title: Yeast homologous recombination-based promoter engineering for the activation of silent natural product biosynthetic gene clusters.
abstract: Large-scale sequencing of prokaryotic (meta)genomic DNA suggests that most bacterial natural product gene clusters are not expressed under common laboratory culture conditions. Silent gene clusters represent a promising resource for natural product discovery and the development of a new generation of therapeutics. Unfortunately, the characterization of molecules encoded by these clusters is hampered owing to our inability to express these gene clusters in the laboratory. To address this bottleneck, we have developed a promoter-engineering platform to transcriptionally activate silent gene clusters in a model heterologous host. Our approach uses yeast homologous recombination, an auxotrophy complementation-based yeast selection system and sequence orthogonal promoter cassettes to exchange all native promoters in silent gene clusters with constitutively active promoters. As part of this platform, we constructed and validated a set of bidirectional promoter cassettes consisting of orthogonal promoter sequences, Streptomyces ribosome binding sites, and yeast selectable marker genes. Using these tools we demonstrate the ability to simultaneously insert multiple promoter cassettes into a gene cluster, thereby expediting the reengineering process. We apply this method to model active and silent gene clusters (rebeccamycin and tetarimycin) and to the silent, cryptic pseudogene-containing, environmental DNA-derived Lzr gene cluster. Complete promoter refactoring and targeted gene exchange in this "dead" cluster led to the discovery of potent indolotryptoline antiproliferative agents, lazarimides A and B. This potentially scalable and cost-effective promoter reengineering platform should streamline the discovery of natural products from silent natural product biosynthetic gene clusters.
authors.affiliation: Laboratory of Genetically Encoded Small Molecules, The Rockefeller University, New York, NY 10065.
authors.forename: Daniel
authors.initials: D
authors.lastname: Montiel
authors.affiliation: Laboratory of Genetically Encoded Small Molecules, The Rockefeller University, New York, NY 10065.
authors.forename: Hahk-Soo
authors.initials: HS
authors.lastname: Kang
authors.affiliation: Laboratory of Genetically Encoded Small Molecules, The Rockefeller University, New York, NY 10065.
authors.forename: Fang-Yuan
authors.initials: FY
authors.lastname: Chang
authors.affiliation: Laboratory of Genetically Encoded Small Molecules, The Rockefeller University, New York, NY 10065.
authors.forename: Zachary
authors.initials: Z
authors.lastname: Charlop-Powers
authors.affiliation: Laboratory of Genetically Encoded Small Molecules, The Rockefeller University, New York, NY 10065 sbrady@rockefeller.edu.
authors.forename: Sean F
authors.initials: SF
authors.lastname: Brady
mesh_terms: D001688:Biological Products / Q000378:metabolism*; D053898:Biosynthetic Pathways / Q000235:genetics*; D018506:Gene Expression Regulation, Plant; D005800:Genes, Fungal; D005818:Genetic Engineering; D059765:Homologous Recombination / Q000235:genetics*; D008954:Models, Biological; D008969:Molecular Sequence Data; D005810:Multigene Family; D016254:Mutagenesis, Insertional; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics*
chemical_list: D001688:Biological Products
keywords: environmental DNA; indolotryptoline; promoter engineering