scholarly journals Activation of silent secondary metabolite gene clusters by nucleosome map-guided positioning of the synthetic transcription factor VPR-dCas9

2020 ◽  
Author(s):  
Andreas Schüller ◽  
Lisa Wolansky ◽  
Harald Berger ◽  
Lena Studt ◽  
Agnieszka Gacek-Matthews ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Target Genes ◽  
Negative Impact ◽  
Gene Clusters ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Activation Domains ◽  
Turn On ◽  
Binding Domains ◽  
A Genome

AbstractCurrent methods for forced expression of selected target genes are based on promoter exchange or on overexpressing native or hybrid transcriptional activators in which gene-specific DNA binding domains are coupled to strong activation domains. While these approaches are very useful for promoters with known or synthetically introduced transcription factor binding sites, they are not suitable to turn on genes in biosynthetic gene clusters which often lack pathway-specific activators. To expand the discovery toolbox, we designed a Cas9-based RNA guided synthetic transcription activation system for Aspergillus nidulans based on enzymatically disabled dCas9 fused to three consecutive activation domains (VPR-dCas9). Targeting two biosynthetic gene clusters involved in the production of secondary metabolites, we demonstrate the utility of the system. Especially in silent regions facultative heterochromatin and strictly positioned nucleosomes can constitute a relevant obstacle to the transcriptional machinery. To avoid this negative impact and to facilitate optimal positioning of RNA-guided VPR-dCas9 to our targeted promoters we have created a genome-wide nucleosome map to identify the cognate nucleosome-free-regions (NFRs). Based on these maps, different single-guide RNAs (sgRNA) were designed and tested for their targeting and activation potential. Our results demonstrate that the system can be used to activate silent BGCs in A. nidulans, partially to very high expression levels and also open the opportunity to stepwise turn on individual genes within a BGC that allows to decipher the correlated biosynthetic pathway.

scholarly journals A novel fungal gene regulation system based on inducible VPR-dCas9 and nucleosome map-guided sgRNA positioning

2020 ◽  
Vol 104 (22) ◽  
pp. 9801-9822
Author(s):  
Andreas Schüller ◽  
Lisa Wolansky ◽  
Harald Berger ◽  
Lena Studt ◽  
Agnieszka Gacek-Matthews ◽  
...  
Keyword(s):  
Aspergillus Nidulans ◽  
Target Genes ◽  
Negative Impact ◽  
Constitutive Expression ◽  
Gene Clusters ◽  
Regulation System ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Genome Wide ◽  
A Genome

Abstract Programmable transcriptional regulation is a powerful tool to study gene functions. Current methods to selectively regulate target genes are mainly based on promoter exchange or on overexpressing transcriptional activators. To expand the discovery toolbox, we designed a dCas9-based RNA-guided synthetic transcription activation system for Aspergillus nidulans that uses enzymatically disabled “dead” Cas9 fused to three consecutive activation domains (VPR-dCas9). The dCas9-encoding gene is under the control of an estrogen-responsive promoter to allow induction timing and to avoid possible negative effects by strong constitutive expression of the highly active VPR domains. Especially in silent genomic regions, facultative heterochromatin and strictly positioned nucleosomes can constitute a relevant obstacle to the transcriptional machinery. To avoid this negative impact and to facilitate optimal positioning of RNA-guided VPR-dCas9 to targeted promoters, we have created a genome-wide nucleosome map from actively growing cells and stationary cultures to identify the cognate nucleosome-free regions (NFRs). Based on these maps, different single-guide RNAs (sgRNAs) were designed and tested for their targeting and activation potential. Our results demonstrate that the system can be used to regulate several genes in parallel and, depending on the VPR-dCas9 positioning, expression can be pushed to very high levels. We have used the system to turn on individual genes within two different biosynthetic gene clusters (BGCs) which are silent under normal growth conditions. This method also opens opportunities to stepwise activate individual genes in a cluster to decipher the correlated biosynthetic pathway. Keypoints • An inducible RNA-guided transcriptional regulator based on VPR-dCas9 was established in Aspergillus nidulans. • Genome-wide nucleosome positioning maps were created that facilitate sgRNA positioning. • The system was successfully applied to activate genes within two silent biosynthetic gene clusters.

