The Potential for Microalgae as Bioreactors to Produce Pharmaceuticals

As photosynthetic organisms, microalgae can efficiently convert solar energy into biomass. Microalgae are currently used as an important source of valuable natural biologically active molecules, such as carotenoids, chlorophyll, long-chain polyunsaturated fatty acids, phycobiliproteins, carotenoids and enzymes. Significant advances have been achieved in microalgae biotechnology over the last decade, and the use of microalgae as bioreactors for expressing recombinant proteins is receiving increased interest. Compared with the bioreactor systems that are currently in use, microalgae may be an attractive alternative for the production of pharmaceuticals, recombinant proteins and other valuable products. Products synthesized via the genetic engineering of microalgae include vaccines, antibodies, enzymes, blood-clotting factors, immune regulators, growth factors, hormones, and other valuable products, such as the anticancer agent Taxol. In this paper, we briefly compare the currently used bioreactor systems, summarize the progress in genetic engineering of microalgae, and discuss the potential for microalgae as bioreactors to produce pharmaceuticals.

[1]  Kyle Botsch,et al.  Production of recombinant enzymes in the marine alga Dunaliella tertiolecta , 2013 .

[2]  C. Walter,et al.  Microalgal Biotechnology: Potential and Production , 2012 .

[3]  C. Meng,et al.  Recombination and heterologous expression of allophycocyanin gene in the chloroplast of Chlamydomonas reinhardtii. , 2005, Acta biochimica et biophysica Sinica.

[4]  Lexun Xue,et al.  Improvement of efficiency of genetic transformation for Dunaliella salina by glass beads method , 2009, Molecular Biology Reports.

[5]  Lei Fang,et al.  Genomic Foundation of Starch-to-Lipid Switch in Oleaginous Chlorella spp.1 , 2015, Plant Physiology.

[6]  S. Prusiner,et al.  Compelling transgenetic evidence for transmission of bovine spongiform encephalopathy prions to humans. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[7]  P. Elzen,et al.  Production of Correctly Processed Human Serum Albumin in Transgenic Plants , 1990, Bio/Technology.

[8]  Richard T. Sayre,et al.  Molecular Mechanisms of Proline-Mediated Tolerance to Toxic Heavy Metals in Transgenic Microalgae , 2002, The Plant Cell Online.

[9]  S. Qiu,et al.  Selenoprotein-Transgenic Chlamydomonas reinhardtii , 2013, Nutrients.

[10]  P. Lefebvre,et al.  Stable nuclear transformation of Chlamydomonas using the Chlamydomonas gene for nitrate reductase , 1989, The Journal of cell biology.

[11]  Esther Vázquez,et al.  Microbial factories for recombinant pharmaceuticals , 2009 .

[12]  Feng Chen,et al.  Biology and Industrial Applications of Chlorella: Advances and Prospects. , 2016, Advances in biochemical engineering/biotechnology.

[13]  Nicole Poulsen,et al.  MOLECULAR GENETIC MANIPULATION OF THE DIATOM THALASSIOSIRA PSEUDONANA (BACILLARIOPHYCEAE) 1 , 2006 .

[14]  Miller Tran,et al.  Robust expression of a bioactive mammalian protein in Chlamydomonas chloroplast. , 2007, Plant biotechnology journal.

[15]  Wolfgang Becker,et al.  Microalgae in human and animal nutrition. , 2007 .

[16]  Stephen M. Miller,et al.  Overexpression of Calvin cycle enzyme fructose 1,6-bisphosphatase in Chlamydomonas reinhardtii has a detrimental effect on growth , 2016 .

[17]  David J. Miller,et al.  Genetic transformation of dinoflagellates (Amphidinium and Symbiodinium): expression of GUS in microalgae using heterologous promoter constructs , 1998 .

[18]  J. Rochaix,et al.  The argininosuccinate lyase gene of Chlamydomonas reinhardtii: an important tool for nuclear transformation and for correlating the genetic and molecular maps of the ARG7 locus. , 1989, The EMBO journal.

[19]  H. Koprowski,et al.  Expression in plants and immunogenicity of plant virus-based experimental rabies vaccine. , 2002, Vaccine.

