title: Enhanced production of β-glucosides by in-situ UDP-glucose regeneration.
abstract: Glycosyltransferase (GT)-mediated methodology is recognized as one of the most practical approaches for large-scale production of glycosides. However, GT enzymes require a sugar nucleotide as donor substrate that must be generated in situ for preparative applications by recycling of the nucleotide moiety, e.g. by sucrose synthase (SUS). Three plant GT genes CaUGT2, VvGT14a, and VvGT15c and the fungal SbUGTA1 were successfully co-expressed with GmSUS from soybean in Escherichia coli BL21 and W cells. In vitro, the crude protein extracts prepared from four GT genes and GmSUS co-expressing cells were able to convert several small molecules to the corresponding glucosides, when sucrose and UDP were supplied. In addition, GmSUS was able to enhance the glucosylation efficiency and reduced the amount of supplying UDP-glucose. In the biotransformation system, co-expression of VvGT15c with GmSUS also improved the glucosylation of geraniol and enhanced the resistance of the cells against the toxic terpenol. GT-EcW and GTSUS-EcW cells tolerated up to 2mM geraniol and converted more than 99% of the substrate into the glucoside at production rates exceeding 40μgml(-1)h(-1). The results confirm that co-expression of SUS allows in situ regeneration of UDP-sugars and avoids product inhibition by UDP.
authors.affiliation: Technische Universität München, Biotechnology of Natural Products, Liesel-Beckmann-Str. 1, D-85354 Freising, Germany.
authors.forename: Fong-Chin
authors.initials: FC
authors.lastname: Huang
authors.affiliation: Technische Universität München, Biotechnology of Natural Products, Liesel-Beckmann-Str. 1, D-85354 Freising, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Hinkelmann
authors.affiliation: Technische Universität München, Biotechnology of Natural Products, Liesel-Beckmann-Str. 1, D-85354 Freising, Germany.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Hermenau
authors.affiliation: Technische Universität München, Biotechnology of Natural Products, Liesel-Beckmann-Str. 1, D-85354 Freising, Germany. Electronic address: schwab@tum.de.
authors.forename: Wilfried
authors.initials: W
authors.lastname: Schwab
mesh_terms: D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D006027:Glycosides / Q000737:chemistry / Q000378:metabolism*; D016695:Glycosyltransferases / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013025:Glycine max / Q000201:enzymology* / Q000235:genetics; D013395:Sucrose / Q000737:chemistry; D014530:Uridine Diphosphate / Q000737:chemistry
chemical_list: D005656:Fungal Proteins; D006027:Glycosides; D010940:Plant Proteins; D011994:Recombinant Proteins; D013395:Sucrose; D014530:Uridine Diphosphate; D016695:Glycosyltransferases
keywords: Glucosylation; Glycosyltransferase; Sucrose synthase; UDP-glucose regeneration