Streptomyces produces diverse secondary metabolites of biopharmaceutical importance, yet the rate of biosynthesis of these metabolites is often hampered by complex transcriptional regulation. Therefore, a fundamental understanding of transcriptional regulation in Streptomyces is key to fully harness its genetic potential. Here, independent component analysis (ICA) of 454 high-quality gene expression profiles of the model species Streptomyces coelicolor is performed, of which 249 profiles are newly generated for S. coelicolor cultivated on 20 different carbon sources and 64 engineered strains with overexpressed sigma factors. ICA of the transcriptome dataset reveals 117 independently modulated groups of genes (iModulons), which account for 81.6% of the variance in the dataset. The genes in each iModulon are involved in specific cellular responses, which are often transcriptionally controlled by specific regulators. Also, iModulons accurately predict 25 secondary metabolite biosynthetic gene clusters encoded in the genome. This systemic analysis leads to reveal the functions of previously uncharacterized genes, putative regulons for 40 transcriptional regulators, including 30 sigma factors, and regulation of secondary metabolism via phosphate- and iron-dependent mechanisms in S. coelicolor. ICA of large transcriptomic datasets thus enlightens a new and fundamental understanding of transcriptional regulation of secondary metabolite synthesis along with interconnected metabolic processes in Streptomyces.

The demand for discovering novel microbial secondary metabolites is growing to address the limitations in bioactivities such as antibacterial, antifungal, anticancer, anthelmintic, and immunosuppressive functions. Among microbes, the genus Streptomyces holds particular significance for secondary metabolite discovery. Each Streptomyces species typically encodes approximately 30 secondary metabolite biosynthetic gene clusters (smBGCs) within its genome, which are mostly uncharacterized in terms of their products and bioactivities. The development of next-generation sequencing has enabled the identification of a large number of potent smBGCs for novel secondary metabolites that are imbalanced in number compared with discovered secondary metabolites. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system has revolutionized the translation of enormous genomic potential into the discovery of secondary metabolites as the most efficient genetic engineering tool for Streptomyces. In this review, the current status of CRISPR/Cas applications in Streptomyces is summarized, with particular focus on the identification of secondary metabolite biosynthesis gene clusters and their potential applications. This review summarizes the broad range of CRISPR/Cas applications in Streptomyces for natural product discovery and production. The demand for discovering novel microbial secondary metabolites is growing to address the limitations in bioactivities such as antibacterial, antifungal, anticancer, anthelmintic, and immunosuppressive functions. Among microbes, the genus Streptomyces holds particular significance for secondary metabolite discovery. Each Streptomyces species typically encodes approximately 30 secondary metabolite biosynthetic gene clusters (smBGCs) within its genome, which are mostly uncharacterized in terms of their products and bioactivities. The development of next-generation sequencing has enabled the identification of a large number of potent smBGCs for novel secondary metabolites that are imbalanced in number compared with discovered secondary metabolites. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system has revolutionized the translation of enormous genomic potential into the discovery of secondary metabolites as the most efficient genetic engineering tool for Streptomyces. In this review, the current status of CRISPR/Cas applications in Streptomyces is summarized, with particular focus on the identification of secondary metabolite biosynthesis gene clusters and their potential applications. This review summarizes the broad range of CRISPR/Cas applications in Streptomyces for natural product discovery and production.One-Sentence Summary This review summarizes the broad range of CRISPR/Cas applications in Streptomyces for natural product discovery and production.

