Optimizing the Expression of Bacterial Cellulases in Chloroplast Transgenic
Plants
Dr. Maureen Hanson, Liberty Hyde Bailey Professor, Department of Molecular Biology Genetics, Cornell
University
ABSTRACT
Enzymatic hydrolysis of biomass to fermentable sugars is a key step in the sustainable production of
cellulosic ethanol. Efficient methods for enzyme synthesis are necessary to reduce the cost of
bioethanol. Biosynthesis of biomass-degrading enzymes by feedstock plants could be part of an effective
strategy for generation of bioenergy from lignocellulosic materials. Because high levels of various
therapeutic proteins have been obtained through transformation of the chloroplast genome, we
investigated what features of chloroplast transgenes are important for transgenic synthesis of celluloseconverting enzymes encoded by genes from the thermophilic bacterium Thermobifida fusca. We placed
coding regions for an endoglucanase and beta-glucosidase under the control of a phage 5’ untranslated
region, the plastid 16S rRNA promoter, and a plastid terminator, and introduced constructs into the
tobacco chloroplast genome by particle bombardment. Sequences were fused at the 5’ end of the coding
regions to express proteins carrying different 14 amino acid N-terminal fusions. In transgenic plants, the
most effective transgenes resulted in plants that accumulated the enzymes to as much as 10-12% of total
leaf soluble protein (TSP), while less than 0.3% TSP was obtained from leaves of plants carrying the least
effective transgenes. Enzyme accumulation was stable during plant development. Endoglucanase could
be purified on cellulose affinity columns and enzyme activity correlated well with protein quantification by
immunoblot. The chloroplast-expressed β-glucosidase was active against cellobiose. This study
demonstrates the feasibility of high-level chloroplast-based synthesis of biomass-degrading enzymes.
Supported by USDA grant NRI 2007-02133 and an NSF Fellowship to BNG.
BIOGRAPHY
Maureen Hanson is Liberty Hyde Bailey Professor in the Department of Molecular Biology and Genetics
at Cornell University. She has previously served as Director of the NSF/DOE/USDA Cornell Plant
Science Center and Associate Director of the Cornell Biotechnology Program. She has an active
research program in organelle genetics, intracellular dynamics of organelles, and genetic engineering of
plants, currently funded by the National Science Foundation, the Department of Energy, and the U.S.
Department of Agriculture. Her laboratory collaborates with Prof. Beth Ahner in the Biological and
Environmental Engineering Department on a project to produce cellulases for lignocellulosic biomass
processing.
Dr. Hanson is a Fellow of the American Society for Advancement of Science and the Cornell Center for a
Sustainable Future. She has received the 2006 Lawrence Bogorad Award of the American Society of
Plant Biologists, a 2008 SUNY Chancellor’s Award, and the 2009 Award for Outstanding
Accomplishments in Basic Research from the Cornell College of Agriculture and Life Sciences. Before
joining the faculty at Cornell University, Dr. Hanson held a faculty appointment at the Department of
Biology at the University of Virginia. Dr. Hanson was awarded a NIH postdoctoral fellowship and an NSF
predoctoral fellowship at Harvard University, where she received a Ph.D. in Biology. She has a B.S.
degree, summa cum laude, from Duke University.