Ag. Heyer et R. Wendenburg, Gene cloning and functional characterization by heterologous expression ofthe fructosyltransferase of Aspergillus sydowi IAM 2544, APPL ENVIR, 67(1), 2001, pp. 363-370
We have purified a fructosyltransferase from conidia of the inulin-producin
g fungus Aspergillus sydowi IAM 2544 and obtained peptide sequences from pr
oteolytic fragments of the protein. With degenerated primers, we amplified
a PCR fragment that was used to screen a cDNA library, The fructosyltransfe
rase gene from Aspergillus sydowi (EMBL accession no. AJ289046) is expresse
d in conidia, while no expression could be detected in mycelia by Northern
blot analysis of mycelial RNA. The gene encodes a protein with a calculated
molecular mass of 75 kDa that is different from all fructosyltransferases
in the databases. The only homology that could be detected was to the inver
tase of Aspergillus niger (EMBL accession no. L06844), The gene was functio
nally expressed in Escherichia coli, yeast, and potato plants. With protein
extracts from transgenic bacteria and yeast, fructooligosaccharides could
be produced in vitro. In transgenic potato plants, inulin molecules of up t
o 40 hexose units were synthesized in vivo. While in vitro experiments with
protein extracts from conidia of Aspergillus sydowi yielded the same patte
rn of oligosaccharides as extracts from transformed bacteria and yeast, in
vivo inulin synthesis with fungal conidia leads to the production of a high
-molecular-weight polymer.