Bacteria belonging to Streptomyces have the ability to produce a wide range of secondary metabolites through a shift from primary to secondary metabolism regulated by complex networks activated after vegetative growth terminates. Despite considerable effort to understand the regulatory elements governing gene expression related to primary and secondary metabolism in Streptomyces, system-level information remains limited. In this study, we integrated four multi-omics datasets from Streptomyces griseus NBRC 13350: RNA-seq, ribosome profiling, dRNA-seq, and Term-Seq, to analyze the regulatory elements of transcription and translation of differentially expressed genes during cell growth. With the functional enrichment of gene expression in different growth phases, one sigma factor regulon and four transcription factor regulons governing differential gene transcription patterns were found. In addition, the regulatory elements of transcription termination and post-transcriptional processing at transcript 3′-end positions were elucidated, including their conserved motifs, stem-loop RNA structures, and non-terminal locations within the polycistronic operons, and the potential regulatory elements of translation initiation and elongation such as 5′-UTR length, RNA structures at ribosome-bound sites, and codon usage were investigated. This comprehensive genetic information provides a foundational genetic resource for strain engineering to enhance secondary metabolite production in Streptomyces.

Abstract Background The gram-positive bacterium, Streptomyces avermitilis, holds industrial importance as the producer of avermectin, a widely used anthelmintic agent, and a heterologous expression host of secondary metabolite-biosynthetic gene clusters. Despite its industrial importance, S. avermitilis’ genome organization and regulation of gene expression remain poorly understood. In this study, four different types of Next-Generation Sequencing techniques, including dRNA-Seq, Term-Seq, RNA-Seq and ribosome profiling, were applied to S. avermitilis to determine transcription units of S. avermitilis at a genome-wide level and elucidate regulatory elements for transcriptional and translational control of individual transcription units. Result By applying dRNA-Seq and Term-Seq to S. avermitilis MA-4680, a total of 2361 transcription start sites and 2017 transcript 3′-end positions were identified, respectively, leading to determination of 1601 transcription units encoded in S. avermitilis’ genome. Cataloguing the transcription units and integrated analysis of multiple high-throughput data types revealed the presence of diverse regulatory elements for gene expression, such as promoters, 5′-UTRs, terminators, 3′-UTRs and riboswitches. The conserved promoter motifs were identified from 2361 transcription start sites as 5′-TANNNT and 5′-BTGACN for the − 10 and − 35 elements, respectively. The − 35 element and spacer lengths between − 10 and − 35 elements were critical for transcriptional regulation of functionally distinct genes, suggesting the involvement of unique sigma factors. In addition, regulatory sequences recognized by antibiotic regulatory proteins were identified from the transcription start site information. Analysis of the 3′-end of RNA transcript revealed that stem structure formation is a major determinant for transcription termination of most transcription units. Conclusions The transcription unit architecture elucidated from the transcripts’ boundary information provides insights for unique genetic regulatory mechanisms of S. avermitilis. Our findings will elevate S. avermitilis’ potential as a production host for a diverse set of secondary metabolites.

Covering: 2010 to 2020 Over the last few decades, Streptomyces have been extensively investigated for their ability to produce diverse bioactive secondary metabolites. Recent advances in Streptomyces research have been largely supported by improvements in high-throughput technology 'omics'. From genomics, numerous secondary metabolite biosynthetic gene clusters were predicted, increasing their genomic potential for novel bioactive compound discovery. Additional omics, including transcriptomics, translatomics, interactomics, proteomics and metabolomics, have been applied to obtain a system-level understanding spanning entire bioprocesses of Streptomyces, revealing highly interconnected and multi-layered regulatory networks for secondary metabolism. The comprehensive understanding derived from this systematic information accelerates the rational engineering of Streptomyces to enhance secondary metabolite production, integrated with the exploitation of the highly efficient 'Design-Build-Test-Learn' cycle in synthetic biology. In this review, we describe the current status of omics applications in Streptomyces research to better understand the organism and exploit its genetic potential for higher production of valuable secondary metabolites and novel secondary metabolite discovery.

Heterologous production of recombinant proteins is gaining increasing interest in biotechnology with respect to productivity, scalability, and wide applicability. The members of genus Streptomyces have been proposed as remarkable hosts for heterologous production due to their versatile nature of expressing various secondary metabolite biosynthetic gene clusters and secretory enzymes. However, there are several issues that limit their use, including low yield, difficulty in genetic manipulation, and their complex cellular features. In this review, we summarize rational engineering approaches to optimizing the heterologous production of secondary metabolites and recombinant proteins in Streptomyces species in terms of genetic tool development and chassis construction. Further perspectives on the development of optimal Streptomyces chassis by the design-build-test-learn cycle in systems are suggested, which may increase the availability of secondary metabolites and recombinant proteins.

