scholarly journals Unraveling the iterative type I polyketide synthases hidden in Streptomyces

2020 ◽  
Vol 117 (15) ◽  
pp. 8449-8454 ◽  
Author(s):  
Bin Wang ◽  
Fang Guo ◽  
Chunshuai Huang ◽  
Huimin Zhao
Keyword(s):  
Assembly Line ◽  
Gene Clusters ◽  
Polyketide Synthases ◽  
Type I ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Computational Tools ◽  
End Products ◽  
Fungal Natural Products ◽  
Biased Distribution

Type I polyketide synthases (T1PKSs) are one of the most extensively studied PKSs, which can act either iteratively or via an assembly-line mechanism. Domains in the T1PKSs can readily be predicted by computational tools based on their highly conserved sequences. However, to distinguish between iterative and noniterative at the module level remains an overwhelming challenge, which may account for the seemingly biased distribution of T1PKSs in fungi and bacteria: small iterative monomodular T1PKSs that are responsible for the enormously diverse fungal natural products exist almost exclusively in fungi. Here we report the discovery of iterative T1PKSs that are unexpectedly both abundant and widespread in Streptomyces. Seven of 11 systematically selected T1PKS monomodules from monomodular T1PKS biosynthetic gene clusters (BGCs) were experimentally confirmed to be iteratively acting, synthesizing diverse branched/nonbranched linear intermediates, and two of them produced bioactive allenic polyketides and citreodiols as end products, respectively. This study indicates the huge potential of iterative T1PKS BGCs from streptomycetes in the discovery of novel polyketides.

scholarly journals GRINS: Genetic elements that recode assembly-line polyketide synthases and accelerate their diversification

2021 ◽  
Vol 118 (26) ◽  
pp. e2100751118 ◽  
Author(s):  
Aleksandra Nivina ◽  
Sur Herrera Paredes ◽  
Hunter B. Fraser ◽  
Chaitan Khosla
Keyword(s):  
Molecular Mechanisms ◽  
Assembly Line ◽  
Gene Families ◽  
Gene Clusters ◽  
Nucleotide Composition ◽  
Polyketide Synthases ◽  
Bacterial Genomes ◽  
Biosynthetic Gene Clusters ◽  
Bacterial Phyla ◽  
Genetic Elements

Assembly-line polyketide synthases (PKSs) are large and complex enzymatic machineries with a multimodular architecture, typically encoded in bacterial genomes by biosynthetic gene clusters. Their modularity has led to an astounding diversity of biosynthesized molecules, many with medical relevance. Thus, understanding the mechanisms that drive PKS evolution is fundamental for both functional prediction of natural PKSs as well as for the engineering of novel PKSs. Here, we describe a repetitive genetic element in assembly-line PKS genes which appears to play a role in accelerating the diversification of closely related biosynthetic clusters. We named this element GRINS: genetic repeats of intense nucleotide skews. GRINS appear to recode PKS protein regions with a biased nucleotide composition and to promote gene conversion. GRINS are present in a large number of assembly-line PKS gene clusters and are particularly widespread in the actinobacterial genus Streptomyces. While the molecular mechanisms associated with GRINS appearance, dissemination, and maintenance are unknown, the presence of GRINS in a broad range of bacterial phyla and gene families indicates that these genetic elements could play a fundamental role in protein evolution.

scholarly journals SeMPI 2.0—A Web Server for PKS and NRPS Predictions Combined with Metabolite Screening in Natural Product Databases

Metabolites ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 13
Author(s):  
Paul F. Zierep ◽  
Adriana T. Ceci ◽  
Ilia Dobrusin ◽  
Sinclair C. Rockwell-Kollmann ◽  
Stefan Günther
Keyword(s):  
Natural Products ◽  
Secondary Metabolites ◽  
Building Block ◽  
Web Server ◽  
Gene Clusters ◽  
Type I ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
A Genome ◽  
Wide Range

Microorganisms produce secondary metabolites with a remarkable range of bioactive properties. The constantly increasing amount of published genomic data provides the opportunity for efficient identification of biosynthetic gene clusters by genome mining. On the other hand, for many natural products with resolved structures, the encoding biosynthetic gene clusters have not been identified yet. Of those secondary metabolites, the scaffolds of nonribosomal peptides and polyketides (type I modular) can be predicted due to their building block-like assembly. SeMPI v2 provides a comprehensive prediction pipeline, which includes the screening of the scaffold in publicly available natural compound databases. The screening algorithm was designed to detect homologous structures even for partial, incomplete clusters. The pipeline allows linking of gene clusters to known natural products and therefore also provides a metric to estimate the novelty of the cluster if a matching scaffold cannot be found. Whereas currently available tools attempt to provide comprehensive information about a wide range of gene clusters, SeMPI v2 aims to focus on precise predictions. Therefore, the cluster detection algorithm, including building block generation and domain substrate prediction, was thoroughly refined and benchmarked, to provide high-quality scaffold predictions. In a benchmark based on 559 gene clusters, SeMPI v2 achieved comparable or better results than antiSMASH v5. Additionally, the SeMPI v2 web server provides features that can help to further investigate a submitted gene cluster, such as the incorporation of a genome browser, and the possibility to modify a predicted scaffold in a workbench before the database screening.