[20]  S. Rosales-Mendoza,et al.  Food-Grade Organisms as Vaccine Biofactories and Oral Delivery Vehicles. , 2016, Trends in biotechnology.

[21]  T. Tonon,et al.  Long chain polyunsaturated fatty acid production and partitioning to triacylglycerols in four microalgae. , 2002, Phytochemistry.

[22]  Nicholas H. Putnam,et al.  The Genome of the Diatom Thalassiosira Pseudonana: Ecology, Evolution, and Metabolism , 2004, Science.

[23]  Juliane Neupert,et al.  Efficient expression of nuclear transgenes in the green alga Chlamydomonas: synthesis of an HIV antigen and development of a new selectable marker , 2016, Plant Molecular Biology.

[24]  C. Ascaso,et al.  CORRELATED BIOCHEMICAL AND ULTRASTRUCTURAL CHANGES IN NITROGEN‐STARVED EUGLENA GRACILIS 1 , 1996 .

[25]  S. Tabata,et al.  Complete genome structure of the unicellular cyanobacterium Synechocystis sp. PCC6803. , 1997, Plant & cell physiology.

[26]  R. Fischer,et al.  Molecular farming of pharmaceutical proteins , 2004, Transgenic Research.

[27]  E. Jin,et al.  Development of a new constitutive expression system for the transformation of the diatom Phaeodactylum tricornutum , 2015 .

[28]  S. Yang,et al.  Development of stable marker-free nuclear transformation strategy in the green microalga Chlorella vulgaris , 2015 .

[29]  T. Kadono,et al.  Nuclear transformation of the diatom Phaeodactylum tricornutum using PCR-amplified DNA fragments by microparticle bombardment. , 2016, Marine genomics.

[30]  Mohammad Hossein Morowvat,et al.  PCR amplification of 18S rRNA, single cell protein production and fatty acid evaluation of some naturally isolated microalgae , 2009 .

[31]  Richard L. Hawkins,et al.  Expression of Human Growth Hormone by the Eukaryotic Alga, Chlorella , 1999, Current Microbiology.

[32]  E. Apostolova,et al.  Plant molecular farming: Opportunities and challenges , 2013 .

[33]  Antonio Villaverde,et al.  Unconventional microbial systems for the cost-efficient production of high-quality protein therapeutics. , 2013, Biotechnology advances.

[34]  S. Briggs,et al.  Heterologous expression of the C-terminal antigenic domain of the malaria vaccine candidate Pfs48/45 in the green algae Chlamydomonas reinhardtii , 2012, Applied Microbiology and Biotechnology.

[35]  S. Vuttipongchaikij,et al.  Increasing the Triacylglycerol Content in Dunaliella tertiolecta through Isolation of Starch-Deficient Mutants. , 2016, Journal of microbiology and biotechnology.

[36]  N. Natarajan,et al.  Immunogenicity in humans of an edible vaccine for hepatitis B. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[37]  J. Boothe,et al.  Transgenic plants for therapeutic proteins: linking upstream and downstream strategies. , 1999, Current topics in microbiology and immunology.

[38]  Jue Ruan,et al.  Overexpression of the soybean transcription factor GmDof4 significantly enhances the lipid content of Chlorella ellipsoidea , 2014, Biotechnology for Biofuels.

[39]  U. Maier,et al.  Distribution of the SELMA Translocon in Secondary Plastids of Red Algal Origin and Predicted Uncoupling of Ubiquitin-Dependent Translocation from Degradation , 2012, Eukaryotic Cell.

[40]  P. Maas,et al.  Time Lag and Nutrient Storage Effects in the Transient Growth Response of Chlamydomonas reinhardii in Nitrogen-limited Batch and Continuous Culture , 1978 .

[41]  Sara L. Zimmer,et al.  The Chlamydomonas Genome Reveals the Evolution of Key Animal and Plant Functions , 2007, Science.

[42]  S. Ball,et al.  Engineering the Chloroplast Targeted Malarial Vaccine Antigens in Chlamydomonas Starch Granules , 2010, PloS one.

