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Antagonistic bacterial interactions often rely on antimicrobial bacteriocins, which attack only a narrow range of target bacteria. However, antimicrobials with broader activity may be advantageous. Here we identify an antimicrobial called epifadin, which is produced by nasal Staphylococcus epidermidis IVK83. It has an unprecedented architecture consisting of a non-ribosomally synthesized peptide, a polyketide component and a terminal modified amino acid moiety. Epifadin combines a wide antimicrobial target spectrum with a short life span of only a few hours. It is highly unstable under in vivo-like conditions, potentially as a means to limit collateral damage of bacterial mutualists. However, Staphylococcus aureus is eliminated by epifadin-producing S. epidermidis during co-cultivation in vitro and in vivo, indicating that epifadin-producing commensals could help prevent nasal S. aureus carriage. These insights into a microbiome-derived, previously unknown antimicrobial compound class suggest that limiting the half-life of an antimicrobial may help to balance its beneficial and detrimental activities., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Soil microbiota can confer fitness advantages to plants and increase crop resilience to drought and other abiotic stressors. However, there is little evidence on the mechanisms correlating a microbial trait with plant abiotic stress tolerance. Here, we report that Streptomyces effectively alleviate drought and salinity stress by producing spiroketal polyketide pteridic acid H (1) and its isomer F (2), both of which promote root growth in Arabidopsis at a concentration of 1.3 nM under abiotic stress. Transcriptomics profiles show increased expression of multiple stress responsive genes in Arabidopsis seedlings after pteridic acids treatment. We confirm in vivo a bifunctional biosynthetic gene cluster for pteridic acids and antimicrobial elaiophylin production. We propose it is mainly disseminated by vertical transmission and is geographically distributed in various environments. This discovery reveals a perspective for understanding plant-Streptomyces interactions and provides a promising approach for utilising beneficial Streptomyces and their secondary metabolites in agriculture to mitigate the detrimental effects of climate change., (© 2023. The Author(s).)

Here, we report the complete, circular genome sequence of a potential novel species from the underexplored Alphaproteobacterial genus Bosea. Bosea sp. NBC_00550 was isolated from a soil sample collected in Lyngby, Denmark. We explore the biosynthetic potential of Bosea sp. NBC_00550 and compare it with that of other Bosea species., Competing Interests: The authors declare no conflict of interest.

CRISPR tools, especially Cas9n-sgRNA guided cytidine deaminase base editors such as CRISPR-BEST, have dramatically simplified genetic manipulation of streptomycetes. One major advantage of CRISPR base editing technology is the possibility to multiplex experiments in genomically instable species. Here, we demonstrate scaled up Csy4 based multiplexed genome editing using CRISPR-mcBEST in Streptomyces coelicolor . We evaluated the system by simultaneously targeting 9, 18, and finally all 28 predicted specialized metabolite biosynthetic gene clusters in a single experiment. We present important insights into the performance of Csy4 based multiplexed genome editing at different scales. Using multiomics analysis, we investigated the systems wide effects of such extensive editing experiments and revealed great potentials and important bottlenecks of CRISPR-mcBEST. The presented analysis provides crucial data and insights toward the development of multiplexed base editing as a novel paradigm for high throughput engineering of Streptomyces chassis and beyond.

Here, we report the complete genome sequences of Methylorubrum extorquens NBC_00036 and Methylorubrum extorquens NBC_00404. The genomes were sequenced using the Oxford Nanopore Technologies MinION and Illumina NovaSeq systems. Both genomes are circular, with sizes of 5,661,342 bp and 5,869,086 bp, respectively., Competing Interests: The authors declare no conflict of interest.

