Due to petroleum energy shortages, the world is looking for energy alternatives such as ethanol. This actually raised concerns of threatening food prices by increasing demand on corn and sugar for ethanol production. The economic and environmental performance of ethanol would likely be improved by producing ethanol from lignocellulosic materials using hydrolysis cellulase enzymes. Although the cost of enzyme production was reduced dramatically in the past few years, it still represents a major cost factor. Cost reduction might be achieved by producing crops that has the inbuilt capacity to sustainably and actively produce the desired hydrolysis enzymes.

Nine Aspergillus niger genes encoding for different cellulases and hemicellulases, in addition to three hyperthermophilic Pyroccoccus cellulases are being used. The hyperthermophilic cellulases represent beta-glucosidase of Pyroccoccus furiosus, endoglucanase of Pyroccoccus horikosii and a bi-functional fusion of both. All genes have been codon optimized and cloned into four different destination vectors using the GATEWAY™ recombination technology. The destination vectors contain along with the promoter and terminator, translational enhancers, signal peptides for ER and Chloroplast targeting as well as protein accumulation enhancers in the ER constructs. Experiments are ongoing to test 48 constructs in the transient expression system of Nicotiana benthamiana for acquiring considerable information and rapidly assaying the expression of these genes in plants before generating stable transgenic alfalfa lines. Preliminary data of different replicates suggest the possibility of accumulating large amounts of these enzymes in plants. More experiments will be performed to generate and characterize transgenic alfalfa plants producing these enzymes.