Breaking the silence: new strategies for discovering novel natural products.

Natural products have been a prolific source of antibacterial and anticancer drugs for decades. One of the major challenges in natural product discovery is that the vast majority of natural product biosynthetic gene clusters (BGCs) have not been characterized, partially due to the fact that they are either transcriptionally silent or expressed at very low levels under standard laboratory conditions. Here we describe the strategies developed in recent years (mostly between 2014-2016) for activating silent BGCs. These strategies can be broadly divided into two categories: approaches in native hosts and approaches in heterologous hosts. In addition, we briefly discuss recent advances in developing new computational tools for identification and characterization of BGCs and high-throughput methods for detection of natural products.

[1]  Guoping Zhao,et al.  Site‐specific recombination for cloning of large DNA fragments in vitro , 2015 .

[2]  Axel Zeeck,et al.  Big Effects from Small Changes: Possible Ways to Explore Nature's Chemical Diversity , 2002, Chembiochem : a European journal of chemical biology.

[3]  B. Moore,et al.  Identification of Thiotetronic Acid Antibiotic Biosynthetic Pathways by Target-directed Genome Mining. , 2015, ACS chemical biology.

[4]  Yaojun Tong,et al.  CRISPR-Cas9 Based Engineering of Actinomycetal Genomes. , 2015, ACS synthetic biology.

[5]  Huimin Zhao,et al.  Refactoring the silent spectinabilin gene cluster using a plug-and-play scaffold. , 2013, ACS synthetic biology.

[6]  G. Challis,et al.  Discovery of microbial natural products by activation of silent biosynthetic gene clusters , 2015, Nature Reviews Microbiology.

[7]  R. Müller,et al.  Heterologous expression of an orphan NRPS gene cluster from Paenibacillus larvae in Escherichia coli revealed production of sevadicin. , 2015, Journal of biotechnology.

[8]  S. Brady,et al.  Yeast homologous recombination-based promoter engineering for the activation of silent natural product biosynthetic gene clusters , 2015, Proceedings of the National Academy of Sciences.

[9]  Identification of AstG1, A LAL Family Regulator that Positively Controls Ansatrienins Production in Streptomyces sp. XZQH13 , 2015, Current Microbiology.

[10]  Michael A. Skinnider,et al.  An automated Genomes-to-Natural Products platform (GNP) for the discovery of modular natural products , 2015, Nature Communications.

[11]  Chad W. Johnston,et al.  Polyketide and nonribosomal peptide retro-biosynthesis and global gene cluster matching. , 2016, Nature chemical biology.

[12]  Q. Tu,et al.  Genetic engineering and heterologous expression of the disorazol biosynthetic gene cluster via Red/ET recombineering , 2016, Scientific Reports.

[13]  Kristian Fog Nielsen,et al.  Sharing and community curation of mass spectrometry data with Global Natural Products Social Molecular Networking , 2016, Nature Biotechnology.

[14]  H. Vlamakis,et al.  Directed natural product biosynthesis gene cluster capture and expression in the model bacterium Bacillus subtilis , 2015, Scientific Reports.

[15]  Wenjun Jiang,et al.  Cas9-Assisted Targeting of CHromosome segments CATCH enables one-step targeted cloning of large gene clusters , 2015, Nature Communications.

[16]  Huimin Zhao,et al.  CRISPR-Cas9 strategy for activation of silent Streptomyces biosynthetic gene clusters , 2017, Nature chemical biology.

[17]  Meng Wang,et al.  Activation and Characterization of a Cryptic Polycyclic Tetramate Macrolactam Biosynthetic Gene Cluster , 2013, Nature Communications.

[18]  Yuemao Shen,et al.  Activating a Cryptic Ansamycin Biosynthetic Gene Cluster To Produce Three New Naphthalenic Octaketide Ansamycins with n-Pentyl and n-Butyl Side Chains. , 2015, Organic letters.

[19]  M. Metsä-Ketelä,et al.  Targeted activation of silent natural product biosynthesis pathways by reporter-guided mutant selection. , 2015, Metabolic engineering.

[20]  Rolf Müller,et al.  Full-length RecE enhances linear-linear homologous recombination and facilitates direct cloning for bioprospecting , 2012, Nature Biotechnology.

[21]  Shu-Lin Chang,et al.  Illuminating the diversity of aromatic polyketide synthases in Aspergillus nidulans. , 2012, Journal of the American Chemical Society.

[22]  G. Niu,et al.  Identification of novel mureidomycin analogues via rational activation of a cryptic gene cluster in Streptomyces roseosporus NRRL 15998 , 2015, Scientific Reports.

[23]  J. Fischer,et al.  Microbial communication leading to the activation of silent fungal secondary metabolite gene clusters , 2015, Front. Microbiol..

[24]  Wenjun Jiang,et al.  Targeted isolation and cloning of 100-kb microbial genomic sequences by Cas9-assisted targeting of chromosome segments , 2016, Nature Protocols.

[25]  D. Newman,et al.  Prospecting for new bacterial metabolites: a glossary of approaches for inducing, activating and upregulating the biosynthesis of bacterial cryptic or silent natural products. , 2016, Natural product reports.

[26]  Q. Tu,et al.  Direct cloning and heterologous expression of the salinomycin biosynthetic gene cluster from Streptomyces albus DSM41398 in Streptomyces coelicolor A3(2) , 2015, Scientific Reports.

[27]  K. Albert,et al.  From single to multiple microcoil flow probe NMR and related capillary techniques: a review , 2011, Analytical and Bioanalytical Chemistry.

[28]  Yi Tang,et al.  Technology development for natural product biosynthesis in Saccharomyces cerevisiae. , 2016, Current opinion in biotechnology.

