antiSMASH 3.0—a comprehensive resource for the genome mining of biosynthetic gene clusters

Weber, Tilmann,Blin, Kai,Duddela, Srikanth,Krug, Daniel,Kim, Hyun Uk,Bruccoleri, Robert,Lee, Sang Yup,Fischbach, Michael A,Mü,ller, Rolf,Wohlleben, Wolfgang,Breitling, Rainer,Takano, Eriko,Medema, Oxford University Press 2015 Nucleic acids research Vol.43 No.w1

<P><B>Abstract</B></P><P>Microbial secondary metabolism constitutes a rich source of antibiotics, chemotherapeutics, insecticides and other high-value chemicals. Genome mining of gene clusters that encode the biosynthetic pathways for these metabolites has become a key methodology for novel compound discovery. In 2011, we introduced antiSMASH, a web server and stand-alone tool for the automatic genomic identification and analysis of biosynthetic gene clusters, available at http://antismash.secondarymetabolites.org. Here, we present version 3.0 of antiSMASH, which has undergone major improvements. A full integration of the recently published ClusterFinder algorithm now allows using this probabilistic algorithm to detect putative gene clusters of unknown types. Also, a new dereplication variant of the ClusterBlast module now identifies similarities of identified clusters to any of 1172 clusters with known end products. At the enzyme level, active sites of key biosynthetic enzymes are now pinpointed through a curated pattern-matching procedure and Enzyme Commission numbers are assigned to functionally classify all enzyme-coding genes. Additionally, chemical structure prediction has been improved by incorporating polyketide reduction states. Finally, in order for users to be able to organize and analyze multiple antiSMASH outputs in a private setting, a new XML output module allows offline editing of antiSMASH annotations within the Geneious software.</P>

The Molecular Treasure Hunt – An ‘Omics-based Approach to Find New Bioactive Compounds

Tilmann WEBER 한국생물공학회 2021 한국생물공학회 학술대회 Vol.2021 No.4

Genome analyses of many microorganisms but also higher organisms indicate that the genetic potential to synthesize specialized metabolites is far beyond the number of molecules observed in traditional screenings. With the availability of cheap and easy-to-obtain whole genome sequences, in silico genome mining has become an indispensable tool to complement the classical chemistry-centered approach to identify and characterize novel secondary / specialized metabolites. Since the initial release in 2011, the open source genome mining pipeline antiSMASH(1) (https://antismash.secondarymetabolites.org), which we develop in collaboration with the group of M. Medema (U. Wageningen, Netherlands) and many international contributors, has become one of the most widely used tools. We recently released version 6 of antiSMASH, including an improved user interface, new detection modules, a new cluster comparison tool, and many internal optimizations. Specialist and non-specialist users can easily analyze genomic sequences for the presence of secondary metabolite biosynthetic gene clusters with antiSMASH. To provide extensive analysis options of the data generated with antiSMASH, we have extended the framework with several databases (2-4). The antiSMASH database, (https://antismash-db.secondarymetabolites.org/)(2), contains 147,517 high quality BGC regions from 388 archaeal, 25,236 bacterial and 177 fungal “high-quality” genomes. These genome mining technologies build the foundation of further in silico studies towards a more comprehensive “Genome Analytics” platform, which we use to streamline our natural product discovery and characterization efforts. Albeit streptomycetes are studied for many decades as proficient producers of bioactive compounds, there are still severe limitations concerning efficiency of mutagenesis protocols that often hamper systems metabolic engineering and Synthetic Biology approaches. We have therefore developed an extensive CRISPR/Cas9-based toolkit (5-7) for streptomycetes that now also includes tools that utilize multiplexing and DSB-free base editing technology to highly effectively engineer actinomycetes.