scholarly journals Distribution and diversity of dimetal-carboxylate halogenases in cyanobacteria

2021 ◽  
Author(s):  
Nadia Eusebio ◽  
Adriana Rego ◽  
Nathaniel R. Glasser ◽  
Raquel Castelo-Branco ◽  
Emily P. Balskus ◽  
...  
Keyword(s):  
Genome Mining ◽  
Gene Clusters ◽  
Culture Collection ◽  
Acid Activation ◽  
Type I ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Genes Encoding ◽  
Fatty Acid Activation ◽  
Biological Catalysis

AbstractHalogenation is a recurring feature in natural products, especially those from marine organisms. The selectivity with which halogenating enzymes act on their substrates renders halogenases interesting targets for biocatalyst development. Recently, CylC – the first predicted dimetal-carboxylate halogenase to be characterized – was shown to regio- and stereoselectively install a chlorine atom onto an unactivated carbon center during cylindrocyclophane biosynthesis. Homologs of CylC are also found in other characterized cyanobacterial secondary metabolite biosynthetic gene clusters. Due to its novelty in biological catalysis, selectivity and ability to perform C-H activation, this halogenase class is of considerable fundamental and applied interest. However, little is known regarding the diversity and distribution of these enzymes in bacteria. In this study, we used both genome mining and PCR-based screening to explore the genetic diversity and distribution of CylC homologs. While we found non-cyanobacterial homologs of these enzymes to be rare, we identified a large number of genes encoding CylC-like enzymes in publicly available cyanobacterial genomes and in our in-house culture collection of cyanobacteria. Genes encoding CylC homologs are widely distributed throughout the cyanobacterial tree of life, within biosynthetic gene clusters of distinct architectures. Their genomic contexts feature a variety of biosynthetic partners, including fatty-acid activation enzymes, type I or type III polyketide synthases, dialkylresorcinol-generating enzymes, monooxygenases or Rieske proteins. Our study also reveals that dimetal-carboxylate halogenases are among the most abundant types of halogenating enzymes in the phylum Cyanobacteria. This work will help to guide the search for new halogenating biocatalysts and natural product scaffolds.Data statementAll supporting data and methods have been provided within the article or through a Supplementary Material file, which includes 14 supplementary figures and 4 supplementary tables.

scholarly journals Microfluidic automated plasmid library enrichment for biosynthetic gene cluster discovery

2020 ◽  
Vol 48 (8) ◽  
pp. e48-e48 ◽  
Author(s):  
Peng Xu ◽  
Cyrus Modavi ◽  
Benjamin Demaree ◽  
Frederick Twigg ◽  
Benjamin Liang ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Polyketide Synthase ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Bioactive Molecules ◽  
Type I ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Plasmid Library ◽  
Polyketide Synthase Gene

Abstract Microbial biosynthetic gene clusters are a valuable source of bioactive molecules. However, because they typically represent a small fraction of genomic material in most metagenomic samples, it remains challenging to deeply sequence them. We present an approach to isolate and sequence gene clusters in metagenomic samples using microfluidic automated plasmid library enrichment. Our approach provides deep coverage of the target gene cluster, facilitating reassembly. We demonstrate the approach by isolating and sequencing type I polyketide synthase gene clusters from an Antarctic soil metagenome. Our method promotes the discovery of functional-related genes and biosynthetic pathways.

scholarly journals Genome Sequencing of Streptomyces olivaceus SCSIO T05 and Activated Production of Lobophorin CR4 via Metabolic Engineering and Genome Mining

Marine Drugs ◽  
2019 ◽  
Vol 17 (10) ◽  
pp. 593 ◽  
Author(s):  
Chunyan Zhang ◽  
Wenjuan Ding ◽  
Xiangjing Qin ◽  
Jianhua Ju
Keyword(s):  
Metabolic Engineering ◽  
Deep Sea ◽  
Genome Mining ◽  
Gene Clusters ◽  
Type I ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Genus Streptomyces ◽  
Disruption Method ◽  
Streptomyces Olivaceus

