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 co-localized 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 auto-resistance. 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.

Antibiotic drug discovery

“…A recently developed method, therefore, combines the search for biosynthesis genes with detecting potential resistance genes. This so‐called target‐directed genome mining (Tang et al ., 2015) is based on the observation that antibiotic‐producing bacteria need to be resistant against the producing compound themselves to avoid suicide. In some cases, a resistant second copy of the target gene is encoded within the biosynthetic gene cluster of the antibiotic allowing a more targeted search for antibiotic compounds with interesting targets.…”

Sequencing rare marine actinomycete genomes reveals high density of unique natural product biosynthetic gene clusters

“…As bioinformatic tools for assessing bacterial secondary metabolite biosynthesis advance, the power in genome mining amplifies, allowing for de-replication of known products, compound class identification, structural predictions and, in some cases, target identification Tang et al, 2015). Likewise, advances in heterologous expression and regulation manipulation allow increased access to silent natural product biosynthetic pathways (Tang et al, 2015;Yamanaka et al, 2014).…”

Breaking the silence: new strategies for discovering novel natural products

“…After further refinement for direct capture of gene clusters and discovery of taromycin A in Streptomyces coelicolor [34 •• ], Moore and coworkers successfully extended this method to the heterologous expression of BGCs in Bacillus subtilis and Escherichia coli (Figure 3) [35,36]. Moreover, by coupling with a target-directed genome mining process, a distinct group of thiotetronic acid natural products was discovered [37]. …”