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.

This review covers current genetic technologies for accessing and manipulating natural product biosynthetic gene clusters through heterologous expression.

Heterologous expression has become a powerful tool for studying microbial biosynthetic gene clusters (BGCs). Here, we extend the transformation-associated recombination cloning and heterologous expression platform for microbial BGCs to include Gram-negative proteobacterial expression hosts. Using a broad-host-range expression platform, we test the implicit assumption that biosynthetic pathways are more successfully expressed in more closely related heterologous hosts. Cloning and expression of the violacein BGC from Pseudoalteromonas luteoviolacea 2ta16 revealed robust production in two proteobacterial hosts, Pseudomonas putida KT2440 and Agrobacterium tumefaciens LBA4404, but very little production of the antibiotic in various laboratory strains of Escherichia coli, despite their closer phylogenetic relationship. We identified a nonclustered LuxR-type quorum-sensing receptor from P. luteoviolacea 2ta16, PviR, that increases pathway transcription and violacein production in E. coli by ∼60-fold independently of acyl-homoserine lactone autoinducers. Although E. coli harbors the most similar homolog of PviR identified from all of the hosts tested, overexpression of various E. coli transcription factors did not result in a statistically significant increase in violacein production, while overexpression of two A. tumefaciens PviR homologs significantly increased production. Thus, this work not only introduces a new genetic platform for the heterologous expression of microbial BGCs, it also challenges the assumption that host phylogeny is an accurate predictor of host compatibility.

Heterologous expression of natural product biosynthetic gene clusters (BGCs) is a robust approach not only to decipher biosynthetic logic behind natural product (NP) biosynthesis, but also to discover new chemicals from uncharacterized BGCs. This approach largely relies on techniques used for cloning large BGCs into suitable expression vectors. Recently, several whole-pathway direct cloning approaches, including full-length RecE-mediated recombination in Escherichia coli, Cas9-assisted in vitro assembly, and transformation-associated recombination (TAR) in Saccharomyces cerevisiae, have been developed to accelerate BGC isolation. In this chapter, we summarize a protocol for TAR cloning large NP BGCs, detailing the process of choosing TAR plasmids, designing pathway-specific TAR vectors, generating yeast spheroplasts, performing yeast transformation, and heterologously expressing BGCs in various host strains. We believe that the established platforms can accelerate the process of discovering new NPs, understanding NP biosynthetic logic, and engineering biosynthetic pathways.