Marine-sourced actinomycete genus Streptomyces continues to be an important source of new natural products. Here we report the complete genome sequence of deep-sea-derived Streptomyces olivaceus SCSIO T05, harboring 37 putative biosynthetic gene clusters (BGCs). A cryptic BGC for type I polyketides was activated by metabolic engineering methods, enabling the discovery of a known compound, lobophorin CR4 (1). Genome mining yielded a putative lobophorin BGC (lbp) that missed the functional FAD-dependent oxidoreductase to generate the d-kijanose, leading to the production of lobophorin CR4 without the attachment of d-kijanose to C17-OH. Using the gene-disruption method, we confirmed that the lbp BGC accounts for lobophorin biosynthesis. We conclude that metabolic engineering and genome mining provide an effective approach to activate cryptic BGCs.

scholarly journals HEx: a heterologous expression platform for the discovery of fungal natural products

2018 ◽  
Author(s):  
Colin JB Harvey ◽  
Mancheng Tang ◽  
Ulrich Schlecht ◽  
Joe Horecka ◽  
Curt R Fischer ◽  
...  
Keyword(s):  
Natural Products ◽  
Synthetic Biology ◽  
Heterologous Expression ◽  
Gene Clusters ◽  
Fungal Species ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Expression Platform ◽  
The World ◽  
Fungal Natural Products

AbstractFor decades, fungi have been a source of FDA-approved natural products such as penicillin, cyclosporine, and the statins. Recent breakthroughs in DNA sequencing suggest that millions of fungal species exist on Earth with each genome encoding pathways capable of generating as many as dozens of natural products. However, the majority of encoded molecules are difficult or impossible to access because the organisms are uncultivable or the genes are transcriptionally silent. To overcome this bottleneck in natural product discovery, we developed the HEx (Heterologous EXpression) synthetic biology platform for rapid, scalable expression of fungal biosynthetic genes and their encoded metabolites in Saccharomyces cerevisiae. We applied this platform to 41 fungal biosynthetic gene clusters from diverse fungal species from around the world, 22 of which produced detectable compounds. These included novel compounds with unexpected biosynthetic origins, particularly from poorly studied species. This result establishes the HEx platform for rapid discovery of natural products from any fungal species, even those that are uncultivable, and opens the door to discovery of the next generation of natural products.SummaryHere we present the largest scale effort reported to date toward the complete refactoring and heterologous expression of fungal biosynthetic gene clusters utilizing HEx, a novel synthetic biology platform.

scholarly journals Distribution and diversity of dimetal-carboxylate halogenases in cyanobacteria

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Nadia Eusebio ◽  
Adriana Rego ◽  
Nathaniel R. Glasser ◽  
Raquel Castelo-Branco ◽  
Emily P. Balskus ◽  
...  
Keyword(s):  
Genome Mining ◽  
Gene Clusters ◽  
Culture Collection ◽  
Type I ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Rieske Proteins ◽  
Number Of Genes ◽  
Genes Encoding ◽  
Biological Catalysis

Abstract Background Halogenation is a recurring feature in natural products, especially those from marine organisms. The selectivity with which halogenating enzymes act on their substrates renders halogenases interesting targets for biocatalyst development. Recently, CylC – the first predicted dimetal-carboxylate halogenase to be characterized – was shown to regio- and stereoselectively install a chlorine atom onto an unactivated carbon center during cylindrocyclophane biosynthesis. Homologs of CylC are also found in other characterized cyanobacterial secondary metabolite biosynthetic gene clusters. Due to its novelty in biological catalysis, selectivity and ability to perform C-H activation, this halogenase class is of considerable fundamental and applied interest. The study of CylC-like enzymes will provide insights into substrate scope, mechanism and catalytic partners, and will also enable engineering these biocatalysts for similar or additional C-H activating functions. Still, little is known regarding the diversity and distribution of these enzymes. Results In this study, we used both genome mining and PCR-based screening to explore the genetic diversity of CylC homologs and their distribution in bacteria. While we found non-cyanobacterial homologs of these enzymes to be rare, we identified a large number of genes encoding CylC-like enzymes in publicly available cyanobacterial genomes and in our in-house culture collection of cyanobacteria. Genes encoding CylC homologs are widely distributed throughout the cyanobacterial tree of life, within biosynthetic gene clusters of distinct architectures (combination of unique gene groups). These enzymes are found in a variety of biosynthetic contexts, which include fatty-acid activating enzymes, type I or type III polyketide synthases, dialkylresorcinol-generating enzymes, monooxygenases or Rieske proteins. Our study also reveals that dimetal-carboxylate halogenases are among the most abundant types of halogenating enzymes in the phylum Cyanobacteria. Conclusions Our data show that dimetal-carboxylate halogenases are widely distributed throughout the Cyanobacteria phylum and that BGCs encoding CylC homologs are diverse and mostly uncharacterized. This work will help guide the search for new halogenating biocatalysts and natural product scaffolds.

scholarly journals Expansion and Conservation of Biosynthetic Gene Clusters in Pathogenic Pyrenophora spp.

