Autohydrolysis of plant xylans by apoplastic expression of thermophilic bacterial endo-xylanases.
The genes encoding the two endo-xylanases XynA and XynB from the thermophilic bacterium Dictyoglomus thermophilum were codon optimized for expression in plants. Both xylanases were designed to be constitutively expressed under the control of the CaMV 35S promoter and targeted to the apoplast. Transient expression in tobacco and stable expression in transgenic Arabidopsis showed that both enzymes were expressed in an active form with temperature optima at 85 degrees C. Transgenic Arabidopsis accumulating heterologous endo-xylanases appeared phenotypically normal and were fully fertile. The highest xylanase activity in Arabidopsis was found in dry stems indicating that the enzymes were not degraded during stem senescence. High levels of enzyme activity were maintained in cell-free extracts from dry transgenic stems during incubation at 85 degrees C for 24 h. Analysis of cell wall polysaccharides after heat treatment of wildtype and transgenic extracts from dry stems showed a decrease in the molecular weight of xylans from transgenic stems.
Borkhardt
Bernhard
B
Department of Genetics and Biotechnology, Faculty of Agricultural Sciences, Aarhus University, Thorvaldsensvej, Frederiksberg C, Denmark. b.borkhardt@dias.kvl.dk
Harholt
Jesper
J
Ulvskov
Peter
P
Ahring
Birgitte K
BK
Jørgensen
Bodil
B
Brinch-Pedersen
Henrik
H
D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics; D001419:Bacteria / Q000201:enzymology; D002473:Cell Wall / Q000378:metabolism; D043364:Endo-1,4-beta Xylanases / Q000378:metabolism*; D005798:Genes, Bacterial; D006056:Golgi Apparatus / Q000201:enzymology; D006868:Hydrolysis; D018547:Plant Stems / Q000737:chemistry / Q000201:enzymology*; D030821:Plants, Genetically Modified / Q000201:enzymology / Q000235:genetics; D013696:Temperature; D014026:Nicotiana / Q000201:enzymology / Q000235:genetics; D014170:Transformation, Genetic; D014990:Xylans / Q000378:metabolism*
D014990:Xylans; D043364:Endo-1,4-beta Xylanases