Streptomyces species have attracted considerable interest as a reservoir of medically important secondary metabolites, which are even diverse and different between strains. Here, we reassess ten Streptomyces venezuelae strains by presenting the highly resolved classification, using 16S rRNA sequencing, MALDI-TOF MS protein profiling, and whole-genome sequencing. The results revealed that seven of the ten strains were misclassified as S. venezuelae species. Secondary metabolite biosynthetic gene cluster (smBGC) mining and targeted LC-MS/MS based metabolite screening of S. venezuelae and misclassified strains identified in total 59 secondary metabolites production. In addition, a comparison of pyrrolamide-type antibiotic BGCs of four misclassified strains, followed by functional genomics, revealed that athv28 is critical in the synthesis of the anthelvencin precursor, 5-amino-3,4-dihydro-2H-pyrrole-2-carboxylate (ADPC). Our findings illustrate the importance of the accurate classification and better utilization of misclassified Streptomyces strains to discover smBGCs and their secondary metabolite products.

Streptomyces sp. is a great source of bioactive secondary metabolites, including antibiotics, antifungal agents, antiparasitic agents, immunosuppressant compounds, and other drugs. Secondary metabolites are synthesized via multistep conversions of the precursor molecules from primary metabolism, governed by multicomplex enzymes from secondary metabolite biosynthetic gene clusters.

A Correction to this paper has been published: https://doi.org/10.1038/s41597-021-00858-2.

Actinomycetes are a rich source of bioactive natural products important for novel drug leads. Recent genome mining approaches have revealed an enormous number of secondary metabolite biosynthetic gene clusters (smBGCs) in actinomycetes. However, under standard laboratory culture conditions, many smBGCs are silent or cryptic. To activate these dormant smBGCs, several approaches, including culture-based or genetic engineering-based strategies, have been developed. Above all, coculture is a promising approach to induce novel secondary metabolite production from actinomycetes by mimicking an ecological habitat where cryptic smBGCs may be activated. In this review, we introduce coculture studies that aim to expand the chemical diversity of actinomycetes, by categorizing the cases by the type of coculture partner. Furthermore, we discuss the current challenges that need to be overcome to support the elicitation of novel bioactive compounds from actinomycetes.

RNA sequencing techniques have enabled the systematic elucidation of gene expression (RNA-Seq), transcription start sites (differential RNA-Seq), transcript 3' ends (Term-Seq), and post-transcriptional processes (ribosome profiling). The main challenge of transcriptomic studies is to remove ribosomal RNAs (rRNAs), which comprise more than 90% of the total RNA in a cell. Here, we report a low-cost and robust bacterial rRNA depletion method, RiboRid, based on the enzymatic degradation of rRNA by thermostable RNase H. This method implemented experimental considerations to minimize nonspecific degradation of mRNA and is capable of depleting pre-rRNAs that often comprise a large portion of RNA, even after rRNA depletion. We demonstrated the highly efficient removal of rRNA up to a removal efficiency of 99.99% for various transcriptome studies, including RNA-Seq, Term-Seq, and ribosome profiling, with a cost of approximately $10 per sample. This method is expected to be a robust method for large-scale high-throughput bacterial transcriptomic studies.

