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 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 CRISPR-mediated activation of biosynthetic gene clusters for bioactive molecule discovery in filamentous fungi

2020 ◽  
Author(s):  
Indra Roux ◽  
Clara Woodcraft ◽  
Jinyu Hu ◽  
Rebecca Wolters ◽  
Cameron L.M. Gilchrist ◽  
...  
Keyword(s):  
Filamentous Fungi ◽  
Transcriptional Activation ◽  
Gene Clusters ◽  
Laboratory Culture ◽  
Culture Conditions ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Fluorescent Reporter ◽  
Artificial Transcription Factors ◽  
Bioactive Secondary Metabolite

AbstractAccessing the full biosynthetic potential encoded in the genomes of fungi is limited by the low expression of most biosynthetic gene clusters (BGCs) under common laboratory culture conditions. CRISPR-mediated transcriptional activation (CRISPRa) of fungal BGC could accelerate genomics-driven bioactive secondary metabolite discovery. In this work, we established the first CRISPRa system for filamentous fungi. First, we constructed a CRISPR/dLbCas12a-VPR-based system and demonstrated the activation of a fluorescent reporter in Aspergillus nidulans. Then, we targeted the native nonribosomal peptide synthetase-like (NRPS-like) gene micA in both chromosomal and episomal contexts, achieving increased production of the compound microperfuranone. Finally, multi-gene CRISPRa led to the discovery of the mic cluster product as dehydromicroperfuranone. Additionally, we demonstrated the utility of the variant dLbCas12aD156R-VPR for CRISPRa at room temperature culture conditions. Different aspects that influence the efficiency of CRISPRa in fungi were investigated, providing a framework for the further development of fungal artificial transcription factors based on CRISPR/Cas.

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 Cre/lox-mediated chromosomal integration of biosynthetic gene clusters for heterologous expression in Aspergillus nidulans

2021 ◽  
Author(s):  
Indra Roux ◽  
Yit-Heng Chooi
Keyword(s):  
Heterologous Expression ◽  
Aspergillus Nidulans ◽  
Filamentous Fungi ◽  
Gene Clusters ◽  
Single Step ◽  
Chromosomal Integration ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Fluorescent Reporter ◽  
Site Specific

Building strains for stable long-term heterologous expression of large biosynthetic pathways in filamentous fungi is limited by the low transformation efficiency or genetic stability of current methods. Here, we developed a system for targeted chromosomal integration of large biosynthetic gene clusters in Aspergillus nidulans based on site-specific recombinase mediated cassette exchange. We built A. nidulans strains harbouring a chromosomal landing pad for Cre/lox-mediated recombination and demonstrated efficient targeted integration of a 21.5 kb heterologous region in a single step. We further evaluated the integration at two loci by analysing the expression of a fluorescent reporter and the production of a heterologous polyketide. We compared chromosomal expression at those landing loci to episomal AMA1-based expression, which also shed light on uncharacterised aspects of episomal expression in filamentous fungi. This is the first demonstration of site-specific recombinase-mediated integration in filamentous fungi, setting the foundations for the further development of this tool.

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 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.

Native and engineered promoters in natural product discovery

2016 ◽  
Vol 33 (8) ◽  
pp. 1006-1019 ◽  
Author(s):  
Maksym Myronovskyi ◽  
Andriy Luzhetskyy
Keyword(s):  
Natural Product ◽  
Transcriptional Activation ◽  
Gene Clusters ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Natural Product Discovery

Transcriptional activation of biosynthetic gene clusters.

scholarly journals Gene Modules Co-regulated with Biosynthetic Gene Clusters for Allelopathy between Rice and Barnyardgrass

2019 ◽  
Vol 20 (16) ◽  
pp. 3846 ◽  
Author(s):  
Most. Humaira Sultana ◽  
Fangjie Liu ◽  
Md. Alamin ◽  
Lingfeng Mao ◽  
Lei Jia ◽  
...  
Keyword(s):  
Gene Networks ◽  
Regulatory Gene ◽  
Crop Protection ◽  
Gene Clusters ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Central Process ◽  
Coordinated Expression ◽  
Gene Modules ◽  
Allelopathic Interactions

Allelopathy is a central process in crop–weed interactions and is mediated by the release of allelochemicals that result in adverse growth effects on one or the other plant in the interaction. The genomic mechanism for the biosynthesis of many critical allelochemicals is unknown but may involve the clustering of non-homologous biosynthetic genes involved in their formation and regulatory gene modules involved in controlling the coordinated expression within these gene clusters. In this study, we used the transcriptomes from mono- or co-cultured rice and barnyardgrass to investigate the nature of the gene clusters and their regulatory gene modules involved in the allelopathic interactions of these two plants. In addition to the already known biosynthetic gene clusters in barnyardgrass we identified three potential new clusters including one for quercetin biosynthesis and potentially involved in allelopathic interaction with rice. Based on the construction of gene networks, we identified one gene regulatory module containing hub transcription factors, significantly positively co-regulated with both the momilactone A and phytocassane clusters in rice. In barnyardgrass, gene modules and hub genes co-expressed with the gene clusters responsible for 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) biosynthesis were also identified. In addition, we found three genes in barnyardgrass encoding indole-3-glycerolphosphate synthase that regulate the expression of the DIMBOA cluster. Our findings offer new insights into the regulatory mechanisms of biosynthetic gene clusters involved in allelopathic interactions between rice and barnyardgrass, and have potential implications in controlling weeds for crop protection.

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