Toxins ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 242
Author(s):  
Paula M. Moolhuijzen ◽  
Mariano Jordi Muria-Gonzalez ◽  
Robert Syme ◽  
Catherine Rawlinson ◽  
Pao Theen See ◽  
...  
Keyword(s):  
Polyketide Synthase ◽  
Gene Clusters ◽  
Type I ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Terpene Synthases ◽  
Type Iii Polyketide Synthase ◽  
Cis Acting ◽  
Surrounding Environment ◽  
Genome Assemblies

Pyrenophora is a fungal genus responsible for a number of major cereal diseases. Although fungi produce many specialised or secondary metabolites for defence and interacting with the surrounding environment, the repertoire of specialised metabolites (SM) within Pyrenophora pathogenic species remains mostly uncharted. In this study, an in-depth comparative analysis of the P. teres f. teres, P teres f. maculata and P. tritici-repentis potential to produce SMs, based on in silico predicted biosynthetic gene clusters (BGCs), was conducted using genome assemblies from PacBio DNA reads. Conservation of BGCs between the Pyrenophora species included type I polyketide synthases, terpene synthases and the first reporting of a type III polyketide synthase in P teres f. maculata. P. teres isolates exhibited substantial expansion of non-ribosomal peptide synthases relative to P. tritici-repentis, hallmarked by the presence of tailoring cis-acting nitrogen methyltransferase domains. P. teres isolates also possessed unique non-ribosomal peptide synthase (NRPS)-indole and indole BGCs, while a P. tritici-repentis phytotoxin BGC for triticone production was absent in P. teres. These differences highlight diversification between the pathogens that reflects their different evolutionary histories, host adaption and lifestyles.

scholarly journals Impact on Multiple Antibiotic Pathways Reveals MtrA as a Master Regulator of Antibiotic Production in Streptomyces spp. and Potentially in Other Actinobacteria

2020 ◽  
Vol 86 (20) ◽  
Author(s):  
Yanping Zhu ◽  
Peipei Zhang ◽  
Jing Zhang ◽  
Jiao Wang ◽  
Yinhua Lu ◽  
...  
Keyword(s):  
Antibiotic Production ◽  
Gene Clusters ◽  
Type I ◽  
Biosynthetic Gene ◽  
Regulatory Factor ◽  
Biosynthetic Gene Clusters ◽  
Master Regulator ◽  
Altered Expression ◽  
Content Type

ABSTRACT Regulation of antibiotic production by Streptomyces is complex. We report that the response regulator MtrA is a master regulator for antibiotic production in Streptomyces. Deletion of MtrA altered production of actinorhodin, undecylprodigiosin, calcium-dependent antibiotic, and the yellow-pigmented type I polyketide and resulted in altered expression of the corresponding gene clusters in S. coelicolor. Integrated in vitro and in vivo analyses identified MtrA binding sites upstream of cdaR, actII-orf4, and redZ and between cpkA and cpkD. MtrA disruption also led to marked changes in chloramphenicol and jadomycin production and in transcription of their biosynthetic gene clusters (cml and jad, respectively) in S. venezuelae, and MtrA sites were identified within cml and jad. MtrA also recognized predicted sites within the avermectin and oligomycin pathways in S. avermitilis and in the validamycin gene cluster of S. hygroscopicus. The regulator GlnR competed for several MtrA sites and impacted production of some antibiotics, but its effects were generally less dramatic than those of MtrA. Additional potential MtrA sites were identified in a range of other antibiotic biosynthetic gene clusters in Streptomyces species and other actinobacteria. Overall, our study suggests a universal role for MtrA in antibiotic production in Streptomyces and potentially other actinobacteria. IMPORTANCE In natural environments, the ability to produce antibiotics helps the producing host to compete with surrounding microbes. In Streptomyces, increasing evidence suggests that the regulation of antibiotic production is complex, involving multiple regulatory factors. The regulatory factor MtrA is known to have additional roles beyond controlling development, and using bioassays, transcriptional studies, and DNA-binding assays, our study identified MtrA recognition sequences within multiple antibiotic pathways and indicated that MtrA directly controls the production of multiple antibiotics. Our analyses further suggest that this role of MtrA is evolutionarily conserved in Streptomyces species, as well as in other actinobacterial species, and also suggest that MtrA is a major regulatory factor in antibiotic production and in the survival of actinobacteria in nature.

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