BACKGROUND:Production of cellulosic ethanol is still expensive compared with corn (maize) grain ethanol due to the high costs of bulk production of microbial cellulases. At least three cellulases including endo-cellulase, exo-cellulase and cellobiase are needed to convert cellulosic biomass into fermentable sugars. All these cellulases could be self-produced within cells of transgenic bio-energy crops. The production of heterologous Acidothermus cellulolyticus (E1) endo-cellulase in endoplasmic reticulum and mitochondria of green tissues of transgenic corn plants was recently reported, and it was confirmed that the heterologous E1 converts cellulose into fermentable sugars. RESULTS:Biologically active A. cellulolyticus E1, Trichoderma reesei 1,4-beta-cellobiohydrolases I (CBH I) exo-cellulase and bovine rumen Butyrivibrio fibrisolvens cellobiase were expressed in corn plant endoplasmic reticulum (ER), apoplast (cell wall areas) and vacuole respectively. Results show that the ratio 1:4:1 (E1:CBH I:cellobiase) of crude heterologous cellulases is ideal for converting ammonia fiber explosion (AFEX) pretreated corn stover into fermentable sugars. CONCLUSIONS:Corn plants that express all three biologically active heterologous cellulases within their cellulosic biomass to facilitate conversion of pretreated corn stover into fermentable sugars is a step forward in the quest for alternatives to the presentmicrobial cellulase mix production for cellulosic biofuels. (C) 2011 Society of Chemical Industry