[43]  Malcolm R. Brown,et al.  Nutritional properties of microalgae for mariculture , 1997 .

[44]  D K Robinson,et al.  Industrial choices for protein production by large-scale cell culture. , 2001, Current opinion in biotechnology.

[45]  R. Meints,et al.  Recombinant viruses as transformation vectors of marine macroalgae , 1994, Journal of Applied Phycology.

[46]  Randor Radakovits,et al.  Draft genome sequence and genetic transformation of the oleaginous alga Nannochloropis gaditana , 2012, Nature Communications.

[47]  José Luis Guil-Guerrero,et al.  Functional properties of the biomass of three microalgal species , 2004 .

[48]  Richard A Lerner,et al.  Expression and assembly of a fully active antibody in algae , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[49]  L. Doré,et al.  N 6-Methyldeoxyadenosine Marks Active Transcription Start Sites in Chlamydomonas , 2015, Cell.

[50]  Christoph Griesbeck,et al.  Influence of codon bias on the expression of foreign genes in microalgae. , 2007, Advances in experimental medicine and biology.

[51]  Huiyun Chang,et al.  Foot-and-mouth disease virus VP1 protein fused with cholera toxin B subunit expressed in Chlamydomonas reinhardtii chloroplast , 2003, Biotechnology Letters.

[52]  Wan-Loy Chu,et al.  Biotechnological applications of microalgae , 2012, International e-Journal of Science, Medicine & Education.

[53]  S. Schillberg,et al.  Molecular farming of recombinant antibodies in plants , 2003, Cellular and Molecular Life Sciences CMLS.

[54]  J. Swartz,et al.  Advances in Escherichia coli production of therapeutic proteins. , 2001, Current opinion in biotechnology.

[55]  M. Sack,et al.  Putting the Spotlight Back on Plant Suspension Cultures , 2016, Front. Plant Sci..

[56]  Richard T. Sayre,et al.  Growth and Heavy Metal Binding Properties of Transgenic Chlamydomonas Expressing a Foreign Metallothionein Gene , 1999 .

[57]  B. Byrne,et al.  Oral delivery of Acid Alpha Glucosidase epitopes expressed in plant chloroplasts suppresses antibody formation in treatment of Pompe mice. , 2015, Plant biotechnology journal.

[58]  Harald Fischer,et al.  TARGETING AND COVALENT MODIFICATION OF CELL WALL AND MEMBRANE PROTEINS HETEROLOGOUSLY EXPRESSED IN THE DIATOM CYLINDROTHECA FUSIFORMIS (BACILLARIOPHYCEAE) , 1999 .

[59]  Zisheng Zhang,et al.  Strategies for high-level recombinant protein expression in transgenic microalgae: a review. , 2010, Biotechnology advances.

[60]  Xiaofeng Wang,et al.  A novel expression platform for the production of diabetes-associated autoantigen human glutamic acid decarboxylase (hGAD65) , 2008, BMC biotechnology.

[61]  K. Kindle Nuclear Transformation: Technology and Applications , 1998 .

[62]  J. Gimpel,et al.  CHLOROPLAST GENETIC TOOL FOR THE GREEN MICROALGAE HAEMATOCOCCUS PLUVIALIS (CHLOROPHYCEAE, VOLVOCALES) 1 , 2012, Journal of phycology.

[63]  Y. Niu,et al.  A new inducible expression system in a transformed green alga, Chlorella vulgaris. , 2011, Genetics and molecular research : GMR.

[64]  Eric C. Henry,et al.  HANDBOOK OF MICROALGAL CULTURE: BIOTECHNOLOGY AND APPLIED PHYCOLOGY , 2004 .

[65]  M. Borowitzka Fats, oils and hydrocarbons , 1988 .

[66]  Fumiko Ohta,et al.  Genome sequence of the ultrasmall unicellular red alga Cyanidioschyzon merolae 10D , 2004, Nature.

[67]  Michael D. Thompson,et al.  Chloroplast transformation in Euglena gracilis: splicing of a group III twintron transcribed from a transgenic psbK operon , 2001, Current Genetics.

