Biofuel production from lignocellulosic biomass depends on technology that efficiently and economically releases fermentable sugars from multi-polymeric cell wall components. Xylan is after cellulose, the most abundant polysaccharide in grass and wood biomass and must be hydrolyzed to its component sugars (xylose or xylobiose) before fermentation to ethanol. Endoxylanases are the main enzymes involved in xylan hydrolysis. In planta production of cell wall degrading enzymes will reduce costs of enzyme production. Sugarcane (Saccharum sp. hybrids) is the main source for production of table sugar and is the most efficient photosynthesizer in the plant kingdom. Stalks from which the juice for sugar crystallization is extracted represent about 75 % of the above ground biomass at harvest. The remaining 25% of the biomass are leaves (10 to 25 tons per hectare) which are typically reduced by open air burning to suppress pests and diseases in the following ratoon and facilitate harvest. Adding value through in planta production of cell wall degrading enzymes offers an environmentally friendly alternative use for this abundant leaf biomass resource.

Constitutive, apoplast or chloroplast targeted expression cassettes of the codon optimized, hypothermostable GH10 xylanase from Thermotoga maritima (xyl10B) were generated for in planta expression. Transgene integration, expression and enzymatic activity were evaluated following biolistic co-transfer of the xyl10B and the selectable nptII expression cassettes by Southern blot analysis, PCR, RT-PCR, ELISA, Western blot analysis, flourometric xylanase activity analysis, Congo red assay and sugar release assay. 17 transgenic sugarcane lines showed clearly detectable xylanase activity. Highest expression was detected in mature leaves. The in planta produced enzyme was purified and sugarcane xylan was used as a substrate. TLC analysis confirmed the superior catalytic activity and stability of the in planta produced enzyme with directly fermentable xylobiose as the main degradation product.