Scientific Report 2008
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
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.
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