Abstract
Glycosylation represents an attractive target for protein engineering of novel antibiotics, because specific attachment of one or more deoxysugars is required for the bioactivity of many antibiotic and antitumour polyketides. However, proper assessment of the potential of these enzymes for such combinatorial biosynthesis requires both more precise information on the enzymology of the pathways and also improvedEscherichia coli-actinomycete shuttle vectors. New replicative vectors have been constructed and used to express independently thednmU gene ofStreptomyces peucetius and theeryBVII gene ofSaccharopolyspora erythraea in aneryBVII deletion mutant ofSac. erythraea. Production of erythromycin A was obtained in both cases, showing that both proteins serve analogous functions in the biosynthetic pathways to dTDP-L-daunosamine and dTDP-L-mycarose, respectively. Over-expression of both proteins was also obtained inS. lividans, paving the way for protein purification and in vitro monitoring of enzyme activity. In a further set of experiments, the putative desosaminyltransferase ofSac. erythraea, EryCIII, was expressed in the picromycin producerStreptomyces sp. 20032, which also synthesises dTDP-D-desosamine. The substrate 3- alpha -mycarosylerythronolide B used for hybrid biosynthesis was found to be glycosylated to produce erythromycin D only when recombinant EryCIII was present, directly confirming the enzymatic role of EryCIII. This convenient plasmid expression system can be readily adapted to study the directed evolution of recombinant glycosyltransferases.