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.

Figure 1

Bioinformatic identification of candidate resistance genes. (a) The workflow for identification of duplicated orthologous groups (OGs) associated with biosynthetic gene cluster (BGC) from 86 Salinispora strains: 1. Application of OrthoMCL to 86 Salinispora genomes resulted in the identification of 12,372 OGs, representing the pan-genome of the strains in the present study; 2. From the 12,372 OGs, application of custom python scripts identified 2707 OGs that are shared among all 86 strains (core genome). The core genome is comprised of 1390 unique COG numbers. Python scripts matched these unique COG numbers to identical COG numbers in the pan-genome, identifying a total of 2393 OGs (duplicate OGs). 3. Application of PHP scripts for identifying duplicated OGs associated with predicted BGCs, resulted in the identification of 912 OGs from 20 COG categories being associated with BGCs. 4. 103 of the 912 OGs were affiliated with COG category “I” (lipid transport and metabolism). These OGs were annotated by BlastP searches, identifying one OG with homology to FabB/F, a known platensimycin and platencin resistance gene. This OG is located within the BGC PKS44 (tlm). (b) Distribution of the total number of duplicated OGs (yellow) and the OGs located within predicted BGC boundaries (green) among the COG categories.

Figure 2

Identification of orphan BGCs with an extra copy of the fabB/F housekeeping gene and their metabolites. (a) Orphan gene cluster annotation. (b) HPLC profile of extracts from (i) S. coelicolor M1152, (ii) S. coelicolor M1152/pMXT13 (tlm), and (iii) S. coelicolor M1152/pMXT16 (ttm). Detection at 239 nm. (c) Structures of thiolactomycin (TLM, 1) and analogues identified in this work.

Figure 3

High-efficiency TAR based direct cloning strategy for capture of natural product gene clusters. (a) The high-efficiency TAR cloning involves three steps. In step 1, the capture vector is constructed in one step by Gibson Assembly using 144 bp synthetic dsDNA. In step 2, the capture vector is linearized by the restriction enzyme PmeI. In step 3, recombination in yeast. (b) Tabulation of direct cloning parameters of two orphan gene clusters.

Figure 4

Representative disk diffusion assay plates for TTM C (300 μg) against S. coelicolor M1152 integrated with (a) the ttm gene cluster (designated as M1152/pMXT16) and (b-d) three mutants: (b) the ttmE deleted mutant S. coelicolor M1152/pMXT16ΔttmEscar, (c) the ttmJ deleted mutant S. coelicolor M1152/pMXT16ΔttmJ, and (d) the ttmE - ttmJ double mutant S. coelicolor M1152/pMXT16ΔttmEscar-ΔttmJ.