Gene cloning and functional characterization by heterologous expression of the fructosyltransferase of Aspergillus sydowi IAM 2544.
We have purified a fructosyltransferase from conidia of the inulin-producing fungus Aspergillus sydowi IAM 2544 and obtained peptide sequences from proteolytic fragments of the protein. With degenerated primers, we amplified a PCR fragment that was used to screen a cDNA library. The fructosyltransferase gene from Aspergillus sydowi (EMBL accession no. AJ289046) is expressed 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 invertase of Aspergillus niger (EMBL accession no. L06844). The gene was functionally 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 to 40 hexose units were synthesized in vivo. While in vitro experiments with protein extracts from conidia of Aspergillus sydowi yielded the same pattern 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.
Heyer
A G
AG
Max-Planck-Institut für Molekulare Pflanzenphysiologie, Am Mühlenberg 1, D-14476 Golm, Germany. heyer@mpimp-golm.mpg.de
Wendenburg
R
R
D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology* / Q000235:genetics; D002845:Chromatography / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006602:Hexosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins
D006602:Hexosyltransferases; C021451:inulosucrase; D001426:Bacterial Proteins