Microorganisms produce small bioactive compounds as part of their secondary or specialised metabolism. Often, such metabolites have antimicrobial, anticancer, antifungal, antiviral or other bio-activities and thus play an important role for applications in medicine and agriculture. In the past decade, genome mining has become a widely-used method to explore, access, and analyse the available biodiversity of these compounds. Since 2011, the 'antibiotics and secondary metabolite analysis shell-antiSMASH' (https://antismash.secondarymetabolites.org/) has supported researchers in their microbial genome mining tasks, both as a free to use web server and as a standalone tool under an OSI-approved open source licence. It is currently the most widely used tool for detecting and characterising biosynthetic gene clusters (BGCs) in archaea, bacteria, and fungi. Here, we present the updated version 7 of antiSMASH. antiSMASH 7 increases the number of supported cluster types from 71 to 81, as well as containing improvements in the areas of chemical structure prediction, enzymatic assembly-line visualisation and gene cluster regulation., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Crosiellidines are intriguing pyrazine-alkylguanidine metabolites isolated from the minor actinomycete genus Crossiella . Their structures present an unprecedented 2-methoxy-3,5,6-trialkyl pyrazine scaffold and uncommon guanidine prenylations, including an exotic O -prenylated N -hydroxyguanidine moiety. The novel substitution pattern of the 2-methoxypyrazine core inaugurates a new class of naturally occurring pyrazine compounds, the biosynthetic implications of which are discussed herein. Isotopic feeding and genome analysis allowed us to propose a biosynthetic pathway from arginine. The crossiellidines exhibited remarkable, broad-spectrum antibacterial activity.

Streptomyces albidoflavus J1074 is a popular platform to discover novel natural products via the expression of heterologous biosynthetic gene clusters (BGCs). There is keen interest in improving the ability of this platform to overexpress BGCs and, consequently, enable the purification of specialized metabolites. Mutations within gene rpoB for the β-subunit of RNA polymerase are known to increase rifampicin resistance and augment the metabolic capabilities of streptomycetes. Yet, the effects of rpoB mutations on J1074 remained unstudied, and we decided to address this issue. A target collection of strains that we studied carried spontaneous rpoB mutations introduced in the background of the other drug resistance mutations. The antibiotic resistance spectra, growth, and specialized metabolism of the resulting mutants were interrogated using a set of microbiological and analytical approaches. We isolated 14 different rpoB mutants showing various degrees of rifampicin resistance; one of them (S433W) was isolated for the first time in actinomycetes. The rpoB mutations had a major effect on antibiotic production by J1074, as evident from bioassays and LC-MS data. Our data support the idea that rpoB mutations are useful tools to enhance the ability of J1074 to produce specialized metabolites.

With an ever-increasing amount of (meta)genomic data being deposited in sequence databases, (meta)genome mining for natural product biosynthetic pathways occupies a critical role in the discovery of novel pharmaceutical drugs, crop protection agents and biomaterials. The genes that encode these pathways are often organised into biosynthetic gene clusters (BGCs). In 2015, we defined the Minimum Information about a Biosynthetic Gene cluster (MIBiG): a standardised data format that describes the minimally required information to uniquely characterise a BGC. We simultaneously constructed an accompanying online database of BGCs, which has since been widely used by the community as a reference dataset for BGCs and was expanded to 2021 entries in 2019 (MIBiG 2.0). Here, we describe MIBiG 3.0, a database update comprising large-scale validation and re-annotation of existing entries and 661 new entries. Particular attention was paid to the annotation of compound structures and biological activities, as well as protein domain selectivities. Together, these new features keep the database up-to-date, and will provide new opportunities for the scientific community to use its freely available data, e.g. for the training of new machine learning models to predict sequence-structure-function relationships for diverse natural products. MIBiG 3.0 is accessible online at https://mibig.secondarymetabolites.org/., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Microorganisms produce a wide variety of secondary/specialized metabolites (SMs), the majority of which are yet to be discovered. These natural products play multiple roles in microbiomes and are important for microbial competition, communication, and success in the environment. SMs have been our major source of antibiotics and are used in a range of biotechnological applications. In silico mining for biosynthetic gene clusters (BGCs) encoding the production of SMs is commonly used to assess the genetic potential of organisms. However, as BGCs span tens to over 200 kb, identifying complete BGCs requires genome data that has minimal assembly gaps within the BGCs, a prerequisite that was previously only met by individually sequenced genomes. Here, we assess the performance of the currently available genome mining platform antiSMASH on 1,080 high-quality metagenome-assembled bacterial genomes (HQ MAGs) previously produced from wastewater treatment plants (WWTPs) using a combination of long-read (Oxford Nanopore) and short-read (Illumina) sequencing technologies. More than 4,200 different BGCs were identified, with 88% of these being complete. Sequence similarity clustering of the BGCs implies that the majority of this biosynthetic potential likely encodes novel compounds, and few BGCs are shared between genera. We identify BGCs in abundant and functionally relevant genera in WWTPs, suggesting a role of secondary metabolism in this ecosystem. We find that the assembly of HQ MAGs using long-read sequencing is vital to explore the genetic potential for SM production among the uncultured members of microbial communities. IMPORTANCE Cataloguing secondary metabolite (SM) potential using genome mining of metagenomic data has become the method of choice in bioprospecting for novel compounds. However, accurate biosynthetic gene cluster (BGC) detection requires unfragmented genomic assemblies, which have been technically difficult to obtain from metagenomes until very recently with new long-read technologies. Here, we determined the biosynthetic potential of activated sludge (AS), the microbial community used in resource recovery and wastewater treatment, by mining high-quality metagenome-assembled genomes generated from long-read data. We found over 4,000 BGCs, including BGCs in abundant process-critical bacteria, with no similarity to the BGCs of characterized products. We show how long-read MAGs are required to confidently assemble complete BGCs, and we determined that the AS BGCs from different studies have very little overlap, suggesting that AS is a rich source of biosynthetic potential and new bioactive compounds.

