan J, Mayfield SP. Improved heterologous protein expression in the chloroplast of Chlamydomonas reinhardtii through promoter and 5' untranslated region optimization. Plant Biotechnol J 2011; 9:674–683 [View Article][PubMed]
    [Google Scholar]
  62. Coragliotti AT, Beligni MV, Franklin SE, Mayfield SP. Molecular factors affecting the accumulation of recombinant proteins in the Chlamydomonas reinhardtii chloroplast. Mol Biotechnol 2011; 48:60–75 [View Article][PubMed]
    [Google Scholar]
  63. Choquet Y, Wollman FA. Translational regulations as specific traits of chloroplast gene expression. FEBS Lett 2002; 529:39–42 [View Article][PubMed]
    [Google Scholar]
  64. Gimpel JA, Hyun JS, Schoepp NG, Mayfield SP. Production of recombinant proteins in microalgae at pilot greenhouse scale. Biotechnol Bioeng 2015; 112:339–345 [View Article][PubMed]
    [Google Scholar]
  65. Specht EA, Mayfield SP. Synthetic oligonucleotide libraries reveal novel regulatory elements in Chlamydomonas chloroplast mRNAs. ACS Synth Biol 2013; 2:34–46 [View Article][PubMed]
    [Google Scholar]
  66. Radzun KA, Wolf J, Jakob G, Zhang E, Stephens E et al. Automated nutrient screening system enables high-throughput optimisation of microalgae production conditions. Biotechnol Biofuels 2015; 8:65 [View Article][PubMed]
    [Google Scholar]
  67. Gupta PL, Lee SM, Choi HJ. A mini review: photobioreactors for large scale algal cultivation. World J Microbiol Biotechnol 2015; 31:1409–1417 [View Article][PubMed]
    [Google Scholar]
  68. Cazzaniga S, dall'osto L, Szaub J, Scibilia L, Ballottari M et al. Domestication of the green alga Chlorella sorokiniana: reduction of antenna size improves light-use efficiency in a photobioreactor. Biotechnol Biofuels 2014; 7:157 [View Article][PubMed]
    [Google Scholar]
  69. Bumbak F, Cook S, Zachleder V, Hauser S, Kovar K. Best practices in heterotrophic high-cell-density microalgal processes: achievements, potential and possible limitations. Appl Microbiol Biotechnol 2011; 91:31–46 [View Article][PubMed]
    [Google Scholar]
  70. Doebbe A, Rupprecht J, Beckmann J, Mussgnug JH, Hallmann A et al. Functional integration of the HUP1 hexose symporter gene into the genome of C. reinhardtii: Impacts on biological H(2) production. J Biotechnol 2007; 131:27–33 [View Article][PubMed]
    [Google Scholar]
  71. Sun M, Qian K, Su N, Chang H, Liu J et al. Foot-and-mouth disease virus VP1 protein fused with cholera toxin B subunit expressed in Chlamydomonas reinhardtii chloroplast. Biotechnol Lett 2003; 25:1087–1092 [View Article][PubMed]
    [Google Scholar]
  72. He DM, Qian KX, Shen GF, Zhang ZF, Li YN et al. Recombination and expression of classical swine fever virus (CSFV) structural protein E2 gene in Chlamydomonas reinhardtii chroloplasts. Colloids Surf B Biointerfaces 2007; 55:26–30 [View Article][PubMed]
    [Google Scholar]
  73. Siripornadulsil S, Dabrowski K, Sayre R. Microalgal vaccines. Adv Exp Med Biol 2007; 616:122–128 [View Article][PubMed]
    [Google Scholar]
  74. Surzycki R, Greenham K, Kitayama K, Dibal F, Wagner R et al. Factors effecting expression of vaccines in microalgae. Biologicals 2009; 37:133–138 [View Article][PubMed]
    [Google Scholar]
  75. Michelet L, Lefebvre-Legendre L, Burr SE, Rochaix JD, Goldschmidt-Clermont M. Enhanced chloroplast transgene expression in a nuclear mutant of Chlamydomonas. Plant Biotechnol J 2011; 9:565–574 [View Article][PubMed]
    [Google Scholar]
  76. Gregory JA, Li F, Tomosada LM, Cox CJ, Topol AB et al. Algae-produced Pfs25 elicits antibodies that inhibit malaria transmission. PLoS One 2012; 7:e37179 [View Article][PubMed]
    [Google Scholar]
  77. Jones CS, Luong T, Hannon M, Tran M, Gregory JA et al. Heterologous expression of the C-terminal antigenic domain of the malaria vaccine candidate Pfs48/45 in the green algae Chlamydomonas reinhardtii . Appl Microbiol Biotechnol 2013; 97:1987–1995 [View Article][PubMed]
    [Google Scholar]
  78. Demurtas OC, Massa S, Ferrante P, Venuti A, Franconi R et al. A Chlamydomonas-derived human papillomavirus 16 E7 vaccine induces specific tumor protection. PLoS One 2013; 8:e61473 [View Article][PubMed]
    [Google Scholar]
  79. Vlasák J, Bøíza J, Š R, Ludvíková V. Alga-based HPV16 E7 vaccine elicits specific immune response in mice. Asian J Plant Sci Res 2013; 3:141–148
    [Google Scholar]
  80. Castellanos-Huerta I, Bañuelos-Hernández B, Téllez G, Rosales-Mendoza S, Brieba LG et al. Recombinant hemagglutinin of avian influenza virus H5 expressed in the chloroplast of Chlamydomonas reinhardtii and evaluation of its immunogenicity in chickens. Avian Dis 2016; 60:784–791 [View Article][PubMed]
    [Google Scholar]
  81. Beltrán-López JI, Romero-Maldonado A, Monreal-Escalante E, Bañuelos-Hernández B, Paz-Maldonado LM et al. Chlamydomonas reinhardtii chloroplasts express an orally immunogenic protein targeting the p210 epitope implicated in atherosclerosis immunotherapies. Plant Cell Rep 2016; 35:1133–1141 [View Article][PubMed]
    [Google Scholar]
  82. Hirschl S, Ralser C, Asam C, Gangitano A, Huber S et al. Expression and characterization of functional recombinant Bet v 1.0101 in the chloroplast of Chlamydomonas reinhardtii . Int Arch Allergy Immunol 2017; 173:44–50 [View Article][PubMed]
    [Google Scholar]
  83. Barrera DJ, Rosenberg JN, Chiu JG, Chang YN, Debatis M et al. Algal chloroplast produced camelid VH H antitoxins are capable of neutralizing botulinum neurotoxin. Plant Biotechnol J 2015; 13:117–124 [View Article][PubMed]
    [Google Scholar]
  84. Campos-Quevedo N, Rosales-Mendoza S, Paz-Maldonado LMT, Martínez-Salgado L, Guevara-Arauza JC et al. Production of milk-derived bioactive peptides as precursor chimeric proteins in chloroplasts of Chlamydomonas reinhardtii . Plant Cell, Tiss Organ Cult 2013; 113:217–225 [View Article]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.000599
Loading
/content/journal/micro/10.1099/mic.0.000599
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error