[68]  S. Mayfield,et al.  Algae-Produced Pfs25 Elicits Antibodies That Inhibit Malaria Transmission , 2012, PloS one.

[69]  Xiaojie Chai,et al.  Expression of soybean Kunitz trypsin inhibitor gene SKTI in Dunaliella salina , 2013, Journal of Applied Phycology.

[70]  P. Spolaore,et al.  Commercial applications of microalgae. , 2006, Journal of bioscience and bioengineering.

[71]  V. Zachleder,et al.  Starch Overproduction by Means of Algae , 2014 .

[72]  Y. Echelard Recombinant protein production in transgenic animals. , 1996, Current opinion in biotechnology.

[73]  S. Mayfield,et al.  Production of unique immunotoxin cancer therapeutics in algal chloroplasts , 2012, Proceedings of the National Academy of Sciences.

[74]  M. Levine,et al.  Human immune responses to a novel norwalk virus vaccine delivered in transgenic potatoes. , 2000, The Journal of infectious diseases.

[75]  J. V. Van Etten,et al.  Giant viruses infecting algae. , 1999, Annual review of microbiology.

[76]  Jeff Shrager,et al.  Chlamydomonas reinhardtii Genome Project. A Guide to the Generation and Use of the cDNA Information1 , 2003, Plant Physiology.

[77]  Bin Zhou,et al.  Synthesis and assembly of a full‐length human monoclonal antibody in algal chloroplasts , 2009, Biotechnology and bioengineering.

[78]  Elizabeth H. Harris,et al.  Further characterization of the respiratory deficient dum-1 mutation of Chlamydomonas reinhardtii and its use as a recipient for mitochondrial transformation , 2004, Molecular and General Genetics MGG.

[79]  M. Calvin,et al.  The path of carbon in photosynthesis. , 1949, Science.

[80]  H. Cerutti,et al.  RNA-Mediated Silencing in Algae: Biological Roles and Tools for Analysis of Gene Function , 2011, Eukaryotic Cell.

[81]  Jeffrey R. Lane,et al.  Corn as a production system for human and animal vaccines. , 2003, Vaccine.

[82]  D. M. Lam,et al.  Expression of hepatitis B surface antigen in transgenic plants. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[83]  Miguel Olaizola,et al.  Commercial development of microalgal biotechnology: from the test tube to the marketplace. , 2003, Biomolecular engineering.

[84]  Brian D Kelley Bioprocessing of therapeutic proteins , 2001 .

[85]  A. Demain,et al.  Production of recombinant proteins by microbes and higher organisms. , 2009, Biotechnology advances.

[86]  H. Soreq,et al.  Expression of recombinant human acetylcholinesterase in transgenic tomato plants. , 2001, Biotechnology and bioengineering.

[87]  Hanhua Hu,et al.  Microalgae as platforms for production of recombinant proteins and valuable compounds: progress and prospects , 2011, Journal of Industrial Microbiology & Biotechnology.

[88]  A. Romero-Maldonado,et al.  Chlamydomonas reinhardtii chloroplasts express an orally immunogenic protein targeting the p210 epitope implicated in atherosclerosis immunotherapies , 2016, Plant Cell Reports.

[89]  Ovidiu Ruecker,et al.  Strategies to facilitate transgene expression in Chlamydomonas reinhardtii , 2009, Planta.

[90]  A. Sheldrake THE PRODUCTION OF HORMONES IN HIGHER PLANTS , 1973 .

[91]  Dunahay Tg,et al.  Transformation of Chlamydomonas reinhardtii with silicon carbide whiskers. , 1993 .

[92]  C. Arntzen,et al.  Transgenic plants as vaccine production systems. , 1995, Trends in biotechnology.

[93]  Jeffrey Philip Obbard,et al.  Two phase microalgae growth in the open system for enhanced lipid productivity , 2011 .

[94]  S. Mayfield,et al.  Alga-Produced Cholera Toxin-Pfs25 Fusion Proteins as Oral Vaccines , 2013, Applied and Environmental Microbiology.

[95]  H. Cerutti,et al.  Gene silencing in microalgae: mechanisms and biological roles. , 2015, Bioresource technology.

