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Optimizing the Expression of Bacterial Cellulases in Chloroplast Transgenic Plants Dr

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Optimizing the Expression of Bacterial Cellulases in Chloroplast Transgenic Plants Dr 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 cellulose- converting 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. .
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