Atolypenes, Tricyclic Bacterial Sesterterpenes Discovered Using a Multiplexed In Vitro Cas9-TAR Gene Cluster Refactoring Approach

Abstract

Most natural product biosynthetic gene clusters identified in bacterial genomic and metagenomic sequencing efforts are silent under laboratory growth conditions. Here, we describe a scalable biosynthetic gene cluster activation method wherein the gene clusters are disassembled at interoperonic regions in vitro using CRISPR/Cas9 and then reassembled with PCR-amplified, short DNAs, carrying synthetic promoters, using transformation assisted recombination (TAR) in yeast. This simple, cost-effective, and scalable method allows for the simultaneous generation of combinatorial libraries of refactored gene clusters, eliminating the need to understand the transcriptional hierarchy of the silent genes. In two test cases, this in vitro disassembly-TAR reassembly method was used to create collections of promoter-replaced gene clusters that were tested in parallel to identify versions that enabled secondary metabolite production. Activation of the atolypene (ato) gene cluster led to the characterization of two unprecedented, bacterial cyclic sesterterpenes, atolypene A (1) and B (2), which are moderately cytotoxic to human cancer cell lines. This streamlined in vitro disassembly-in vivo reassembly method offers a simplified approach for silent gene cluster refactoring that should facilitate the discovery of natural products from silent gene clusters cloned from either metagenomes or cultured bacteria.

Unusual and Highly Bioactive Sesterterpenes Synthesized by Pleurotus ostreatus during Coculture with Trametes robiniophila Murr

“…In contrast, for the biosynthesis of sesterterpenes originating from plants such as Arabidopsis thaliana and Brassicaceae, colocalization of a prenyltransferase and terpene synthase pair instead of one bifunctional sesterterpene synthase has been demonstrated in those steps (41,42). Genes encoding sesterterpene synthases recently discovered from bacteria are in clusters resembling those in plants with genes encoding an independent prenyltransferase and a terpene synthase colocated within the cluster (43)(44)(45). So far, only one linear sesterterpene synthase, Bcl-TS, has been identified from Bacillus clausii, in which the presence of an independent terpene synthase is sufficient to synthesize the linear product in a head-to-tail manner (46).…”

Pseudomonas putida rDNA is a favored site for the expression of biosynthetic genes

“…This might, however, compromise a required balance in transcript levels of individual genes and needs to be tested experimentally. Alternative approaches include systematic gene cluster and promoter refactoring which can serve to install beneficial transcription strengths 41,42 .…”

Recent Advances in Synthetic Biology Approaches to Optimize Production of Bioactive Natural Products in Actinobacteria

“…Next, a variety of in vitro or in vivo DNA assembly methods have been established to reconstruct target BGCs for diverse applications (Li et al, 2017a; Zhang et al, 2019). On the one hand, a panel of in vivo DNA assembly methods for single-marker or marker-free multiplexed promoter engineering of large BGCs have been developed on the basis of powerful homologous recombination capacity in S. cerevisiae , including DNA assembler (Shao et al, 2011), mCRISTAR (multiplexed Cas9-transformation-associated recombination) (Kang et al, 2016) and miCRISTAR (multiplexed in vitro Cas9-transformation-associated recombination) (Kim et al, 2019). For example, using the miCRISTAR strategy, the activation of a silent BGC led to the characterization of two bacterial cyclic sesterterpenes atolypene A and B (Kim et al, 2019).…”

“…On the one hand, a panel of in vivo DNA assembly methods for single-marker or marker-free multiplexed promoter engineering of large BGCs have been developed on the basis of powerful homologous recombination capacity in S. cerevisiae , including DNA assembler (Shao et al, 2011), mCRISTAR (multiplexed Cas9-transformation-associated recombination) (Kang et al, 2016) and miCRISTAR (multiplexed in vitro Cas9-transformation-associated recombination) (Kim et al, 2019). For example, using the miCRISTAR strategy, the activation of a silent BGC led to the characterization of two bacterial cyclic sesterterpenes atolypene A and B (Kim et al, 2019). In another study, yeast-mediated construction of a riboswitch-controlled pathway achieved a 120-fold increase in bottromycin productivity (Eyles et al, 2018).…”