On top of biosynthetic gene clusters: How epigenetic machinery influences secondary metabolism in fungi

Pfannenstiel, Brandon T.; Keller, Nancy P.

doi:10.1016/j.biotechadv.2019.02.001

Cited by 126 publications

(115 citation statements)

References 169 publications

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“…Genes that are involved in secondary metabolism are sequentially distributed in biosynthetic gene clusters (BGCs), providing the coordinated regulation of the genes that are related to any metabolite [89]. The treatment with DNA methylation inhibitors, such as 5-azacytidine (5-AC) and RG-108, which are commonly used to inhibit the activity of DNA MTases, have been successfully confirmed to induce the expression of silent BGCs and influence secondary metabolites in Alternaria species [90], A. niger [91], Cladosporium cladosporioides [92], Diatrype sp.…”

Section: Association Between Dna Methylation and Secondary Metabolismmentioning

confidence: 99%

The Pattern and Function of DNA Methylation in Fungal Plant Pathogens

Zhang

et al. 2020

Microorganisms

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To successfully infect plants and trigger disease, fungal plant pathogens use various strategies that are dependent on characteristics of their biology and genomes. Although pathogenic fungi are different from animals and plants in the genomic heritability, sequence feature, and epigenetic modification, an increasing number of phytopathogenic fungi have been demonstrated to share DNA methyltransferases (MTases) responsible for DNA methylation with animals and plants. Fungal plant pathogens predominantly possess four types of DNA MTase homologs, including DIM-2, DNMT1, DNMT5, and RID. Numerous studies have indicated that DNA methylation in phytopathogenic fungi mainly distributes in transposable elements (TEs), gene promoter regions, and the repetitive DNA sequences. As an important and heritable epigenetic modification, DNA methylation is associated with silencing of gene expression and transposon, and it is responsible for a wide range of biological phenomena in fungi. This review highlights the relevant reports and insights into the important roles of DNA methylation in the modulation of development, pathogenicity, and secondary metabolism of fungal plant pathogens. Recent evidences prove that there are massive links between DNA and histone methylation in fungi, and they commonly regulate fungal development and mycotoxin biosynthesis.

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Section: Association Between Dna Methylation and Secondary Metabolismmentioning

confidence: 99%

The Pattern and Function of DNA Methylation in Fungal Plant Pathogens

Zhang

et al. 2020

Microorganisms

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“…In recent years, epigenetic and consequent histone posttranslational modifications have been shown to play an important role in secondary metabolite production in fungi (Pfannenstiel and Keller, 2019), including regulation of several Aspergillus and Fusarium mycotoxins such as aflatoxin, fumonisin and trichothecenes (Visentin et al, 2012;Liu et al, 2015;Yang et al, 2016;Pfannenstiel et al, 2018). Most studies investigated the role of histone modifying enzymes, such as methylases or histone deacetylases in modulating secondary metabolite synthesis in fungi.…”

Section: Introductionmentioning

confidence: 99%

New Insight Into Pathogenicity and Secondary Metabolism of the Plant Pathogen Penicillium expansum Through Deletion of the Epigenetic Reader SntB

et al. 2020

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“…Genomic surveys have revealed that fungal species typically harbour 30–100 biosynthetic gene clusters (BGCs) each encoding the biosynthetic pathway required to produce a SM(s) 3 . However, the vast majority of BGCs remain uncharacterised or ‘cryptic’ as the products they encode are undetectable under standard culture conditions, often because BGCs remain ‘silent’ or lowly expressed due to tight regulatory control 1,4 . Filamentous fungi, which have yielded a plethora of SMs with pharmaceutical and agricultural applications 5 , thus serve as attractive targets for genome mining of novel molecules.…”

Section: Introductionmentioning

confidence: 99%

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

Roux

Woodcraft

et al. 2020

Preprint

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7Accessing the full biosynthetic potential encoded in the genomes of fungi is limited by the low 8 expression of many biosynthetic gene clusters (BGCs) under standard culture conditions. In 9 this work, we develop a fungal CRISPR activation (CRISPRa) system for targeted upregulation 10 of biosynthetic genes, which could accelerate the emerging genomics-driven approach to 11 bioactive secondary metabolite discovery. We construct a fungal CRISPR/dLbCas12a-VPR 12 system and demonstrate activation of a fluorescent reporter in Aspergillus nidulans. Then, we 13 target the native nonribosomal peptide synthetase-like (NRPS-like) gene micA in both 14 chromosomal and episomal contexts, achieving increases in production of the compound 15 microperfuranone. Finally, multi-gene CRISPRa leads to the discovery of the mic cluster 16 product as dehydromicroperfuranone. Additionally, we demonstrate the utility of the variant 17 LbCas12a D156R -VPR for CRISPRa at lower culture temperatures. This is the first 18 demonstration of CRISPRa in filamentous fungi, providing a framework for CRISPR-mediated 19 transcriptional activation of fungal BGCs. 20 activation (CRISPRa)-mediated approach for BGC activation in filamentous fungi (Fig. 1a). In 49 CRISPRa systems, DNase-deactivated RNA-guided CRISPR/dCas ribonucleoprotein 50 complexes linked to activation effectors are targeted to gene regulatory regions to increase 51 gene expression [17][18][19] . Taking advantage of the streamlined CRISPR RNA (crRNA) cloning 52 and multiplexing capabilities, CRISPRa of BGCs has the potential to greatly accelerate fungal 53 genome mining. CRISPRa has already been used to tune the expression of biosynthetic 54 pathways 20,21 , including in ascomycetous yeasts 22,23 . However, to our knowledge, CRISPRa 55has not yet been demonstrated in filamentous fungi. 56In this work, we develop a suite of fungal CRISPRa vectors based on both dLbCas12a from 57Lachnospiraceae bacterium Cas12a (previously known as Cpf1) 19 , and dSpCas9 from 58Streptococcus pyogenes fused to the tripartite VPR activator 18 , and test them in A. nidulans. 59We further explore the application of our CRISPR/dLbCas12a-VPR system for fungal BGC 60 activation as a tool to fuel bioactive molecule discovery. 61 Results 62 Construction and testing of fungal CRISPRa systems 63To develop a CRISPRa system for filamentous fungi, we constructed and tested 64 CRISPR/dLbCas12a-VPR-and CRISPR/dSpCas9-VPR-based systems in the model 65 organism and chassis A. nidulans. To evaluate alternative strategies for expressing either 66 3 dCas effector, we created parent strains with a chromosomally integrated dCas-VPR 67 expression cassette and compared their performance with entirely AMA1-episomally encoded 68 systems. The AMA1 sequence acts as an extrachromosomal vector replicator and confers 69 increased transformation frequency in several filamentous fungi species 24 . AMA1-bearing 70 vectors are found at multiple copies per nucleus although their genetic stability has been 71 reported to be limited under non...

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On top of biosynthetic gene clusters: How epigenetic machinery influences secondary metabolism in fungi

Cited by 126 publications

References 169 publications

The Pattern and Function of DNA Methylation in Fungal Plant Pathogens

The Pattern and Function of DNA Methylation in Fungal Plant Pathogens

New Insight Into Pathogenicity and Secondary Metabolism of the Plant Pathogen Penicillium expansum Through Deletion of the Epigenetic Reader SntB

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

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