Actinomycetes make a wealth of complex, structurally diverse natural products, and a key challenge is to link them to their biosynthetic gene clusters and delineate the reactions catalyzed by each of the enzymes. Here, we report the biosynthetic gene cluster for pyracrimycin A, a set of nine genes that includes a core nonribosomal peptide synthase ( pymB ) that utilizes serine and proline as precursors and a monooxygenase ( pymC ) that catalyzes Baeyer-Villiger oxidation. The cluster is similar to the one for brabantamide A; however, pyracrimycin A biosynthesis differs in that feeding experiments with isotope-labeled serine and proline suggest that a ring opening reaction takes place and a carbon is lost from serine downstream of the oxidation reaction. Based on these data, we propose a full biosynthesis pathway for pyracrimycin A.

The actinomycete Streptomyces sp. strain Gö40/10 has the potential to produce a range of secondary metabolites, one of which is collinolactone, a compound with neuroprotective properties and potential for pharmaceutical applications. The genome was sequenced with Oxford Nanopore Technologies MinION and Illumina MiSeq systems and consists of a single 9,635,564-nucleotide linear chromosome.

An ever-increasing demand for novel antimicrobials to treat life-threatening infections caused by the global spread of multidrug-resistant bacterial pathogens stands in stark contrast to the current level of investment in their development, particularly in the fields of natural-product-derived and synthetic small molecules. New agents displaying innovative chemistry and modes of action are desperately needed worldwide to tackle the public health menace posed by antimicrobial resistance. Here, our consortium presents a strategic blueprint to substantially improve our ability to discover and develop new antibiotics. We propose both short-term and long-term solutions to overcome the most urgent limitations in the various sectors of research and funding, aiming to bridge the gap between academic, industrial and political stakeholders, and to unite interdisciplinary expertise in order to efficiently fuel the translational pipeline for the benefit of future generations., (© 2021. Springer Nature Limited.)

We report the sequencing, assembly, and annotation of the genome of Amycolatopsis sp. CA-230715, a potentially interesting producer of natural products. The genome of CA-230715 was sequenced using PacBio, Illumina, and Nanopore technologies. It consists of a circular 10,363,158-nucleotide (nt) chromosome and a circular 12,080-nt plasmid.

We report the major conclusions of the online open-access workshop "Computational Applications in Secondary Metabolite Discovery (CAiSMD)" that took place from 08 to 10 March 2021. Invited speakers from academia and industry and about 200 registered participants from five continents (Africa, Asia, Europe, South America, and North America) took part in the workshop. The workshop highlighted the potential applications of computational methodologies in the search for secondary metabolites (SMs) or natural products (NPs) as potential drugs and drug leads. During 3 days, the participants of this online workshop received an overview of modern computer-based approaches for exploring NP discovery in the "omics" age. The invited experts gave keynote lectures, trained participants in hands-on sessions, and held round table discussions. This was followed by oral presentations with much interaction between the speakers and the audience. Selected applicants (early-career scientists) were offered the opportunity to give oral presentations (15 min) and present posters in the form of flash presentations (5 min) upon submission of an abstract. The final program available on the workshop website ( https://caismd.indiayouth.info/ ) comprised of 4 keynote lectures (KLs), 12 oral presentations (OPs), 2 round table discussions (RTDs), and 5 hands-on sessions (HSs). This meeting report also references internet resources for computational biology in the area of secondary metabolites that are of use outside of the workshop areas and will constitute a long-term valuable source for the community. The workshop concluded with an online survey form to be completed by speakers and participants for the goal of improving any subsequent editions., (© 2021. The Author(s).)

