Insights about the biosynthesis of the avermectin deoxysugar L-oleandrose through heterologous expression of Streptomyces avermitilis deoxysugar genes in Streptomyces lividans

Chem Biol. 2001 Jul;8(7):681-700. doi: 10.1016/s1074-5521(01)00043-6.

Abstract

Background: The avermectins, produced by Streptomyces avermitilis, are potent anthelminthic agents with a polyketide-derived macrolide skeleton linked to a disaccharide composed of two alpha-linked L-oleandrose units. Eight contiguous genes, avrBCDEFGHI (also called aveBI-BVIII), are located within the avermectin-producing gene cluster and have previously been mapped to the biosynthesis and attachment of thymidinediphospho-oleandrose to the avermectin aglycone. This gene cassette provides a convenient way to study the biosynthesis of 2,6-dideoxysugars, namely that of L-oleandrose, and to explore ways to alter the biosynthesis and structures of the avermectins by combinatorial biosynthesis.

Results: A Streptomyces lividans strain harboring a single plasmid with the avrBCDEFGHI genes in which avrBEDC and avrIHGF were expressed under control of the actI and actIII promoters, respectively, correctly glycosylated exogenous avermectin A1a aglycone with identical oleandrose units to yield avermectin A1a. Modified versions of this minimal gene set produced novel mono- and disaccharide avermectins. The results provide further insight into the biosynthesis of L-oleandrose.

Conclusions: The plasmid-based reconstruction of the avr deoxysugar genes for expression in a heterologous system combined with biotransformation has led to new information about the mechanism of 2,6-deoxysugar biosynthesis. The structures of the di-demethyldeoxysugar avermectins accumulated indicate that in the oleandrose pathway the stereochemistry at C-3 is ultimately determined by the 3-O-methyltransferase and not by the 3-ketoreductase or a possible 3,5-epimerase. The AvrF protein is therefore a 5-epimerase and not a 3,5-epimerase. The ability of the AvrB (mono-)glycosyltransferase to accommodate different deoxysugar intermediates is evident from the structures of the novel avermectins produced.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Anthelmintics / metabolism*
  • Combinatorial Chemistry Techniques
  • Deoxy Sugars / biosynthesis*
  • Deoxy Sugars / genetics
  • Deoxy Sugars / metabolism*
  • Gene Expression
  • Hexoses / metabolism*
  • Ivermectin / analogs & derivatives
  • Ivermectin / metabolism*
  • Multigene Family / genetics
  • Plasmids
  • Protein Engineering
  • Racemases and Epimerases / metabolism
  • Stereoisomerism
  • Streptomyces / chemistry*
  • Streptomyces / genetics
  • Streptomyces / metabolism

Substances

  • Anthelmintics
  • Deoxy Sugars
  • Hexoses
  • oleandrose
  • Ivermectin
  • avermectin
  • Racemases and Epimerases