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Plant cell walls provide a vast, untapped source of sugars in the form of cellulose that can be fermented for the production of ethanol and other biofuels. However, there are several roadblocks to developing an economically viable cellulosic ethanol production process based on biochemical conversion, one of which is the cost of the enzymes required to change the cellulose to glucose. Historically, cellulase enzymes for digesting plant biomass have been produced and purified using bacteria. An alternative and potentially more economic approach is the production of the enzymes within the plant itself. The goal of this research was to verify a transgenic technology (In Plant Activation Technology ? INPACT) in poplar which allows for the controlled high level accumulation of enzymes within the plant (Dugdale et al., 2013), and to assess the impact of the resulting cellulases on the efficiency of converting cellulose to fermentable sugars. INPACT is an inducible, gene amplification system which is unique in two ways. First, the INPACT gene cassette is designed so that the gene of interest is only transcribed in the presence of an initiation protein and as a result, no functional protein expression can occur without the initiation protein. Second, expression of the initiation protein is regulated by an inducible promoter such that INPACT acts as a switch to turn on high-level expression of proteins within the plant, and is particularly suited to proteins that may be toxic or hinder plant development as expression is tightly controlled by the INPACT trigger. The INPACT expression system combines the advantages of both stable and transient expression, and significantly, can be used to produce ?difficult? proteins that cannot be expressed within the plant using conventional approaches.<
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The primary outcome of the project involved confirmation that the base INPACT system is not effective at producing extreme levels of gene expression in poplar. Expression of the GUS reporter gene under control of the INPACT system containing the CesA8 vascular specific promoter and the Rbc promoter was inducible, but did not result in extreme expression. The same results were seen with the expression of cellulases under the control of this system, expression was inducible, but in general expression levels were in line with the expression seen in traditional transgenics. A secondary outcome of the project was the identification of a number of traditional overexpression lines that displayed marked improvement in glucose release by enzyme hydrolysis when heat-treated immediately following harvest. Most of the cellulases of interest used in this project were hyperthermophilic, requiring high heat (>
75�C for full activity), and so the inclusion of a heat treatment was necessary to observe the full effect of the transgene expression.<
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This work has contributed to our understanding of transgene expression under the INPACT system, which has previously worked well in model systems. It has identified areas for further improvement of the system, which is currently underway. In addition, this work has contributed to the understanding of the capability of overexpressing hyperthermophilic enzymes in the biofuels feedstock directly. While the capability exists to do so, the enzyme yield achieved through traditional overexpression cannot meet the required need for these enzymes for full hydrolysis of the feedstock. Further work is required to develop systems for high levels of expression of enzymes in plant feedstocks.<
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