Microbial coculture to mimic the ecological habitat has been suggested as an approach to elucidate the effect of microbial interaction on secondary metabolite biosynthesis of Streptomyces. However, because of chemical complexity during coculture, underlying mechanisms are largely unknown. Here, we found that iron competition triggered antibiotic biosynthesis in Streptomyces coelicolor during coculture with Myxococcus xanthus. During coculture, M. xanthus enhanced the production of a siderophore, myxochelin, leading M. xanthus to dominate iron scavenging and S. coelicolor to experience iron-restricted conditions. This chemical competition, but not physical contact, activated the actinorhodin biosynthetic gene cluster and the branched-chain amino acid degradation pathway which imply the potential to produce precursors, along with activation of a novel actinorhodin export system. Furthermore, we found that iron restriction increased the expression of 21 secondary metabolite biosynthetic gene clusters (smBGCs) in other Streptomyces species. These findings suggested that the availability for key ions stimulates specific smBGCs, which had the potential to enhance secondary metabolite biosynthesis in Streptomyces.

The composition of anti-rRNA oligo mix utilized for depletion of rRNA

Streptomyces are Gram-positive bacteria of significant industrial importance due to their ability to produce a wide range of antibiotics and bioactive secondary metabolites. Recent advances in genome mining have revealed that Streptomyces genomes possess a large number of unexplored silent secondary metabolite biosynthetic gene clusters (smBGCs). This indicates that Streptomyces genomes continue to be an invaluable source for new drug discovery. Here, we present high-quality genome sequences of 22 Streptomyces species and eight different Streptomyces venezuelae strains assembled by a hybrid strategy exploiting both long-read and short-read genome sequencing methods. The assembled genomes have more than 97.4% gene space completeness and total lengths ranging from 6.7 to 10.1 Mbp. Their annotation identified 7,000 protein coding genes, 20 rRNAs, and 68 tRNAs on average. In silico prediction of smBGCs identified a total of 922 clusters, including many clusters whose products are unknown. We anticipate that the availability of these genomes will accelerate discovery of novel secondary metabolites from Streptomyces and elucidate complex smBGC regulation.

Streptomyces species are gram-positive bacteria with GC-rich linear genomes and they serve as dominant reservoirs for producing clinically and industrially important secondary metabolites. Genome mining of Streptomyces revealed that each Streptomyces species typically encodes 20–50 secondary metabolite biosynthetic gene clusters (smBGCs), emphasizing their potential for novel compound discovery. Unfortunately, most of smBGCs are uncharacterized in terms of their products and regulation since they are silent under laboratory culture conditions. To translate the genomic potential of Streptomyces to practical applications, it is essential to understand the complex regulation of smBGC expression and to identify the underlying regulatory elements. To progress towards these goals, we applied two Next-Generation Sequencing methods, dRNA-Seq and Term-Seq, to industrially relevant Streptomyces species to reveal the 5´ and 3´ boundaries of RNA transcripts on a genome scale. This data provides a fundamental resource to aid our understanding of Streptomyces ’ regulation of smBGC expression and to enhance their potential for secondary metabolite synthesis.

Streptomyces venezuelaeis well known to produce various secondary metabolites, including chloramphenicol, jadomycin, and pikromycin. Although many strains have been classified asS. venezuelaespecies, only a limited number of strains have been explored extensively for their genomic contents. Moreover, genomic differences and diversity in secondary metabolite production between the strains have never been compared. Here, we report complete genome sequences of three S. venezuelae strains (ATCC 10712, ATCC 10595, and ATCC 21113) harboring chloramphenicol and jadomycin biosynthetic gene clusters (BGC). With these high-quality genome sequences, we revealed that the three strains share more than 85% of total genes and most of the secondary metabolite biosynthetic gene clusters (smBGC). Despite such conservation, the strains produced different amounts of chloramphenicol and jadomycin, indicating differential regulation of secondary metabolite production at the strain level. Interestingly, antagonistic production of chloramphenicol and jadomycin was observed in these strains. Through comparison of the chloramphenicol and jadomycin BGCs among the three strains, we found sequence variations in many genes, the non-coding RNA coding regions, and binding sites of regulators, which affect the production of the secondary metabolites. We anticipate that these genome sequences of closely related strains would serve as useful resources for understanding the complex secondary metabolism and for designing an optimal production process using Streptomyces strains.

