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

Abstract

Xylanases are used in various agricultural and industrial applications. A synthetic, modified, codonoptimized 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.

Production of hyperthermostable GH10 xylanase Xyl10B from Thermotoga maritima in transplastomic plants enables complete hydrolysis of methylglucuronoxylan to fermentable sugars for biofuel production

“…X 1 xylose, X 2 xylobiose, X 3 xylotriose, X 4 xylotetraose, MeGX 1 methylglucuronoxylose, MeGX 2 methylglucuronoxylobiose, MeGX 3 methylglucuronoxylotriose, MeGX 4 methylglucuronoxylotetraose transformation approach (Laliberté et al 1992). Maximum xylanase expression levels of 3-5% of the total soluble protein in mature leaves were reported following gene transfer to the nucleus (Herbers et al 1995;Bae et al 2006;Yang et al 2007;Chatterjee et al 2010). However, an excessive line to line variation exists in nuclear transformed plants due to copy number, position effects and gene silencing mechanisms (Eike et al 2005;Vaucheret 2006;Fischer et al 2008), therefore requiring the screening of a large number of transgenic plants to identify those with highest expression.…”

Overexpression of the Synthetic Chimeric Native-T-phylloplanin-GFP Genes Optimized for Monocot and Dicot Plants Renders Enhanced Resistance to Blue Mold Disease in Tobacco (N. tabacumL.)

“…Genomic DNA from untransformed control plants, control plants transformed with the empty vector, and the pKM24-ibm8 and pKM24-ibm10 transformants was isolated using a DNeasy Plant Mini Kit (Qiagen, Valencia, CA) as described earlier [23]. …”

Cell Wall Engineering by Heterologous Expression of Cell Wall-Degrading Enzymes for Better Conversion of Lignocellulosic Biomass into Biofuels

“…b Gene coding for cell wall-degrading enzymes were cloned, and suitable constructs are made with a signal peptide for secretion and c introduced into the plant under control of a constitutive promoter and depending on the substrate type, the secreted enzyme alters the plant cell wall structure (lignin in brown, hemicellulose in red, pectin in green, cellulose in blue) and e boosts bioethanol yields [120,121]. Large numbers of xylanases have been found in a variety of bacteria and fungi, some of which have been cloned, characterized [121][122][123], and expressed in plants [86,87]. The reduction of xylan content in plants to increase cell wall degradability by heterologous expression of xylanases was shown by several research groups [84,86].…”

“…Engineered plants showed a decrease in molecular weight of xylans compared to WT lines; however, sugar release of these plants has not been reported [86]. Similarly, Chatterjee et al [87] successfully expressed a thermophilic xylanase in tobacco without any deleterious effect. In this study, the enzyme activity and transcript analysis have been identified, but there was no information regarding the cell wall composition.…”