[96]  M. Kawachi,et al.  Complete genome sequence of cyanobacterium Nostoc sp. NIES-3756, a potentially useful strain for phytochrome-based bioengineering. , 2016, Journal of biotechnology.

[97]  Eric E. Jarvis,et al.  Manipulation of microalgal lipid production using genetic engineering , 1996 .

[98]  Feng Wang,et al.  Scale-up cultivation of Chlorella ellipsoidea from indoor to outdoor in bubble column bioreactors. , 2014, Bioresource technology.

[99]  S. Purton,et al.  Microalgae as bioreactors , 2005, Plant Cell Reports.

[100]  D. Quist,et al.  Transgenic DNA introgressed into traditional maize landraces in Oaxaca, Mexico , 2001, Nature.

[101]  J. Teixeira,et al.  Mixotrophic cultivation of Chlorella vulgaris using industrial dairy waste as organic carbon source. , 2012, Bioresource technology.

[102]  Sanxing Guo,et al.  Preparation of transgenic Dunaliella salina for immunization against white spot syndrome virus in crayfish , 2013, Archives of Virology.

[103]  Jack Bui,et al.  Production of anti‐cancer immunotoxins in algae: Ribosome inactivating proteins as fusion partners , 2013, Biotechnology and bioengineering.

[104]  Lewis M. Brown,et al.  Transient expression of firefly luciferase in protoplasts of the green alga Chlorella ellipsoidea , 1991, Current Genetics.

[105]  Elizabeth A Specht,et al.  Algae-based oral recombinant vaccines , 2014, Front. Microbiol..

[106]  Dong Li,et al.  Expression of human soluble TRAIL in Chlamydomonas reinhardtii chloroplast , 2006 .

[107]  U. Sonnewald,et al.  Production of new/modified proteins in transgenic plants. , 1999, Current opinion in biotechnology.

[108]  S. Mayfield,et al.  Selenocystamine improves protein accumulation in chloroplasts of eukaryotic green algae , 2015, AMB Express.

[109]  Y. Chisti Biodiesel from microalgae. , 2007, Biotechnology advances.

[110]  T. Dunahay,et al.  Transformation of Chlamydomonas reinhardtii with silicon carbide whiskers. , 1993, BioTechniques.

[111]  M. El-sheekh Stable Transformation of the Intact Cells of Chlorella Kessleri with High Velocity Microprojectiles , 1999, Biologia Plantarum.

[112]  K. Hefferon Clinical Trials Fuel the Promise of Plant-Derived Vaccines , 2010 .

[113]  Zhengkai Xu,et al.  Functional complementation of a nitrate reductase defective mutant of a green alga Dunaliella viridis by introducing the nitrate reductase gene. , 2006, Gene.

[114]  I. Raskin,et al.  Production of recombinant proteins in plant root exudates , 1999, Nature Biotechnology.

[115]  K. Ohnishi,et al.  Stable nuclear transformation of the diatom Chaetoceros sp. , 2011 .

[116]  Yunqi Weng,et al.  Plants as Factories for Human Pharmaceuticals: Applications and Challenges , 2015, International journal of molecular sciences.

[117]  M. Posewitz,et al.  Critical role of Chlamydomonas reinhardtii ferredoxin-5 in maintaining membrane structure and dark metabolism , 2015, Proceedings of the National Academy of Sciences.

[118]  Fu-Quan Yang,et al.  A New Strategy to Produce a Defensin: Stable Production of Mutated NP-1 in Nitrate Reductase-Deficient Chlorella ellipsoidea , 2013, PloS one.

[119]  Wei Liu,et al.  Inhibitory effect of 13 taxane diterpenoids from Chinese yew (Taxus chinensis var. mairei) on the proliferation of HeLa cervical cancer cells , 2016, Bioscience, biotechnology, and biochemistry.

[120]  Xin-Hui Xing,et al.  Introduction of foreign DNA into Chlorella saccharophila by electroporation , 1994 .

