Monday, May 4, 2009
5-82
Biochemical Characterization of Bacterial and Fungal Hemicellulases and Heterologous Expression in planta
Transgenic expression of microbial carbohydrate-active enzymes will be evaluated as a means to decrease the load and cost of enzymes used to refine pulp fibre for the production of value-added biomaterials, or hydrolyze plant biomass to platform sugars. Over twenty bacterial gene targets have been isolated from fourteen commercially available bacterial genomes. The corresponding enzyme activities included: α-arabinofuranosidase, α-glucuronidase, α-galactosidase, α-fucosidase, and mannanase. Purified enzymes will be biochemically characterized to measure pH optimum, temperature stability, and substrate specificity using both synthetic and natural substrates. In addition to bacterial genes, a glucuronoyl esterase gene from the white-rot fungus Phanerochaete carnosa (PcGE1) will be included in our studies. PcGE1 was recombinantly expressed in Pichia pastoris, and the purified enzyme will be biochemically characterized in vitro using 4-O-methyl-D-glucopyranuronate as the substrate. The transient expression of these microbial enzymes in tobacco leaves were validated using green fluorescence protein. These data may reveal correlations between microbial gene features, such as codon usage or AT content, and functional enzyme expression in plants. Transiently expressed microbial genes will then be targeted for transgenic expression in Arabidopsis. Initial experiments will constitutively express the microbial genes in Arabidopsis using the 35S promoter. We are also developing a transcription activation system based on the LhGR/pOp vector pair, which will facilitate tissue-specific and time-dependent expression of microbial genes in Arabidopsis. Resulting transgenic plants will be characterized in terms of morphology, cell wall structure, and total sugar composition.
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