Search

With the advent of rapid automatedin silicoidentification of biosynthetic gene clusters (BGCs), genomics presents vast opportunities to accelerate natural product (NP) discovery. However, prolific NP producers,Streptomyces, are exceptionally GC-rich (>80%) and highly repetitive within BGCs. These pose challenges in sequencing and high-quality genome assembly which are currently circumventedviaintensive sequencing. Here, we outline a more cost-effective workflow using multiplex Illumina and Oxford Nanopore sequencing with hybrid long-short read assembly algorithms to generate high quality genomes. Our protocol involves subjecting long read-derived assemblies to up to 4 rounds of polishing with short reads to yield accurate BGC predictions. We successfully sequenced and assembled 8 GC-richStreptomycesgenomes whose lengths range from 7.1 to 12.1 Mb at an average N50 of 5.9 Mb. Taxonomic analysis revealed previous misrepresentation among these strains and allowed us to propose a potentially new species,Streptomyces sydneybrenneri. Further comprehensive characterization of their biosynthetic, pan-genomic and antibiotic resistance features especially for molecules derived from type I polyketide synthase (PKS) BGCs reflected their potential as NP chassis. Thus, the genome assemblies and insights presented here are envisioned to serve as gateway for the scientific community to expand their avenues in NP discovery.Graphic abstractSchematic of hybrid long- and short read assembly workflow for genome sequencing of GC-richStreptomyces. Boxes shaded blue and grey correspond to experimental andin silicoworkflows, respectively.HighlightsA cost-effective genome sequencing approach for GC-richStreptomycesis presentedHybrid assembly improves BGC annotation and identificationA new species,Streptomyces sydneybrenneri, identified by taxonomic analysisGenomes of 8Streptomycesspecies are reported and analysed in this study

In recent years, CRISPR-Cas toolboxes for Streptomyces editing have rapidly accelerated natural product discovery and engineering. However, Cas efficiencies are also oftentimes strain dependent, subsequently a variety of Cas proteins would allow for flexibility and enable genetic manipulation within a wider range of Streptomyces strains. In this work, we have further expanded the Cas toolbox by presenting the first example of Cas12j mediated editing in Streptomyces sp. A34053. In our study, we have also observed significantly improved editing efficiencies with Acidaminococcus sp. Cas12j compared to Cas12a, Francisella tularensis subsp. novicida U112’s type V-A Cas (FnCpf1).