title: Identification of Thiotetronic Acid Antibiotic Biosynthetic Pathways by Target-directed Genome Mining.
abstract: Recent genome sequencing efforts have led to the rapid accumulation of uncharacterized or "orphaned" secondary metabolic biosynthesis gene clusters (BGCs) in public databases. This increase in DNA-sequenced big data has given rise to significant challenges in the applied field of natural product genome mining, including (i) how to prioritize the characterization of orphan BGCs and (ii) how to rapidly connect genes to biosynthesized small molecules. Here, we show that by correlating putative antibiotic resistance genes that encode target-modified proteins with orphan BGCs, we predict the biological function of pathway specific small molecules before they have been revealed in a process we call target-directed genome mining. By querying the pan-genome of 86 Salinispora bacterial genomes for duplicated house-keeping genes colocalized with natural product BGCs, we prioritized an orphan polyketide synthase-nonribosomal peptide synthetase hybrid BGC (tlm) with a putative fatty acid synthase resistance gene. We employed a new synthetic double-stranded DNA-mediated cloning strategy based on transformation-associated recombination to efficiently capture tlm and the related ttm BGCs directly from genomic DNA and to heterologously express them in Streptomyces hosts. We show the production of a group of unusual thiotetronic acid natural products, including the well-known fatty acid synthase inhibitor thiolactomycin that was first described over 30 years ago, yet never at the genetic level in regards to biosynthesis and autoresistance. This finding not only validates the target-directed genome mining strategy for the discovery of antibiotic producing gene clusters without a priori knowledge of the molecule synthesized but also paves the way for the investigation of novel enzymology involved in thiotetronic acid natural product biosynthesis.
authors.affiliation: Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA, USA.
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Tang
authors.affiliation: Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA, USA.
authors.forename: Jie
authors.initials: J
authors.lastname: Li
authors.affiliation: Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA, USA.
authors.forename: Natalie
authors.initials: N
authors.lastname: Millán-Aguiñaga
authors.affiliation: Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA, USA.
authors.forename: Jia Jia
authors.initials: JJ
authors.lastname: Zhang
authors.affiliation: Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA, USA.
authors.forename: Ellis C
authors.initials: EC
authors.lastname: O'Neill
authors.affiliation: Centro de Genómica y Bioinformática. Facultad de Ciencias. Universidad Mayor. Campus Huechuraba, Camino a la Pirámide 5750. Santiago, Chile.
authors.forename: Juan A
authors.initials: JA
authors.lastname: Ugalde
authors.affiliation: Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA, USA.
authors.forename: Paul R
authors.initials: PR
authors.lastname: Jensen
authors.affiliation: Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA, USA.
authors.forename: Simone M
authors.initials: SM
authors.lastname: Mantovani
authors.affiliation: Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA, USA.|Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA, USA.
authors.forename: Bradley S
authors.initials: BS
authors.lastname: Moore
mesh_terms: D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D053898:Biosynthetic Pathways / Q000235:genetics*; D019295:Computational Biology; D018390:Gene Targeting; D016680:Genome, Bacterial; D006885:Hydroxybutyrates / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D015394:Molecular Structure; D005810:Multigene Family; D013302:Streptomyces / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D013876:Thiophenes / Q000737:chemistry / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D006885:Hydroxybutyrates; D013438:Sulfhydryl Compounds; D013876:Thiophenes; C000606611:thiotetronic acid; C035068:thiolactomycin