Functional screening of metagenome and genome libraries for detection of novel flavonoid-modifying enzymes

Link:
Autor/in:
Erscheinungsjahr:
2013
Medientyp:
Text
Schlagworte:
  • Amino Acid Sequence
  • Bacteria/genetics
  • Chromatography, Thin Layer/methods
  • Cloning, Molecular
  • DNA, Bacterial/genetics
  • Flavonoids/metabolism
  • Genome, Bacterial
  • Genomic Library
  • Metagenome
  • Molecular Sequence Data
  • Polymerase Chain Reaction
  • Sequence Alignment
  • Sequence Analysis, DNA
Beschreibung:
  • The functional detection of novel enzymes other than hydrolases from metagenomes is limited since only a very few reliable screening procedures are available that allow the rapid screening of large clone libraries. For the discovery of flavonoid-modifying enzymes in genome and metagenome clone libraries, we have developed a new screening system based on high-performance thin-layer chromatography (HPTLC). This metagenome extract thin-layer chromatography analysis (META) allows the rapid detection of glycosyltransferase (GT) and also other flavonoid-modifying activities. The developed screening method is highly sensitive, and an amount of 4 ng of modified flavonoid molecules can be detected. This novel technology was validated against a control library of 1,920 fosmid clones generated from a single Bacillus cereusisolate and then used to analyze more than 38,000 clones derived from two different metagenomic preparations. Thereby we identified two novel UDP glycosyltransferase (UGT) genes. The metagenome-derived gtfCgene encoded a 52-kDa protein, and the deduced amino acid sequence was weakly similar to sequences of putative UGTs from Fibrisomaand Dyadobacter. GtfC mediated the transfer of different hexose moieties and exhibited high activities on flavones, flavonols, flavanones, and stilbenes and also accepted isoflavones and chalcones. From the control library we identified a novel macroside glycosyltransferase (MGT) with a calculated molecular mass of 46 kDa. The deduced amino acid sequence was highly similar to sequences of MGTs from Bacillus thuringiensis. Recombinant MgtB transferred the sugar residue from UDP-glucose effectively to flavones, flavonols, isoflavones, and flavanones. Moreover, MgtB exhibited high activity on larger flavonoid molecules such as tiliroside. © 2013, American Society for Microbiology. All Rights Reserved.
Lizenz:
  • info:eu-repo/semantics/restrictedAccess
Quellsystem:
Forschungsinformationssystem der UHH
Interne Metadaten
Quelldatensatz
oai:www.edit.fis.uni-hamburg.de:publications/5056334f-021e-4a1f-9301-9434de06ef76