Production of xylanase in transgenic tobacco for industrial use in bioenergy and biofuel applications

Xylanases are used in various agricultural and industrial applications. A synthetic, modified, codon-optimized xylanase gene (XynZ) from Clostridium thermocellum was expressed in transgenic tobacco plants. The coding sequence of XynZ was placed between the modified Mirabilis mosaic virus full-length transcript promoter with duplicated enhancer domains and the terminator sequence from the rbcSE9 gene. Three constructs were developed to evaluate XynZ expression levels by targeting gene products into the cytosol, intercellular space, or endoplasmic reticulum in transgenic plants. These chimeric genes, expressed in transgenic tobacco (Nicotiana tabacum cv. Samsun NN) were stably inherited in successive plant generations (R0, R1, and R2 progeny; primary, second, and third generation) as shown by molecular characterization (RT-PCR and qRT-PCR) and enzymatic assays. A Western blot analysis of plant extracts showed presence of a polypeptide of the expected size that cross-reacted with xylanase-specific antibodies. Transgenic plants were morphologically similar to wild-type plants and showed no deleterious effect due to transgene expression. The expressed xylanase was heat-stable, having optimum activity between 55°C and 75°C over a pH range of 5 to 5.6.