Streptomyces are efficient producers of various bioactive compounds, which are mostly synthesized by their secondary metabolite biosynthetic gene clusters (smBGCs). The smBGCs are tightly controlled by complex regulatory systems at transcriptional and translational levels to effectively utilize precursors that are supplied by primary metabolism. Thus, dynamic changes in gene expression in response to cellular status at both the transcriptional and translational levels should be elucidated to directly reflect protein levels, rapid downstream responses, and cellular energy costs. In this study, RNA-Seq and ribosome profiling were performed for five industrially important Streptomyces species at different growth phases, for the deep sequencing of total mRNA, and only those mRNA fragments that are protected by translating ribosomes, respectively. Herein, 12.0 to 763.8 million raw reads were sufficiently obtained with high quality of more than 80% for the Phred score Q30 and high reproducibility. These data provide a comprehensive understanding of the transcriptional and translational landscape across the Streptomyces species and contribute to facilitating the rational engineering of secondary metabolite production.

The gram-positive bacterium, Streptomyces, is noticed for its ability to produce a wide array of pharmaceutically active compounds through secondary metabolism. To discover novel bioactive secondary metabolites and increase the production, Streptomyces species have been extensively studied for the past decades. Among the cellular components, RNA molecules play important roles as the messengers for gene expression and diverse regulations taking place at the RNA level. Thus, the analysis of RNA-level regulation is critical to understanding the regulation of Streptomyces’ metabolism and secondary metabolite production. A dramatic advance in Streptomyces research was made recently, by exploiting high-throughput technology to systematically understand RNA levels. In this review, we describe the current status of the system-wide investigation of Streptomyces in terms of RNA, toward expansion of its genetic potential for secondary metabolite synthesis.

Streptomyces are attractive microbial cell factories that have industrial capability to produce a wide array of bioactive secondary metabolites. However, the genetic potential of the Streptomyces species has not been fully utilized because most of their secondary metabolite biosynthetic gene clusters (SM-BGCs) are silent under laboratory culture conditions. In an effort to activate SM-BGCs encoded in Streptomyces genomes, synthetic biology has emerged as a robust strategy to understand, design, and engineer the biosynthetic capability of Streptomyces secondary metabolites. In this regard, diverse synthetic biology tools have been developed for Streptomyces species with technical advances in DNA synthesis, sequencing, and editing. Here, we review recent progress in the development of synthetic biology tools for the production of novel secondary metabolites in Streptomyces, including genomic elements and genome engineering tools for Streptomyces, the heterologous gene expression strategy of designed biosynthetic gene clusters in the Streptomyces chassis strain, and future directions to expand diversity of novel secondary metabolites.

Abstract Determining transcriptional and translational regulatory elements in GC-rich Streptomyces genomes is essential to elucidating the complex regulatory networks that govern secondary metabolite biosynthetic gene cluster (BGC) expression. However, information about such regulatory elements has been limited for Streptomyces genomes. To address this limitation, a high-quality genome sequence of β-lactam antibiotic-producing Streptomyces clavuligerus ATCC 27 064 is completed, which contains 7163 newly annotated genes. This provides a fundamental reference genome sequence to integrate multiple genome-scale data types, including dRNA-Seq, RNA-Seq and ribosome profiling. Data integration results in the precise determination of 2659 transcription start sites which reveal transcriptional and translational regulatory elements, including −10 and −35 promoter components specific to sigma (σ) factors, and 5′-untranslated region as a determinant for translation efficiency regulation. Particularly, sequence analysis of a wide diversity of the −35 components enables us to predict potential σ-factor regulons, along with various spacer lengths between the −10 and −35 elements. At last, the primary transcriptome landscape of the β-lactam biosynthetic pathway is analyzed, suggesting temporal changes in metabolism for the synthesis of secondary metabolites driven by transcriptional regulation. This comprehensive genetic information provides a versatile genetic resource for rational engineering of secondary metabolite BGCs in Streptomyces.