The technology of converting lignocellulose to biofuels has advanced swiftly over the past few years, and enzymes are a significant constituent of this technology. In this regard, cost effective production of cellulases has been the focus of research for many years. One approach to reach cost targets of these enzymes involves the use of plants as bio-factories. The application of this technology to plant biomass conversion for biofuels and biobased products has the potential for significantly lowering the cost of these products due to lower enzyme production costs. Cel6A, one of the two cellobiohydrolases (CBH II) produced by Hypocrea jecorina, is an exoglucanase that cleaves primarily cellobiose units from the non-reducing end of cellulose microfibrils. In this work we describe the expression of Cel6A in maize endosperm as part of the process to lower the cost of this dominant enzyme for the bioconversion process. The enzyme is active on microcrystalline cellulose as exponential microbial growth was observed in the mixture of cellulose, cellulases, yeast and Cel6A, Cel7A (endoglucanase), and Cel5A (cellobiohydrolase I) expressed in maize seeds. We quantify the amount accumulated and the activity of the enzyme. Cel6A expressed in maize endosperm was purified to homogeneity and verified using peptide mass finger printing.

Transgenic plants in the US and abroad generated using genetic engineering technology are regulated with respect to release into the environment and inclusion into diets of humans and animals. For crops incorporating pharmaceuticals or industrial enzymes regulations are even more stringent. Notifications are not allowed for movement and release, therefore a permit is required. However, growing under permit is cumbersome and more expensive than open, non- regulated growth. Thus, when the genetically engineered pharmaceutical or industrial crop is ready for scale-up, achieving non-regulated status is critical. Regulatory compliance in the US comprises petitioning the appropriate agencies for permission for environmental release and feeding trials. For release without yearly permits, a petition for allowing non-regulated status can be filed with the United States Department of Agriculture with consultations that include the Food and Drug Administration and possibly the Environmental Protection Agency, the latter if the plant includes an incorporated pesticide. The data package should ensure that the plants are substantially equivalent in every parameter except for the engineered trait. We undertook a preliminary study on transgenic maize field-grown hybrids that express one of two cellulase genes, an exo-cellulase or an endo-cellulase. We performed field observations of whole plants and numerous in vitro analyses of grain. Although some minor differences were observed when comparing genetically engineered hybrid plants to control wild type hybrids, no significant differences were seen.

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