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Microalgae have been largely cultured and commercialized as food and feed additives, and their potential as a source of high-added value compounds is well known. Yet, in contrast to the large number of genetically modified bacteria, yeast and even higher plants, only a few species of microalgae have been genetically transformed with efficiency.
More work is needed to transform new species of microalgae, specially those that have commercial value, so that it would be possible to increase the productivity of traditional compounds or synthesize novel ones. Silencing transgenes remains as an important limitation for stable expression of foreign genes. This problem is not unique to microalgae since it has also been observed in plants, animals and fungi. A better understanding of the mechanisms that control the regulation of gene expression in eukaryotes is therefore needed.
In this book a group of outstanding researchers working on different areas of microalgae biotechnology offer a global vision of the genetic manipulation of microalgae and their applications.
CONTENTS 1. NUCLEAR TRANSFORMATION OF EUKARYOTIC MICROALGAE: HISTORICAL OVERVIEW, ACHIEVEMENTS AND PROBLEMS ... 1 Rosa Leon and Emilio Fernandez Abstract ... 1 Introduction ... 1 Microalgae Groups Transformed ... 2 Methods for Microalgae Transformation ... 4 Characteristics of the Transformation Process ... 6 DNA Constructions Used in Transformation ... 6 Difficulties for Stable Expression of the Transgenes ... 8 Concluding Remarks ... 8 2. TRANSFORMATION OF CYANOBACTERIA... 12 Agustin Vioque Abstract ... 12 Introduction ... 12 Transformation of Cyanobacteria ... 13 Applications ... 14 3. MOLECULAR BIOLOGY AND THE BIOTECHNOLOGICAL POTENTIAL OF DIATOMS ... 23 Peter Kroth Abstract ... 23 Diatom Biology ... 23 Genetic Manipulation of Diatoms ... 25 Biochemistry of Diatoms and Technological Applications ... 29 Synthesis of Fatty Acids ... 29 Biomineralization ... 30 Concluding Remarks ... 31 00LeonAEMB 9 8/13/07, 9:53 AM x Contents 4. TOOLS AND TECHNIQUES FOR CHLOROPLAST TRANSFORMATION OF CHLAMYDOMONAS... 34 Saul Purton Abstract ... 34 Introduction ... 34 Delivery of DNA into the Chloroplast Compartment ... 36 Integration of Transforming DNA ... 37 Polyploidy and the Problems of Heteroplasmy ... 39 Selection Strategies ... 41 Reverse-Genetic Studies of the Chlamydomonas Plastome ... 42 Expression of Foreign Genes in the Chlamydomonas Chloroplast ... 42 Future Prospects ... 43 5. INFLUENCE OF CODON BIAS ON THE EXPRESSION OF FOREIGN GENES IN MICROALGAE ... 46 Markus Heitzer, Almut Eckert, Markus Fuhrmann and Christoph Griesbeck Abstract ... 46 General Aspects of Codon Bias in Pro- and Eukaryotic Expression Hosts ... 46 Phaeodactylum tricornutum ... 47 Chlamydomonas reinhardtii -- Expression from Chloroplast and Nucleus ... 48 Concluding Remarks ... 52 6. IN THE GRIP OF ALGAL GENOMICS ... 54 Arthur R. Grossman Abstract ... 54 Introduction ... 54 Which Organisms Should Have Their Genomes Sequenced? ... 56 Full Genome Sequences ... 56 cDNA and Partial Genome Sequences ... 64 Viral Genomes ... 65 Concluding Remarks ... 67 7. INSERTIONAL MUTAGENESIS AS A TOOL TO STUDY GENES/FUNCTIONS IN CHLAMYDOMONAS ... 77 Aurora Galvan, David Gonzalez-Ballester and Emilio Fernandez Abstract ... 77 Chlamydomonas as a Model for Translational Biology ... 77 Mutants as a Tool for Functional Genomics ... 78 Future Perspectives ... 86 00LeonAEMB 10 8/13/07, 9:53 AM Contents xi 8. OPTIMIZATION OF RECOMBINANT PROTEIN EXPRESSION IN THE CHLOROPLASTS OF GREEN ALGAE ... 90 Samuel P. Fletcher, Machiko Muto and Stephen P. Mayfield Abstract ... 90 Introduction ... 90 Expression of Recombinant Proteins in the Chlamydomonas Chloroplast ... 92 Strategies for Increasing Recombinant Protein Expression in Algal Chloroplast... 94 Conclusion and Prospectus ... 96 9. PHYCOREMEDIATION OF HEAVY METALS USING TRANSGENIC MICROALGAE... 99 Sathish Rajamani, Surasak Siripornadulsil, Vanessa Falcao, Moacir Torres, Pio Colepicolo and Richard Sayre Abstract ... 99 Metals in the Environment ... 99 The Role of the Algal Cell Wall in Heavy Metal Binding and Tolerance ... 100 The Plasma Membrane and Heavy Metal Flux ... 101 Heavy Metal Metabolism in the Cytoplasm of Algae ... 102 Algal Heavy Metal Biosensors ... 103 Application of Engineered Algae for Bioremediation: The Risks and Benefits ... 106 10. HYDROGEN FUEL PRODUCTION BY TRANSGENIC MICROALGAE ... 110 Anastasios Melis, Michael Seibert and Maria L. Ghirardi Summary ... 110 Overview ... 110 Sulfur-Nutrient Deprivation Attenuates Photosystem-II Repair and Promotes H2-Production in Unicellular Green Algae ... 111 Genetic Engineering of Sulfate Uptake in Microalgae for H2-Production ... 113 Application of the Hydrogenase Assembly Genes in Conferring H2-Production Capacity in a Variety of Organisms ... 113 Engineering O2 Tolerance to the Green Algal Hydrogenase ... 115 Engineering Starch Accumulation in Microalgae for H2-Production ... 116 Engineering Optimal Light Utilization in Microalgae for H2-Production ... 117 Future Directions ... 118 11. MICROALGAL VACCINES ... 122 Surasak Siripornadulsil, Konrad Dabrowski and Richard Sayre Abstract ... 122 Introduction ... 122 Oral Vaccines ... 123 Microalgal Vaccines ... 123 Recent Progress ... 124
