Engineered urdamycin glycosyltransferases are broadened and altered in substrate specificity.
Combinatorial biosynthesis is a promising technique used to provide modified natural products for drug development. To enzymatically bridge the gap between what is possible in aglycon biosynthesis and sugar derivatization, glycosyltransferases are the tools of choice. To overcome limitations set by their intrinsic specificities, we have genetically engineered the protein regions governing nucleotide sugar and acceptor substrate specificities of two urdamycin deoxysugar glycosyltransferases, UrdGT1b and UrdGT1c. Targeted amino acid exchanges reduced the number of amino acids potentially dictating substrate specificity to ten. Subsequently, a gene library was created such that only codons of these ten amino acids from both parental genes were independently combined. Library members displayed parental and/or a novel specificity, with the latter being responsible for the biosynthesis of urdamycin P that carries a branched saccharide side chain hitherto unknown for urdamycins.
Hoffmeister
Dirk
D
Department of Pharmaceutical Biology, Albert-Ludwigs-Universität Freiburg, Stefan-Meier-Strasse 19, 79104 Freiburg, Germany.
Wilkinson
Barrie
B
Foster
Graham
G
Sidebottom
Philip J
PJ
Ichinose
Koji
K
Bechthold
Andreas
A
D000595:Amino Acid Sequence; D000617:Aminoglycosides; D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D001426:Bacterial Proteins; D020650:Combinatorial Chemistry Techniques; D015723:Gene Library; D005798:Genes, Bacterial; D016695:Glycosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D015202:Protein Engineering; D016415:Sequence Alignment; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity
D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D016695:Glycosyltransferases; C436974:UrdGT1b protein, Streptomyces fradiae; C436975:UrdGT1c protein, Streptomyces fradiae