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Scientific Report 2008


Cell Biology




Engineering Eukaryotic Algae for Production of Human Therapeutic Proteins and of Biofuels

S.P. Mayfield, A. Manuell, J. Marìn-Navarro, M. Muto, M. Tran, P. Pettersson, P. Lee, B. Rasala

Eukaryotic algae are an excellent system for biotechnology applications, including the production of human therapeutic proteins, industrial enzymes, and biofuels. Algae can produce biomass at more than 10 times the rate of terrestrial plants on a unit-area basis, making the microorganisms a potentially important and economically practical source of both proteins and biofuel molecules. Algal biomass contains starches, lipids, and other hydrocarbon-rich molecules, all of which are fuel precursors. Along with producing biofuel molecules, algae are extremely efficient at expressing proteins and can be used to produce proteins at a fraction of the cost of traditional expression systems and at a scale not achievable with traditional fermentation methods.

To realize the potential of algae, we must understand and control gene expression to optimize the production of proteins and biofuel molecules. In algae, a number of proteins and most biofuel molecules are produced in chloroplasts, and understanding chloroplast translation is essential for understanding chloroplast gene expression. The core translational apparatus of chloroplasts is highly conserved with that of bacteria. However, chloroplasts have incorporated novel protein components that allow for complex regulatory mechanisms. Some of these novel components are found on the plastid ribosomes; others are translation factors that are not found in bacterial systems. Chloroplast mRNAs also contain unique regulatory elements that interact with plastid ribosomes and translation factors to enable the complex regulation.

To better understand translation in algae, we are characterizing the structure of both chloroplasts and cytoplasmic ribosomes from Chlamydomonas reinhardtii, a unicellular photosynthetic alga. Using electron cryomicroscopy and single-particle reconstruction, we determined the structure of the C reinhardtii cytoplasmic 80S ribosome and found that it is nearly identical to 80S ribosomes from animals. We also determined the structure of the chloroplast ribosome to 15 Å and found that although it is conserved with bacterial 70S ribosomes, it has large unique structural domains. These domains likely are involved in unique aspects of chloroplast translation, including light-activated translation, which occurs in all photosynthetic organisms.

Light-activated translation in chloroplasts is achieved through reducing potential, derived from photosynthesis, which is used to activate the binding of a protein complex to plastid mRNAs. Binding of this protein complex to the 5′ untranslated region of mRNAs enables ribosome association of the mRNA and hence increased translation. How the proteins involved in light activation interact with the unique chloroplast ribosomal proteins is unknown, and understanding these interactions will be an important aspect of our research in the coming years.

In addition to these basic studies on translation, we have developed a system for the expression of recombinant proteins, including human therapeutic agents and enzymes involved in biofuel production in C reinhardtii chloroplasts. We have expressed a number of mammalian proteins, including monoclonal antibodies and mammalian growth factors, and have shown that this alga-based system can produce human therapeutic proteins at high levels. Most recently, we have focused on producing antibody-toxin fusion proteins in which a targeting antibody domain is linked to a cell-killing toxin. Using this technology, we have produced an antibody-toxin fusion protein that binds and kills human B-cell lymphomas, and cell-based assays have shown the usefulness and specificity of this molecule. These proteins have great potential for the treatment of cancers and infectious diseases, and chloroplasts offer perhaps the only system in which these types of proteins can be produced.

We have also begun to engineer algae for the production of hydrocarbon molecules that can be used as biofuels. Introducing enzymes from other organisms that can increase the accumulation of isoprenoids and fatty acids should allow for the use of microalgae as a biological source of these fuel precursors. Because algae can be grown by using sunlight and carbon dioxide as primary inputs, the potential of algae as a sustainable energy source is obvious. We have shown the tremendous potential of eukaryotic algae for the expression of recombinant human therapeutic proteins and for the production of biofuels. Our continued genetic, biochemical, and structural studies should lead to a greater understanding of the mechanism of chloroplast translation. With this understanding, we should be able to design appropriate transgenes to affect higher levels of expression of therapeutic proteins and allow algae to become a practical source for sustainable production of biofuels.

Publications

Beligni, M., Mayfield, S.P. Arabidopsis thaliana mutants reveal a role for CSP41a and CSP41b, two ribosome-associated endonucleases, in chloroplast ribosomal RNA metabolism. Plant Mol. Biol. 67:389, 2008.

Manuell, A.l., Quispe, J., Mayfield, S.P. Structure of the chloroplast ribosome: novel domains for translation regulation. PLoS Biol. 8:e209, 2007.

Marìn-Navarro, J., Manuell, A.L, Wu, J., Mayfield, S.P. Chloroplast translation regulation. Photosynth. Res. 94:359, 2007.

Merchant, S.S., Prochnik, S.E., Vallon, O., et al. The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science 318:245, 2007.

Stephen P. Mayfield, Ph.D.
Associate Professor



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