[121]  V. Zachleder,et al.  Production of lipids in 10 strains of Chlorella and Parachlorella, and enhanced lipid productivity in Chlorella vulgaris , 2012, Applied Microbiology and Biotechnology.

[122]  U. Maier,et al.  A Single Peroxisomal Targeting Signal Mediates Matrix Protein Import in Diatoms , 2011, PloS one.

[123]  J. Cregg,et al.  Applications of yeast in biotechnology: protein production and genetic analysis. , 1999, Current opinion in biotechnology.

[124]  A. Chandrashekar,et al.  AGROBACTERIUM‐MEDIATED TRANSFORMATION IN THE GREEN ALGA HAEMATOCOCCUS PLUVIALIS (CHLOROPHYCEAE, VOLVOCALES) 1 , 2009, Journal of phycology.

[125]  T M Klein,et al.  Chloroplast transformation in Chlamydomonas with high velocity microprojectiles. , 1988, Science.

[126]  P. Lerouge,et al.  N-Glycosylation of a mouse IgG expressed in transgenic tobacco plants. , 1999, Glycobiology.

[127]  F B Metting,et al.  Biodiversity and application of microalgae , 1996, Journal of Industrial Microbiology.

[128]  Peng Wang,et al.  Stable expression of hepatitis B surface antigen gene in Dunaliella salina (Chlorophyta) , 2003, Journal of Applied Phycology.

[129]  A. Falciatore,et al.  Transformation of Nonselectable Reporter Genes in Marine Diatoms , 1999, Marine Biotechnology.

[130]  H. Daniell,et al.  Overexpression of the Bt cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals , 2001, Nature Biotechnology.

[131]  R. Tisch,et al.  Immune response to glutamic acid decarboxylase correlates with insulitis in non-obese diabetic mice , 1993, Nature.

[132]  D. Weeks,et al.  Molecular analysis of the acetolactate synthase gene of Chlamydomonas reinhardtii and development of a genetically engineered gene as a dominant selectable marker for genetic transformation. , 2002, The Plant journal : for cell and molecular biology.

[133]  H. Cerutti,et al.  Epigenetic silencing of a foreign gene in nuclear transformants of Chlamydomonas. , 1997, The Plant cell.

[134]  U. Maier,et al.  An engineered diatom acting like a plasma cell secreting human IgG antibodies with high efficiency , 2012, Microbial Cell Factories.

[135]  M. Ayala,et al.  Expression of a single-chain Fv antibody fragment specific for the Hepatitis B surface antigen in transgenic tobacco plants , 2002, Transgenic Research.

[136]  S. Mayfield,et al.  Photosynthetic biomanufacturing in green algae; production of recombinant proteins for industrial, nutritional, and medical uses , 2014, Photosynthesis Research.

[137]  Henri G. Gerken,et al.  Genetic engineering of the green alga Chlorella zofingiensis: a modified norflurazon-resistant phytoene desaturase gene as a dominant selectable marker , 2014, Applied Microbiology and Biotechnology.

[138]  I. Hwang,et al.  Stable Integration and Functional Expression of Flounder Growth Hormone Gene in Transformed Microalga, Chlorella ellipsoidea , 2002, Marine Biotechnology.

[139]  K. Apt,et al.  COMMERCIAL DEVELOPMENTS IN MICROALGAL BIOTECHNOLOGY , 1999 .

[140]  B. Ru,et al.  Survival of human metallothionein-2 transplastomic Chlamydomonas reinhardtii to ultraviolet B exposure. , 2006, Acta biochimica et biophysica Sinica.

[141]  Attilio Converti,et al.  Batch and fed-batch cultivations of Spirulina platensis using ammonium sulphate and urea as nitrogen sources , 2005 .

[142]  W. Müller,et al.  Nuclear transformation of Volvox carteri. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[143]  O. Pulz,et al.  Biotechnology with Cyanobacteria and Microalgae , 2001 .

[144]  Gordon Allison,et al.  Transgenic plants as factories for biopharmaceuticals , 2000, Nature Biotechnology.

[145]  E. Jarvis,et al.  GENETIC TRANSFORMATION OF THE DIATOMS CYCLOTELLA CRYPTICA AND NAVICULA SAPROPHILA , 1995 .

