title: Cloning and Heterologous Expression of the Grecocycline Biosynthetic Gene Cluster.
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.
authors.affiliation: Helmholtz-Institute for Pharmaceutical Research Saarland, Saarland University Campus, Building C2.3, 66123 Saarbrücken, Germany.
authors.forename: Oksana
authors.initials: O
authors.lastname: Bilyk
authors.affiliation: Department of Pharmacognosy, University of Vienna, 1090 Vienna, Austria.
authors.forename: Olga N
authors.initials: ON
authors.lastname: Sekurova
authors.affiliation: Department of Pharmacognosy, University of Vienna, 1090 Vienna, Austria.
authors.forename: Sergey B
authors.initials: SB
authors.lastname: Zotchev
authors.affiliation: Helmholtz-Institute for Pharmaceutical Research Saarland, Saarland University Campus, Building C2.3, 66123 Saarbrücken, Germany.|Universität des Saarlandes, Pharmazeutische Biotechnologie, Building C2.3, 66123 Saarbrücken, Germany.
authors.forename: Andriy
authors.initials: A
authors.lastname: Luzhetskyy
mesh_terms: D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D001551:Benz(a)Anthracenes / Q000737:chemistry* / Q000378:metabolism; D022202:Chromosomes, Artificial, Bacterial; D003001:Cloning, Molecular / Q000379:methods*; D005798:Genes, Bacterial / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006027:Glycosides / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D005810:Multigene Family / Q000235:genetics; D016133:Polymerase Chain Reaction; D012093:Replicon / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013302:Streptomyces / Q000235:genetics* / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D001551:Benz(a)Anthracenes; D006027:Glycosides