CRISPR base editing is a powerful method to engineer bacterial genomes. However, it restricts editing to single-nucleotide substitutions. Here, to address this challenge, we adapt a CRISPR-Prime Editing-based, DSB-free, versatile, and single-nucleotide resolution genetic manipulation toolkit for prokaryotes. It can introduce substitutions, deletions, insertions, and the combination thereof, both in plasmids and the chromosome of E. coli with high fidelity. Notably, under optimal conditions, the efficiency of 1-bp deletions reach up to 40%. Moreover, deletions of up to 97 bp and insertions up to 33 bp were successful with the toolkit in E. coli, however, efficiencies dropped sharply with increased fragment sizes. With a second guide RNA, our toolkit can achieve multiplexed editing albeit with low efficiency. Here we report not only a useful addition to the genome engineering arsenal for E. coli, but also a potential basis for the development of similar toolkits for other bacteria., (© 2021. The Author(s).)

Actinobacteria have been a rich source of novel, structurally complex natural products for many decades. Although the largest genus is Streptomyces , from which the majority of antibiotics in current and past clinical use were originally isolated, other less common genera also have the potential to produce a wealth of novel secondary metabolites. One example is the Kutzneria genus, which currently contains only five reported species. One of these species is Kutzneria albida DSM 43870T, which has 46 predicted biosynthetic gene clusters and is known to produce the macrolide antibiotic aculeximycin. Here, we report the isolation and structural characterization of two novel 30-membered glycosylated macrolides, epemicins A and B, that are structurally related to aculeximycin, from a rare Kutzneria sp. The absolute configuration for all chiral centers in the two compounds is proposed based on extensive 1D and 2D NMR studies and bioinformatics analysis of the gene cluster. Through heterologous expression and genetic inactivation, we have confirmed the link between the biosynthetic gene cluster and the new molecules. These findings show the potential of rare Actinobacteria to produce new, structurally diverse metabolites. Furthermore, the gene inactivation represents the first published report to genetically manipulate a representative of the Kutzneria genus.

Here, we report the sequencing, assembly, and annotation of the genome of the rare actinobacterium Kutzneria sp. strain CA-103260. The genome of CA-103260 was sequenced using PacBio and Illumina technologies and it consists of a circular 11,609,901-bp chromosome.

Streptomycetes are producers of a wide range of specialized metabolites of great medicinal and industrial importance, such as antibiotics, antifungals, or pesticides. Having been the drivers of the golden age of antibiotics in the 1950s and 1960s, technological advancements over the last two decades have revealed that very little of their biosynthetic potential has been exploited so far. Given the great need for new antibiotics due to the emerging antimicrobial resistance crisis, as well as the urgent need for sustainable biobased production of complex molecules, there is a great renewed interest in exploring and engineering the biosynthetic potential of streptomycetes. Here, we describe the Design-Build-Test-Learn (DBTL) cycle for metabolic engineering experiments in streptomycetes and how it can be used for the discovery and production of novel specialized metabolites., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Many microorganisms produce natural products that form the basis of antimicrobials, antivirals, and other drugs. Genome mining is routinely used to complement screening-based workflows to discover novel natural products. Since 2011, the "antibiotics and secondary metabolite analysis shell-antiSMASH" (https://antismash.secondarymetabolites.org/) has supported researchers in their microbial genome mining tasks, both as a free-to-use web server and as a standalone tool under an OSI-approved open-source license. It is currently the most widely used tool for detecting and characterising biosynthetic gene clusters (BGCs) in bacteria and fungi. Here, we present the updated version 6 of antiSMASH. antiSMASH 6 increases the number of supported cluster types from 58 to 71, displays the modular structure of multi-modular BGCs, adds a new BGC comparison algorithm, allows for the integration of results from other prediction tools, and more effectively detects tailoring enzymes in RiPP clusters., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Here, we report the sequencing, assembly, and annotation of the genome of Streptomyces sp. strain CA-256286. The genome consists of a linear 7,726,360-nucleotide chromosome and a linear 466,817-nucleotide putative plasmid. This strain is predicted to produce a range of novel secondary metabolites.