[29]  Clay C C Wang,et al.  VeA and MvlA repression of the cryptic orsellinic acid gene cluster in Aspergillus nidulans involves histone 3 acetylation , 2013, Molecular microbiology.

[30]  Mingzi M. Zhang,et al.  Engineering microbial hosts for production of bacterial natural products. , 2016, Natural product reports.

[31]  M. Seyedsayamdost High-throughput platform for the discovery of elicitors of silent bacterial gene clusters , 2014, Proceedings of the National Academy of Sciences.

[32]  D. Newman,et al.  Natural Products as Sources of New Drugs from 1981 to 2014. , 2016, Journal of natural products.

[33]  Roger G. Linington,et al.  Insights into Secondary Metabolism from a Global Analysis of Prokaryotic Biosynthetic Gene Clusters , 2014, Cell.

[34]  A. Clatworthy,et al.  Targeting virulence: a new paradigm for antimicrobial therapy , 2007, Nature Chemical Biology.

[35]  K. Ochi,et al.  Activating the expression of bacterial cryptic genes by rpoB mutations in RNA polymerase or by rare earth elements , 2014, Journal of Industrial Microbiology & Biotechnology.

[36]  Vladimir Larionov,et al.  Selective isolation of genomic loci from complex genomes by transformation-associated recombination cloning in the yeast Saccharomyces cerevisiae , 2008, Nature Protocols.

[37]  F. Barona-Gómez,et al.  Phylogenomic Analysis of Natural Products Biosynthetic Gene Clusters Allows Discovery of Arseno-Organic Metabolites in Model Streptomycetes , 2016, bioRxiv.

[38]  Jakob Weber,et al.  Functional Reconstitution of a Fungal Natural Product Gene Cluster by Advanced Genome Editing. , 2017, ACS synthetic biology.

[39]  Emmanuel Mikros,et al.  Recent advances and new strategies in the NMR-based identification of natural products. , 2014, Current opinion in biotechnology.

[40]  M. Fischbach,et al.  Small molecules from the human microbiota , 2015, Science.

[41]  Tilmann Weber,et al.  The secondary metabolite bioinformatics portal: Computational tools to facilitate synthetic biology of secondary metabolite production , 2016, Synthetic and systems biotechnology.

[42]  Magnus Lundgren,et al.  Efficient programmable gene silencing by Cascade , 2014, Nucleic acids research.

[43]  Huimin Zhao,et al.  Recent advances in DNA assembly technologies. , 2014, FEMS yeast research.

[44]  A. Stewart,et al.  RecET direct cloning and Redαβ recombineering of biosynthetic gene clusters, large operons or single genes for heterologous expression , 2016, Nature Protocols.

[45]  S. Brady,et al.  Multiplexed CRISPR/Cas9- and TAR-Mediated Promoter Engineering of Natural Product Biosynthetic Gene Clusters in Yeast. , 2016, ACS synthetic biology.

[46]  Hosein Mohimani,et al.  Dereplication, sequencing and identification of peptidic natural products: from genome mining to peptidogenomics to spectral networks. , 2016, Natural product reports.

[47]  Huimin Zhao,et al.  High-Efficiency Multiplex Genome Editing of Streptomyces Species Using an Engineered CRISPR/Cas System , 2014, ACS synthetic biology.

[48]  P. Dorrestein,et al.  Direct cloning and refactoring of a silent lipopeptide biosynthetic gene cluster yields the antibiotic taromycin A , 2014, Proceedings of the National Academy of Sciences.

[49]  Yunkun Liu,et al.  In Vitro CRISPR/Cas9 System for Efficient Targeted DNA Editing , 2015, mBio.

[50]  M. Bibb,et al.  Discovery of Unusual Biaryl Polyketides by Activation of a Silent Streptomyces venezuelae Biosynthetic Gene Cluster , 2016, Chembiochem : a European journal of chemical biology.

[51]  Gang Liu,et al.  Molecular Regulation of Antibiotic Biosynthesis in Streptomyces , 2013, Microbiology and Molecular Reviews.

[52]  Frank Buchholz,et al.  A new logic for DNA engineering using recombination in Escherichia coli , 1998, Nature Genetics.

[53]  Neil L Kelleher,et al.  Modern mass spectrometry for synthetic biology and structure-based discovery of natural products. , 2016, Natural product reports.

[54]  Kai Blin,et al.  antiSMASH 3.0—a comprehensive resource for the genome mining of biosynthetic gene clusters , 2015, Nucleic Acids Res..

[55]  X. Chen,et al.  Specific cloning of human DNA as yeast artificial chromosomes by transformation-associated recombination. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[56]  A. Stewart,et al.  “Cre/loxP plus BAC”: a strategy for direct cloning of large DNA fragment and its applications in Photorhabdus luminescens and Agrobacterium tumefaciens , 2016, Scientific Reports.

[57]  K. Murphy,et al.  Use of Bacteriophage λ Recombination Functions To Promote Gene Replacement in Escherichia coli , 1998, Journal of bacteriology.

[58]  Martin A. Nowak,et al.  Evolution and emergence of infectious diseases in theoretical and real-world networks , 2015, Nature Communications.

[59]  Nuno Bandeira,et al.  MS/MS networking guided analysis of molecule and gene cluster families , 2013, Proceedings of the National Academy of Sciences.

[60]  Yong-Quan Li,et al.  Genome Mining‐Directed Activation of a Silent Angucycline Biosynthetic Gene Cluster in Streptomyces chattanoogensis , 2015, Chembiochem : a European journal of chemical biology.

[61]  M. Seyedsayamdost,et al.  Antibiotic dialogues: induction of silent biosynthetic gene clusters by exogenous small molecules , 2017, FEMS microbiology reviews.