Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Analysis of genes involved in 6-deoxyhexose biosynthesis and transfer in Saccharopolyspora erythraea

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 improved Escherichia coli-actinomycete shuttle vectors. New replicative vectors have been constructed and used to express independently the dnmU gene of Streptomyces peucetius and the eryBVII gene of Saccharopolyspora erythraea in an eryBVII deletion mutant of Sac. 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 in S. lividans, paving the way for protein purification and in vitro monitoring of enzyme activity. In a further set of experiments, the putative desosaminyltransferase of Sac. erythraea, EryCIII, was expressed in the picromycin producer Streptomyces sp. 20032, which also synthesises dTDP-D-desosamine. The substrate 3-α-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.

This is a preview of subscription content, log in to check access.

Author information

Additional information

Electronic Publication

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Doumith, M., Weingarten, P., Wehmeier, U. et al. Analysis of genes involved in 6-deoxyhexose biosynthesis and transfer in Saccharopolyspora erythraea . Mol Gen Genet 264, 477–485 (2000). https://doi.org/10.1007/s004380000329

Download citation

  • Polyketide antibiotics Glycosylation Pathway engineering Erythromycin Saccharopolyspora erythraea