Production of 1-kestose in transgenic yeast expressing a fructosyltransferase from Aspergillus foetidus.
Sucrose-inducible secretory sucrose:sucrose 1-fructosyltransferase (1-SST) from Aspergillus foetidus has been purified and subjected to N-terminal amino acid sequence determination. The enzyme is extensively glycosylated, and the active form is probably represented by a dimer of identical subunits with an apparent molecular mass of 180 kDa as judged from mobility in seminative acrylamide gels. The enzyme catalyzes fructosyl transfer from sucrose to sucrose producing glucose and 1-kestose. Oligosaccharides with a higher degree of polymerization are not obtained with sucrose as the substrate. The cDNA encoding the A. foetidus 1-SST has been cloned and sequenced. Sequence homology was found to be highest to levanases, but no hydrolytic activity was observed when levan was incubated with the enzyme. Expression of the cloned gene in an invertase-deficient mutant of Saccharomyces cerevisiae resulted in 1-kestose production, with 6-kestose and neokestose being side products of the reaction. Products were well distinguishable from those formed by yeast transformants expressing a cytosolic invertase.
Rehm
J
J
Max-Planck-Institut für Molekulare Pflanzenphysiologie, Golm, Germany.
Willmitzer
L
L
Heyer
A G
AG
D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D003001:Cloning, Molecular; D019281:Dimerization; D005800:Genes, Fungal; D005947:Glucose / Q000378:metabolism; D006031:Glycosylation; D006602:Hexosyltransferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D013395:Sucrose / Q000378:metabolism*; D014170:Transformation, Genetic; D014312:Trisaccharides / Q000096:biosynthesis*; D001426:Bacterial Proteins
D005947:Glucose; D006602:Hexosyltransferases; D011994:Recombinant Proteins; D013395:Sucrose; D014312:Trisaccharides; C072433:1-kestose; C021451:inulosucrase; D001426:Bacterial Proteins