scholarly journals CRISPR-based transcriptional activation tool for silent genes in filamentous fungi

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
László Mózsik ◽  
Mirthe Hoekzema ◽  
Niels A. W. de Kok ◽  
Roel A. L. Bovenberg ◽  
Yvonne Nygård ◽  
...  
Keyword(s):  
Filamentous Fungi ◽  
Transcriptional Activation ◽  
Core Promoter ◽  
Crop Protection ◽  
Gene Clusters ◽  
Growth Conditions ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Fluorescent Reporter ◽  
A Genome

AbstractFilamentous fungi are historically known to be a rich reservoir of bioactive compounds that are applied in a myriad of fields ranging from crop protection to medicine. The surge of genomic data available shows that fungi remain an excellent source for new pharmaceuticals. However, most of the responsible biosynthetic gene clusters are transcriptionally silent under laboratory growth conditions. Therefore, generic strategies for activation of these clusters are required. Here, we present a genome-editing-free, transcriptional regulation tool for filamentous fungi, based on the CRISPR activation (CRISPRa) methodology. Herein, a nuclease-defective mutant of Cas9 (dCas9) was fused to a highly active tripartite activator VP64-p65-Rta (VPR) to allow for sgRNA directed targeted gene regulation. dCas9-VPR was introduced, together with an easy to use sgRNA “plug-and-play” module, into a non-integrative AMA1-vector, which is compatible with several filamentous fungal species. To demonstrate its potential, this vector was used to transcriptionally activate a fluorescent reporter gene under the control of the penDE core promoter in Penicillium rubens. Subsequently, we activated the transcriptionally silent, native P. rubens macrophorin biosynthetic gene cluster by targeting dCas9-VPR to the promoter region of the transcription factor macR. This resulted in the production of antimicrobial macrophorins. This CRISPRa technology can be used for the rapid and convenient activation of silent fungal biosynthetic gene clusters, and thereby aid in the identification of novel compounds such as antimicrobials.

scholarly journals Whole-genome sequence of bioactive streptomycete derived from mangrove forest in Malaysia, Streptomyces sp. MUSC 14

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Hooi-Leng Ser ◽  
Loh Teng-Hern Tan ◽  
Wen-Si Tan ◽  
Wai-Fong Yin ◽  
Kok-Gan Chan
Keyword(s):  
East Coast ◽  
Genetic Manipulation ◽  
Genome Mining ◽  
Gene Clusters ◽  
Peninsular Malaysia ◽  
Whole Genome Sequence ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Streptomyces Sp ◽  
A Genome

The contribution of streptomycetes to human health is undeniably important and significant, given that these filamentous microbes can produce interesting compounds that can be used to cure deadly infections and even cancer. Isolated from the east coast of Peninsular Malaysia, Streptomyces sp. MUSC 14 has shown significant antioxidant capacity. The current study explores the genomic potential of MUSC 14 via a genome mining approach. The genome size of MUSC 14 is 10,274,825 bp with G + C content of 71.3 %. AntiSMASH analysis revealed a total of nine biosynthetic gene clusters (with more than 80 % similarities to known gene clusters). This information serves as an important foundation for subsequent studies, particularly the purification and isolation of bioactive compounds by genetic manipulation techniques.

scholarly journals CRISPR-Based Transcriptional Activation Tool for Silent Genes in Filamentous Fungi

2020 ◽  
Author(s):  
László Mózsik ◽  
Mirthe Hoekzema ◽  
Niels A.W. de Kok ◽  
Roel A.L. Bovenberg ◽  
Yvonne Nygård ◽  
...  
Keyword(s):  
Filamentous Fungi ◽  
Transcriptional Activation ◽  
Core Promoter ◽  
Crop Protection ◽  
Gene Clusters ◽  
Growth Conditions ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Fluorescent Reporter ◽  
A Genome

