Yeast homologous recombination-based promoter engineering for the activation of silent natural product biosynthetic gene clusters

• Published in: Proceedings of the National Academy of Sciences - PNAS 2015-07, Vol.112 (29), p.8953-8958
• Main Authors: Montiel, Daniel, Hahk-Soo Kang, Fang-Yuan Chang, Zachary Charlop-Powers, Sean F. Brady
• Format: Article
• Language: English
• Subjects: Biological Products - metabolism; Biological Sciences; Biosynthesis; Biosynthetic Pathways - genetics; cost effectiveness; data collection; Deoxyribonucleic acid; DNA; engineering; environmental DNA; fermentation; Gene expression; Gene Expression Regulation, Plant; Genes, Fungal; Genetic Engineering; Genomics; Homologous Recombination - genetics; indolotryptoline; metabolites; Models, Biological; Molecular Sequence Data; Multigene Family; Mutagenesis, Insertional; Natural products; nucleotide sequences; Physical Sciences; Prokaryotes; promoter engineering; Promoter Regions, Genetic; Saccharomyces cerevisiae - genetics; sequence analysis; Streptomyces; yeasts
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1507606112

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. A rapidly growing number of cryptic natural product biosynthetic gene clusters have been identified in bacterial DNA sequencing datasets. The metabolites encoded by most of these gene clusters remain uncharacterized because they are not readily activated using monoculture fermentation methods. The development of generic gene cluster activation strategies is needed to access molecules encoded by this rapidly growing collection of sequenced gene clusters. The promoter engineering platform outlined here provides a simple, cost-effective, and potentially scalable tool for the characterization of molecules encoded by gene clusters found in sequenced microbial (meta)genomes. We believe