ε-Poly-l-lysine is a potent antimicrobial produced through fermentation of Streptomyces and used in many Asian countries as a food preservative. It is synthesized and excreted by a special nonribosomal peptide synthetase (NRPS)-like enzyme called Pls. In this study, we discovered a gene from cheese bacterium Corynebacterium variabile that showed high similarity to the Pls from Streptomyces in terms of domain architecture and gene context. By cloning it into Streptomyces coelicolor with a Streptomyces albulus Pls promoter, we confirmed that its product is indeed ε-poly-l-lysine. A comprehensive sequence analysis suggested that Pls genes are widely spread among coryneform actinobacteria isolated from cheese and human skin; 14 out of 15 Brevibacterium isolates and 10 out of 12 Corynebacterium isolates contain it in their genomes. This finding raises the possibility that ε-poly-l-lysine as a bioactive secondary metabolite might be produced and play a role in the cheese and skin ecosystems. IMPORTANCE Every year, microbial contamination causes billions of tons of food wasted and millions of cases of illness. ε-Poly-l-lysine has potent, wide-spectrum inhibitory activity and is heat stable and biodegradable. It has been approved for food preservation by an increasing number of countries. ε-Poly-l-lysine is produced from soil bacteria of the genus Streptomyces , also producers of various antibiotic drugs and toxins and not considered to be a naturally occurring food component. The frequent finding of pls in cheese and skin bacteria suggests that ε-poly-l-lysine may naturally exist in cheese and on our skin, and ε-poly-l-lysine producers are not limited to filamentous actinobacteria., (Copyright © 2021 Jiang et al.)

Microorganisms produce natural products that are frequently used in the development of antibacterial, antiviral, and anticancer drugs, pesticides, herbicides, or fungicides. In recent years, genome mining has evolved into a prominent method to access this potential. antiSMASH is one of the most popular tools for this task. Here, we present version 3 of the antiSMASH database, providing a means to access and query precomputed antiSMASH-5.2-detected biosynthetic gene clusters from representative, publicly available, high-quality microbial genomes via an interactive graphical user interface. In version 3, the database contains 147 517 high quality BGC regions from 388 archaeal, 25 236 bacterial and 177 fungal genomes and is available at https://antismash-db.secondarymetabolites.org/., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Computational analysis of biosynthetic gene clusters (BGCs) has revolutionized natural product discovery by enabling the rapid investigation of secondary metabolic potential within microbial genome sequences. Grouping homologous BGCs into Gene Cluster Families (GCFs) facilitates mapping their architectural and taxonomic diversity and provides insights into the novelty of putative BGCs, through dereplication with BGCs of known function. While multiple databases exist for exploring BGCs from publicly available data, no public resources exist that focus on GCF relationships. Here, we present BiG-FAM, a database of 29,955 GCFs capturing the global diversity of 1,225,071 BGCs predicted from 209,206 publicly available microbial genomes and metagenome-assembled genomes (MAGs). The database offers rich functionalities, such as multi-criterion GCF searches, direct links to BGC databases such as antiSMASH-DB, and rapid GCF annotation of user-supplied BGCs from antiSMASH results. BiG-FAM can be accessed online at https://bigfam.bioinformatics.nl., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Streptomycetes are prominent sources of bioactive natural products, but metabolic engineering of the natural products of these organisms is greatly hindered by relatively inefficient genetic manipulation approaches. New advances in genome editing techniques, particularly CRISPR-based tools, have revolutionized genetic manipulation of many organisms, including actinomycetes. We have developed a comprehensive CRISPR toolkit that includes several variations of 'classic' CRISPR-Cas9 systems, along with CRISPRi and CRISPR-base editing systems (CRISPR-BEST) for streptomycetes. Here, we provide step-by-step protocols for designing and constructing the CRISPR plasmids, transferring these plasmids to the target streptomycetes, and identifying correctly edited clones. Our CRISPR toolkit can be used to generate random-sized deletion libraries, introduce small indels, generate in-frame deletions of specific target genes, reversibly suppress gene transcription, and substitute single base pairs in streptomycete genomes. Furthermore, the toolkit includes a Csy4-based multiplexing option to introduce multiple edits in a single experiment. The toolkit can be easily extended to other actinomycetes. With our protocol, it takes <10 d to inactivate a target gene, which is much faster than alternative protocols.