Genome-Scale Metabolic Reconstruction of Actinomycetes for Antibiotics Production

Mohite, Omkar S.,Weber, Tilmann,Kim, Hyun Uk,Lee, Sang Yup Wiley (John WileySons) 2019 Biotechnology journal Vol.14 No.1

Toward Systems Metabolic Engineering of Streptomycetes for Secondary Metabolites Production

Robertsen, Helene Lunde,Weber, Tilmann,Kim, Hyun Uk,Lee, Sang Yup Wiley Blackwell (John Wiley Sons) 2018 BIOTECHNOLOGY JOURNAL Vol.13 No.1

Recent Advances in Re-engineering Modular PKS and NRPS Assembly Lines

Charlotte Beck,Jaime Felipe Guerrero Garzón,Tilmann Weber 한국생물공학회 2020 Biotechnology and Bioprocess Engineering Vol.25 No.6

Polyketides such as the antibiotic erythromycin or the immunosuppressant rapamycin, and non-ribosomal peptides, such as the antibiotics penicillin or vancomycin, are important classes of natural products. The core of these molecules are biosynthesized by large polyketide synthases (PKS) and non-ribosomal peptide synthetases (NRPS), respectively. The modular architecture of these enzymatic assembly lines makes them interesting candidates for synthetic biology approaches. The re-engineering efforts aim to understand the molecular structure, produce new compounds, produce analogs of known compounds, tag the products or improve activity and/or yield. Here, we first consider the definition of PKS and NRPS modules, then give an overview of different strategies for re-engineering and finally review recent examples of PKS and NRPS reengineering.

Metabolic engineering with systems biology tools to optimize production of prokaryotic secondary metabolites

Kim, Hyun Uk,Charusanti, Pep,Lee, Sang Yup,Weber, Tilmann The Royal Society of Chemistry 2016 Natural product reports Vol.33 No.8

<P>Metabolic engineering using systems biology toots is increasingly applied to overproduce secondary metabolites for their potential industrial production. In this Highlight, recent relevant metabolic engineering studies are analyzed with emphasis on host selection and engineering approaches for the optimal production of various prokaryotic secondary metabolites: native versus heterologous hosts (e.g., Escherichia cols) and rational versus random approaches. This comparative analysis is followed by discussions on systems biology tools deployed in optimizing the production of secondary metabolites. The potential contributions of additional systems biology tools are also discussed in the context of current challenges encountered during optimization of secondary metabolite production.</P>

The antiSMASH database, a comprehensive database of microbial secondary metabolite biosynthetic gene clusters

Blin, Kai,Medema, Marnix H.,Kottmann, Renzo,Lee, Sang Yup,Weber, Tilmann Oxford University Press 2017 Nucleic acids research Vol.45 No.d1

<P>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 <I>de novo</I> 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/.</P>

Highly efficient DSB-free base editing for streptomycetes with CRISPR-BEST

Tong, Yaojun,Whitford, Christopher M.,Robertsen, Helene L.,Blin, Kai,Jørgensen, Tue S.,Klitgaard, Andreas K.,Gren, Tetiana,Jiang, Xinglin,Weber, Tilmann,Lee, Sang Yup National Academy of Sciences 2019 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF Vol.116 No.41

<P><B>Significance</B></P><P>Although CRISPR-Cas9 tools dramatically simplified the genetic manipulation of actinomycetes, significant concerns of genome instability caused by the DNA double-strand breaks (DSBs) and common off-target effects remain. To address these concerns, we developed CRISPR-BEST, a DSB-free and high-fidelity single-nucleotide–resolution base editing system for streptomycetes and validated its use by determining editing properties and genome-wide off-target effects. Furthermore, our CRISPR-BEST toolkit supports Csy4-based multiplexing to target multiple genes of interest in parallel. We believe that our CRISPR-BEST approach is a significant improvement over existing genetic manipulation methods to engineer streptomycetes, especially for those strains that cannot be genome-edited using normal DSB-based genome editing systems, such as CRISPR-Cas9.</P><P>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 <I>Streptomyces</I> species. Particularly in nonmodel actinomycete <I>Streptomyces collinus</I> Tu¨365, CRISPR-cBEST efficiently inactivated the 2 copies of <I>kirN</I> 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.</P>