Cloning and Heterologous Expression of the Grecocycline Biosynthetic Gene Cluster

PLoS One. 2016 Jul 13;11(7):e0158682. doi: 10.1371/journal.pone.0158682. eCollection 2016.

Abstract

Transformation-associated recombination (TAR) in yeast is a rapid and inexpensive method for cloning and assembly of large DNA fragments, which relies on natural homologous recombination. Two vectors, based on p15a and F-factor replicons that can be maintained in yeast, E. coli and streptomycetes have been constructed. These vectors have been successfully employed for assembly of the grecocycline biosynthetic gene cluster from Streptomyces sp. Acta 1362. Fragments of the cluster were obtained by PCR and transformed together with the "capture" vector into the yeast cells, yielding a construct carrying the entire gene cluster. The obtained construct was heterologously expressed in S. albus J1074, yielding several grecocycline congeners. Grecocyclines have unique structural moieties such as a dissacharide side chain, an additional amino sugar at the C-5 position and a thiol group. Enzymes from this pathway may be used for the derivatization of known active angucyclines in order to improve their desired biological properties.

MeSH terms

  • Anti-Bacterial Agents / biosynthesis*
  • Benz(a)Anthracenes / chemistry*
  • Benz(a)Anthracenes / metabolism
  • Chromosomes, Artificial, Bacterial
  • Cloning, Molecular / methods*
  • Genes, Bacterial / genetics
  • Genetic Vectors / genetics
  • Glycosides / biosynthesis
  • Glycosides / chemistry
  • Glycosides / genetics*
  • Multigene Family / genetics
  • Polymerase Chain Reaction
  • Replicon / genetics
  • Saccharomyces cerevisiae / genetics*
  • Streptomyces / genetics*
  • Streptomyces / metabolism*

Substances

  • Anti-Bacterial Agents
  • Benz(a)Anthracenes
  • Glycosides

Grants and funding

This work was supported by the ERC starting grant EXPLOGEN No. 281623 to AL, https://erc.europa.eu/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.