To accelerate the shift to bio-based production and overcome complicated functional implementation of natural and artificial biosynthetic pathways to industry relevant organisms, development of new, versatile, bio-based production platforms is required. Here we present a novel yeast-based platform for biosynthesis of bacterial aromatic polyketides. The platform is based on a synthetic polyketide synthase system enabling a first demonstration of bacterial aromatic polyketide biosynthesis in a eukaryotic host., Competing Interests: Jay D. Keasling has commercial interests in Amyris, Lygos, Demetrix, Napigen, Apertor Labs, Berkeley Brewing Sciences, and Ansa Biosciences., (© 2020 Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd.)

Fueled by the explosion of (meta)genomic data, genome mining of specialized metabolites has become a major technology for drug discovery and studying microbiome ecology. In these efforts, computational tools like antiSMASH have played a central role through the analysis of Biosynthetic Gene Clusters (BGCs). Thousands of candidate BGCs from microbial genomes have been identified and stored in public databases. Interpreting the function and novelty of these predicted BGCs requires comparison with a well-documented set of BGCs of known function. The MIBiG (Minimum Information about a Biosynthetic Gene Cluster) Data Standard and Repository was established in 2015 to enable curation and storage of known BGCs. Here, we present MIBiG 2.0, which encompasses major updates to the schema, the data, and the online repository itself. Over the past five years, 851 new BGCs have been added. Additionally, we performed extensive manual data curation of all entries to improve the annotation quality of our repository. We also redesigned the data schema to ensure the compliance of future annotations. Finally, we improved the user experience by adding new features such as query searches and a statistics page, and enabled direct link-outs to chemical structure databases. The repository is accessible online at https://mibig.secondarymetabolites.org/., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Streptomycetes serve as major producers of various pharmacologically and industrially important natural products. Although CRISPR-Cas9 systems have been developed for more robust genetic manipulations, concerns of genome instability caused by the DNA double-strand breaks (DSBs) and the toxicity of Cas9 remain. To overcome these limitations, here we report development of the DSB-free, single-nucleotide-resolution genome editing system CRISPR-BEST (CRISPR-Base Editing SysTem), which comprises a cytidine (CRISPR-cBEST) and an adenosine (CRISPR-aBEST) deaminase-based base editor. Specifically targeted by an sgRNA, CRISPR-cBEST can efficiently convert a C:G base pair to a T:A base pair and CRISPR-aBEST can convert an A:T base pair to a G:C base pair within a window of approximately 7 and 6 nucleotides, respectively. CRISPR-BEST was validated and successfully used in different Streptomyces species. Particularly in nonmodel actinomycete Streptomyces collinus Tü365, CRISPR-cBEST efficiently inactivated the 2 copies of kirN gene that are in the duplicated kirromycin biosynthetic pathways simultaneously by STOP codon introduction. Generating such a knockout mutant repeatedly failed using the conventional DSB-based CRISPR-Cas9. An unbiased, genome-wide off-target evaluation indicates the high fidelity and applicability of CRISPR-BEST. Furthermore, the system supports multiplexed editing with a single plasmid by providing a Csy4-based sgRNA processing machinery. To simplify the protospacer identification process, we also updated the CRISPy-web (https://crispy.secondarymetabolites.org), and now it allows designing sgRNAs specifically for CRISPR-BEST applications., Competing Interests: The authors declare no competing interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Natural products originating from microorganisms are frequently used in antimicrobial and anticancer drugs, pesticides, herbicides or fungicides. In the last years, the increasing availability of microbial genome data has made it possible to access the wealth of biosynthetic clusters responsible for the production of these compounds by genome mining. antiSMASH is one of the most popular tools in this field. The antiSMASH database provides pre-computed antiSMASH results for many publicly available microbial genomes and allows for advanced cross-genome searches. The current version 2 of the antiSMASH database contains annotations for 6200 full bacterial genomes and 18,576 bacterial draft genomes and is available at https://antismash-db.secondarymetabolites.org/.