[146]  S. Mayfield,et al.  Production of therapeutic proteins in algae, analysis of expression of seven human proteins in the chloroplast of Chlamydomonas reinhardtii. , 2010, Plant biotechnology journal.

[147]  J. Vlak,et al.  The white spot syndrome virus DNA genome sequence. , 2001, Virology.

[148]  M. Miyahara,et al.  Highly Efficient Transformation of the Diatom Phaeodactylum tricornutum by Multi-Pulse Electroporation , 2013, Bioscience, biotechnology, and biochemistry.

[149]  P. Hegemann,et al.  A Streptomyces rimosus aphVIII gene coding for a new type phosphotransferase provides stable antibiotic resistance to Chlamydomonas reinhardtii. , 2001, Gene.

[150]  Humberto J Morris,et al.  Utilisation of Chlorella vulgaris cell biomass for the production of enzymatic protein hydrolysates. , 2008, Bioresource technology.

[151]  K. Ohnishi,et al.  Research note: High efficiency transformation of the diatom Phaeodactylum tricornutum with a promoter from the diatom Cylindrotheca fusiformis , 2009 .

[152]  K. Ishida,et al.  Internal plastid-targeting signal found in a RubisCO small subunit protein of a chlorarachniophyte alga. , 2010, The Plant journal : for cell and molecular biology.

[153]  Xiaomei Wang,et al.  Low cost industrial production of coagulation factor IX bioencapsulated in lettuce cells for oral tolerance induction in hemophilia B. , 2015, Biomaterials.

[154]  S. Kim,et al.  Transgenic microalgae expressing Escherichia coli AppA phytase as feed additive to reduce phytate excretion in the manure of young broiler chicks , 2011, Applied Microbiology and Biotechnology.

[155]  Nicole Poulsen,et al.  A new molecular tool for transgenic diatoms , 2005 .

[156]  Martin Fussenegger,et al.  Heat-stable oral alga-based vaccine protects mice from Staphylococcus aureus infection. , 2010, Journal of biotechnology.

[157]  Phang Siew Moi,et al.  Handbook of Microalgal Culture. Biotechnology and Applied Phycology , 2004, Journal of Applied Phycology.

[158]  J. Fox First plant-made biologic approved , 2012, Nature Biotechnology.

[159]  Yoram Tekoah,et al.  Plant-based oral delivery of β-glucocerebrosidase as an enzyme replacement therapy for Gaucher's disease. , 2015, Plant biotechnology journal.

[160]  Expression of an HBcAg-based antigen carrying angiotensin II in Chlamydomonas reinhardtii as a candidate hypertension vaccine , 2014, Plant Cell, Tissue and Organ Culture (PCTOC).

[161]  A. Demain,et al.  Enzymes and Bioconversions of Industrial, Pharmaceutical, and Biotechnological Significance , 2011 .

[162]  C. Arntzen,et al.  Plant‐made pharmaceuticals: from ‘Edible Vaccines’ to Ebola therapeutics , 2015, Plant biotechnology journal.

[163]  Stefan Surzycki,et al.  Factors effecting expression of vaccines in microalgae. , 2009, Biologicals : journal of the International Association of Biological Standardization.

[164]  P. Lefebvre,et al.  Isolation and characterization of the nitrate reductase structural gene of Chlamydomonas reinhardtii. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[165]  A. Grossman,et al.  Stable nuclear transformation of the diatomPhaeodactylum tricornutum , 1996, Molecular and General Genetics MGG.

[166]  L. Alhonen,et al.  Transgenic bioreactors. , 1998, Biotechnology Annual Review.

[167]  Michal Shapira,et al.  Transgene Expression in Microalgae—From Tools to Applications , 2016, Front. Plant Sci..

[168]  O. Dym,et al.  Production of glucocerebrosidase with terminal mannose glycans for enzyme replacement therapy of Gaucher's disease using a plant cell system. , 2007, Plant biotechnology journal.

[169]  Yinü Li,et al.  Recombination and expression of classical swine fever virus (CSFV) structural protein E2 gene in Chlamydomonas reinhardtii chroloplasts. , 2007, Colloids and surfaces. B, Biointerfaces.

