Functional cloning of an endo-arabinanase from Aspergillus aculeatus and its heterologous expression in A. or oryzae and tobacco.

Functional cloning in yeast has been used to isolate full-length cDNAs encoding an endo-alpha-1,5-L-arabinanase from the filamentous fungus Aspergillus aculeatus. Screening of a cDNA library constructed in a yeast expression vector for transformants that hydrolysed AZCL-arabinan identified 44 Saccharomyces cerevisiae clones all harbouring the same arabinanase-encoding cDNA. The cloned cDNA was expressed in A. oryzae and the recombinant enzyme was purified and characterized. The mode of action of the enzyme was studied by analysis of the digestion pattern towards debranched arabinan. The digestion profile obtained strongly suggests that the enzyme is an endo-arabinanase. In addition, the feasibility using Nicotiana tabacum as an alternative host for arabinanase expression was investigated.

Pectin engineering: modification of potato pectin by in vivo expression of an endo-1,4-beta-D-galactanase.

Potato tuber pectin is rich in galactan (oligomer of beta-1,4-linked galactosyl residues). We have expressed a fungal endo-galactanase cDNA in potato under control of the granule bound starch synthase promoter to obtain expression of the enzyme in tubers during growth. The transgenic plants displayed no altered phenotype compared with the wild type. Fungal endo-galactanase activity was quantified in the transgenic tubers, and its expression was verified by Western blot analysis. The effect of the endo-galactanase activity on potato tuber pectin was studied by Fourier transform infrared microspectroscopy, immuno-gold labeling, and sugar analysis. All analyses revealed alterations in pectin composition. Monosaccharide composition of total cell walls and isolated rhamnogalacturonan I fragments showed a reduction in galactosyl content to 30% in the transformants compared with the wild type. Increased solubility of pectin from transgenic cell walls by endo-polygalacturonase/pectin methylesterase digestion points to other changes in wall architecture.