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

Abstract

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.promoter engineering | indolotryptoline | environmental DNA

De novo Biosynthesis of “Non‐Natural” Thaxtomin Phytotoxins

“…However there are many microorganisms that produce useful secondary metabolites, which are not amenable to such genetic manipulation. The rise of synthetic biology has provided access to larger synthetic DNA constructs, rapid DNA capture,7, 8, 9 editing,10, 11 assembly,12, 13 and other advances 14, 15. The prospect of using these new tools to assemble de novo biosynthetic pathways in well‐characterized heterologous host strains16, 17 for diversification and optimization of natural products, derived from less tractable microorganisms, is an attractive goal.…”

Breaking the silence: new strategies for discovering novel natural products

“…The more commonly used refactoring strategy is to replace the native promoters by the well-characterized constitutive promoters. One way to accomplish this is through HR in S. cerevisiae [49 • ]. The target BGC is first cloned into a shuttle vector and then co-transformed with promoter cassettes into S. cerevisiae.…”

Prospecting for new bacterial metabolites: a glossary of approaches for inducing, activating and upregulating the biosynthesis of bacterial cryptic or silent natural products

“…In such a manner the group used a series of single cassette insertions to replace each of the three natural bidirectional promoter regions involved in indolotryptoline biosynthesis with synthetic promoter cassettes, whilst at the same time repairing a base deletion in one of the genes that had rendered it non functional; the product of the previously dead cluster has been named lazarimide. 76 In Streptomycetes, overexpression of the pathway-specific regulators SARP (Antibiotic Regulatory Protein family) has been shown to trigger the production of secondary metabolites. The pathway specific regulator ccaR, a member of the SARP family, is responsible for the regulation of clavulanic acid (48) in Streptomyces clavuligerus.…”

“…A growing number of different approaches, with the aim of providing a more global solution to unlocking bacterial cryptic pathways, have been published in recent years, including studies with a focus on unlocking the treasures of the megasynthases through increasing transcription levels and "reviving dead" genes. 74,76 Streptomycetaceae are known to be a prolific source of bioactive compounds: so far over 200 drugs based on products isolated from these microorganisms are in clinical trials or FDA approved 12 and have been used for treating infections, cancer circulation and immune system disorders and many other disease states. When the genome sequence of the archetypal Streptomycetaceae Streptomyces coelicolor A3(2) was published in 2002, 13 the sequence data revealed, along with the known secondary metabolite clusters, a plethora of genes encoding for as yet unobserved molecules, including nonribosomal peptides and polyketides.…”