AbstractFilamentous fungi are historically known to be a rich reservoir of bioactive compounds that are applied in a myriad of fields ranging from crop protection to medicine. The surge of genomic data available shows that fungi remain an excellent source for new pharmaceuticals. However, most of the responsible biosynthetic gene clusters are transcriptionally silent under laboratory growth conditions. Therefore, generic strategies for activation of these clusters are required. Here, we present a genome-editing-free, transcriptional regulation tool for filamentous fungi, based on the CRISPR activation (CRISPRa) methodology. Herein, a nuclease-defective mutant of Cas9 (dCas9) was fused to a highly active tripartite activator VP64-p65-Rta (VPR) to allow for sgRNA directed targeted gene regulation. dCas9-VPR was introduced, together with an easy to use sgRNA “plug-and-play” module, into an AMA1-vector, which is compatible with several filamentous fungal species. To demonstrate its potential, this vector was used to transcriptionally activate a fluorescent reporter gene under the control of the penDE core promoter in Penicillium rubens. Subsequently, we activated the transcriptionally silent, native P. rubens macrophorin biosynthetic gene cluster by targeting dCas9-VPR to the promoter region of the transcription factor macR. This resulted in the production of antimicrobial macrophorins. This CRISPRa technology can be used for the rapid and convenient activation of silent fungal biosynthetic gene clusters, and thereby aid in the identification of novel compounds such as antimicrobials.

scholarly journals Transcription Factor Repurposing Offers Insights into Evolution of Biosynthetic Gene Cluster Regulation

mBio ◽  
2021 ◽  
Author(s):  
Wenjie Wang ◽  
Milton Drott ◽  
Claudio Greco ◽  
Dianiris Luciano-Rosario ◽  
Pinmei Wang ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Secondary Metabolites ◽  
Gene Cluster ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
The Other ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Metabolite Production ◽  
Fungal Secondary Metabolites

Fungal secondary metabolites (SMs) are an important source of pharmaceuticals on one hand and toxins on the other. Efforts to identify the biosynthetic gene clusters (BGCs) that synthesize SMs have yielded significant insights into how variation in the genes that compose BGCs may impact subsequent metabolite production within and between species.

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 luxRHomolog-Linked Biosynthetic Gene Clusters inProteobacteria

mSystems ◽  
2018 ◽  
Vol 3 (3) ◽  
Author(s):  
Carolyn A. Brotherton ◽  
Marnix H. Medema ◽  
E. Peter Greenberg
Keyword(s):  
Genome Mining ◽  
Gene Clusters ◽  
Growth Conditions ◽  
Bioactive Metabolites ◽  
Growth Media ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Specialized Metabolites ◽  
A Genome ◽  
Functionally Diverse

ABSTRACTMicrobes are a major source of antibiotics, pharmaceuticals, and other bioactive compounds. The production of many specialized microbial metabolites is encoded in biosynthetic gene clusters (BGCs). A challenge associated with natural product discovery is that many BGCs are not expressed under laboratory growth conditions. Here we report a genome-mining approach to discover BGCs withluxR-type quorum sensing (QS) genes, which code for regulatory proteins that control gene expression. Our results show that BGCs linked to genes coding for LuxR-like proteins are widespread inProteobacteria. In addition, we show that associations betweenluxRhomolog genes and BGCs have evolved independently many times, with functionally diverse gene clusters. Overall, these clusters may provide a source of new natural products for which there is some understanding about how to elicit production.IMPORTANCEBacteria biosynthesize specialized metabolites with a variety of ecological functions, including defense against other microbes. Genes that code for specialized metabolite biosynthetic enzymes are frequently clustered together. These BGCs are often regulated by a transcription factor encoded within the cluster itself. These pathway-specific regulators respond to a signal or indirectly through other means of environmental sensing. Many specialized metabolites are not produced under laboratory growth conditions, and one reason for this issue is that laboratory growth media lack environmental cues necessary for BGC expression. Here, we report a bioinformatics study that reveals that BGCs are frequently linked to genes coding for LuxR family QS-responsive transcription factors in the phylumProteobacteria. The products of theseluxRhomolog-associated gene clusters may serve as a practical source of bioactive metabolites.

scholarly journals Activation of silent biosynthetic gene clusters using transcription factor decoys

2018 ◽  
Vol 15 (2) ◽  
pp. 111-114 ◽  
Author(s):  
Bin Wang ◽  
Fang Guo ◽  
Shi-Hui Dong ◽  
Huimin Zhao
Keyword(s):  
Transcription Factor ◽  
Gene Clusters ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters
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