Bacteria of the order Actinomycetales are one of the most important sources of bioactive natural products, which are the source of many drugs. However, many of them still lack efficient genome editing methods, some strains even cannot be manipulated at all. This restricts systematic metabolic engineering approaches for boosting known and discovering novel natural products. In order to facilitate the genome editing for actinomycetes, we developed a CRISPR-Cas9 toolkit with high efficiency for actinomyces genome editing. This basic toolkit includes a software for spacer (sgRNA) identification, a system for in-frame gene/gene cluster knockout, a system for gene loss-of-function study, a system for generating a random size deletion library, and a system for gene knockdown. For the latter, a uracil-specific excision reagent (USER) cloning technology was adapted to simplify the CRISPR vector construction process. The application of this toolkit was successfully demonstrated by perturbation of genomes of Streptomyces coelicolor A3(2) and Streptomyces collinus Tü 365. The CRISPR-Cas9 toolkit and related protocol described here can be widely used for metabolic engineering of actinomycetes.

Secondary metabolites produced by microorganisms are the main source of bioactive compounds that are in use as antimicrobial and anticancer drugs, fungicides, herbicides and pesticides. In the last decade, the increasing availability of microbial genomes has established genome mining as a very important method for the identification of their biosynthetic gene clusters (BGCs). One of the most popular tools for this task is antiSMASH. However, so far, antiSMASH is limited to de novo computing results for user-submitted genomes and only partially connects these with BGCs from other organisms. Therefore, we developed the antiSMASH database, a simple but highly useful new resource to browse antiSMASH-annotated BGCs in the currently 3907 bacterial genomes in the database and perform advanced search queries combining multiple search criteria. antiSMASH-DB is available at http://antismash-db.secondarymetabolites.org/., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

This chapter helps in the use of bioinformatics tools relevant to the discovery of new nonribosomal peptides (NRPs) produced by microorganisms. The strategy described can be applied to draft or fully assembled genome sequences. It relies on the identification of the synthetase genes and the deciphering of the domain architecture of the nonribosomal peptide synthetases (NRPSs). In the next step, candidate peptides synthesized by these NRPSs are predicted in silico, considering the specificity of incorporated monomers together with their isomery. To assess their novelty, the two-dimensional structure of the peptides can be compared with the structural patterns of all known NRPs. The presented workflow leads to an efficient and rapid screening of genomic data generated by high throughput technologies. The exploration of such sequenced genomes may lead to the discovery of new drugs (i.e., antibiotics against multi-resistant pathogens or anti-tumors).

Polyketide synthases construct polyketides with diverse structures and biological activities via the condensation of extender units and acyl thioesters. Although a growing body of evidence suggests that polyketide synthases might be tolerant to non-natural extender units, in vitro and in vivo studies aimed at probing and utilizing polyketide synthase specificity are severely limited to only a small number of extender units, owing to the lack of synthetic routes to a broad variety of acyl-CoA extender units. Here, we report the construction of promiscuous malonyl-CoA synthetase variants that can be used to synthesize a broad range of malonyl-CoA extender units substituted at the C2-position, several of which contain handles for chemoselective ligation and are not found in natural biosynthetic systems. We highlighted utility of these enzymes by probing the acyl-CoA specificity of several trans-acyltransferases, leading to the unprecedented discovery of poly specificity toward non-natural extender units, several of which are not found in naturally occurring biosynthetic pathways. These results reveal that polyketide biosynthetic machinery might be more tolerant to non-natural substrates than previously established, and that mutant synthetases are valuable tools for probing the specificity of biosynthetic machinery. Our data suggest new synthetic biology strategies for harnessing this promiscuity and enabling the regioselective modification of polyketides.

Kirromycin is a complex linear polyketide that acts as a protein biosynthesis inhibitor by binding to the bacterial elongation factor Tu. The kirromycin biosynthetic gene cluster was isolated from the producer, Streptomyces collinus Tü 365, and confirmed by targeted disruption of essential biosynthesis genes. Kirromycin is synthesized by a large hybrid polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) encoded by the genes kirAI-kirAVI. This complex involves some very unusual features, including the absence of internal acyltransferase (AT) domains in KirAI-KirAV, multiple split-ups of PKS modules on separate genes, and swapping in the domain organization. Interestingly, one PKS enzyme, KirAVI, contains internal AT domains. Based on in silico analysis, a route to pyridone formation involving PKS and NRPS steps was postulated. This hypothesis was experimentally proven by feeding studies with [U-13C3(15)N]beta-alanine and NMR and MS analyses of the isolated pure kirromycin.