Efficient Production of Microbial Cellulase Within Recombinant Maize Biomass Converts AFEX-Pretreated Corn Stover into Fermentable Sugars for Alcohol Fuels.
Callista Ransom, Venkatesh Balan, Gadab Biswas, Bruce Dale, and Mariam Sticklen. Michigan State University, 361 Plant And Soil Science Building, East Lansing, MI 48824, United States of America
Commercial conversion of lignocellulosic biomass to fermentable sugars requires inexpensive bulk production of biologically active cellulase enzymes, which might be achieved through direct production of these enzymes within the biomass crops. Transgenic corn plants containing the catalytic domain of Acidothermus cellulolyticus E1 endo-1,4-β glucanase (E1) and the bar Bialaphos resistance coding sequences were generated after BiolisticTM bombardment of immature embryo-derived cells. E1 sequences were regulated under the control of the Cauliflower Mosaic Virus (CaMV) 35S promoter and Tobacco Mosaic Virus (TMV) translational enhancer, and E1 protein was targeted to the apoplast using the signal peptide of tobacco pathogenesis-related protein (Pr1a) to achieve accumulation of this enzyme. The integration, expression and segregation of E1 and bar transgenes were demonstrated respectively via Southern and Western blotting, and progeny analyses. Accumulation of up to 1.13% of transgenic plant total soluble proteins was detected as biologically active E1 by enzymatic activity assay. The corn-produced heterologous E1 could successfully convert Ammonia Fiber Explosion (AFEX)-pretreated corn stover polysaccharides into glucose as a fermentable sugar for ethanol production, confirming that the E1 enzyme is produced in its active form.