Cellulosic biomass is considered to be a very good source for bioethanol production mainly because it has abundant supply and is environmental-friendly.  However, some steps of producing cellulosic bioethanol are expensive and limiting its industrial application, such as the severity of pre-treatment and the production of cellulose-degrading enzymes in microbial reactors.  Researchers are now turning their attentions to express the plant cell-wall hydrolysis enzymes in plants in order to reduce the capital cost of bioethanol production.  To increase the accumulation of cell wall degrading enzymes, we studied the effects of subcellular targeting, elastin-like polypeptide (ELP) protein fusion and hydrophobin (HFBI) fusion on the accumulation level of these enzymes.              

      The 10 A. niger cell wall degrading genes were cloned into Gateway-compatible plant binary expression vectors.  All 10 genes were targeted to the cytoplasm, chloroplast, ER, vacuole, and apoplast in order to conduct localization study.  Each of the 50 expression constructs were infiltrated into 10 independent Nicotiana benthamiana plants and transiently expressed.  Quantitative analysis on the 10 genes of 5 localizations were performed.  The utility of ELP protein and HFBI fusions of the 10 genes was determined. 

       Our results showed that for transiently-agroinfiltrated N. Benthamiana leaves, the secretory pathway (i.e. ER, vacuole, and apoplast) is the ideal local to accumulate these proteins (the ER is best).  And ELP tag is better for increasing the accumulation of these proteins compared with HFBI.  Future study will be focusing on increasing hemicellulose-degrading enzymes accumulation levels and characterizing their activities.