[170]  M. Borowitzka Commercial production of microalgae: ponds, tanks, tubes and fermenters , 1999 .

[171]  Randor Radakovits,et al.  Corrigendum: Draft genome sequence and genetic transformation of the oleaginous alga Nannochloropsis gaditana , 2013, Nature Communications.

[172]  Christopher Preston,et al.  Pollen-Mediated Movement of Herbicide Resistance Between Commercial Canola Fields , 2002, Science.

[173]  U. Maier,et al.  Algae as Protein Factories: Expression of a Human Antibody and the Respective Antigen in the Diatom Phaeodactylum tricornutum , 2011, PloS one.

[174]  C. Hollak An evidence-based review of the potential benefits of taliglucerase alfa in the treatment of patients with Gaucher disease , 2012, Core evidence.

[175]  F. Takaiwa,et al.  Rice seed for delivery of vaccines to gut mucosal immune tissues. , 2015, Plant biotechnology journal.

[176]  M. Hirono,et al.  Recovery of flagellar dynein function in a Chlamydomonas actin/dynein-deficient mutant upon introduction of muscle actin by electroporation. , 2001, Cell motility and the cytoskeleton.

[177]  G. Giuliano,et al.  A Chlamydomonas-Derived Human Papillomavirus 16 E7 Vaccine Induces Specific Tumor Protection , 2013, PloS one.

[178]  H. Daniell,et al.  Advances in molecular farming: key technologies, scaled up production and lead targets. , 2015, Plant biotechnology journal.

[179]  R. Burlingame,et al.  Stable Transformation of Chlorella: Rescue of Nitrate Reductase-Deficient Mutants with the Nitrate Reductase Gene , 1997, Current Microbiology.

[180]  Michal Shapira,et al.  Stable Chloroplast Transformation of the Unicellular Red AlgaPorphyridium Species1 , 2002, Plant Physiology.

[181]  A. Grossman,et al.  Transformation of the diatom Phaeodactylum tricornutum (Bacillariophyceae) with a variety of selectable marker and reporter genes , 2000 .

[182]  Developing inexpensive malaria vaccines from plants and algae , 2014, Applied Microbiology and Biotechnology.

[183]  Jacob M. Robertson,et al.  An Indexed, Mapped Mutant Library Enables Reverse Genetics Studies of Biological Processes in Chlamydomonas reinhardtii[OPEN] , 2016, Plant Cell.

[184]  H. Cerutti,et al.  A eubacterial gene conferring spectinomycin resistance on Chlamydomonas reinhardtii: integration into the nuclear genome and gene expression. , 1997, Genetics.

[185]  A. Hallmann,et al.  Genetic engineering of the multicellular green alga Volvox: a modified and multiplied bacterial antibiotic resistance gene as a dominant selectable marker. , 1999, The Plant journal : for cell and molecular biology.

[186]  Wenbin Li,et al.  Highly efficient expression of rabbit neutrophil peptide-1 gene in Chlorella ellipsoidea cells , 2001, Current Genetics.

[187]  A. Hiatt,et al.  Production of antibodies in transgenic plants , 1989, Nature.

[188]  N. Pauw,et al.  Optimisation of T-ISO biomass production rich in essential fatty acids: I. Effect of different light regimes on growth and biomass production , 2003 .

[189]  H. Daniell,et al.  Plant-made oral vaccines against human infectious diseases-Are we there yet? , 2015, Plant biotechnology journal.

[190]  S. Briggs,et al.  Robust Expression and Secretion of Xylanase1 in Chlamydomonas reinhardtii by Fusion to a Selection Gene and Processing with the FMDV 2A Peptide , 2012, PloS one.

[191]  A. Grossman,et al.  High-efficiency transformation of Chlamydomonas reinhardtii by electroporation. , 1998, Genetics.

[192]  R. Ranjith Kumar,et al.  Enzymatic and non-enzymatic antioxidant potentials of Chlorella vulgaris grown in effluent of a confectionery industry , 2014, Journal of Food Science and Technology.