Discovery of McrA, a master regulator of <i>Aspergillus</i> secondary metabolism

Oakley, C. Elizabeth; Ahuja, Manmeet; Sun, Weiwen; Entwistle, Ruth; Akashi, Tomohiro; Yaegashi, Junko; Guo, Chun-Jun; Cerqueira, Gustavo C.; Wortman, Jennifer; Wang, Clay C. C.; Chiang, Yun‐Wei; Oakley, Berl R.

doi:10.1111/mmi.13562

Cited by 66 publications

(85 citation statements)

References 90 publications

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“…Multiple approaches have been devised over the past decades to identify and produce fungal SMs [17][18] . More recent approaches take advantage of the wealth of information garnered through whole genome sequencing and genome annotation to accelerate novel compound discovery 12,15,[19][20] . The accumulated body of genomic information from many different organisms has allowed for the bioinformatic prediction of SM gene clusters.…”

Section: Introductionmentioning

confidence: 99%

Biosynthesis of alkylcitric acids inAspergillus nigerinvolves both co-localized and unlinked genes

Palys

Pham

Tsang

2019

Preprint

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Filamentous fungi are an abundant source of bioactive secondary metabolites (SMs). In many cases, the biosynthetic processes of SMs are not well understood. This work focuses on a group of SMs, the alkylcitric acids, each of which contains a saturated alkyl "tail" and a citratederived "head". We initially identified their biosynthetic gene cluster and the transcriptional regulator (akcR) involved in the biosynthesis of alkylcitrates in the filamentous fungus Aspergillus niger by examining the functional annotation of SM gene clusters predicted from genomic data.We overexpressed the transcription regulator gene akcR and obtained from a litre of culture filtrate 8.5 grams of extract containing seven alkylcitric acids as determined by NMR. Hexylaconitic acid A comprised ~ 95% of the total production, and four of the seven identified alkylcitrates have not been reported previously. Analysis of orthologous alkylcitrate gene clusters in the Aspergilli revealed an in-cluster cis-aconitate decarboxylase gene (cadA) in A. oryzae and A. flavus, which in A. niger is located on a different chromosome. Overexpression of the A. niger cadA and akcR genes together shifted the profile of alkylcitrates production from primarily hexylaconitic acids to mainly hexylitaconic acids. We also detected two additional, previously unreported, alkylcitric acids in the double overexpression strain. This study shows that phylogenomic analysis together with experimental manipulations can be used to reconstruct a more complete biosynthetic pathway in generating a broader spectrum of alkylcitric compounds. The approach adopted here has the potential of elucidating the complexity of other SM biosynthetic pathways in fungi.

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Section: Introductionmentioning

confidence: 99%

Biosynthesis of alkylcitric acids inAspergillus nigerinvolves both co-localized and unlinked genes

Palys

Pham

Tsang

2019

Preprint

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“…With the relatively small size of most fungal genomes and the continually declining price of whole-genome sequencing, there is now an opportunity for the revitalization of forward genetic screening for identifying and characterization of novel genetic pathways. Next-generation sequencing represents a solution to old genetic problems and can be used to move the field toward a more complete picture of gene regulation, biochemistry, and cellular biology (31,55,56). The use of Ion Torrent and Illumina sequencing technology allowed us to generate whole-genome sequences and successfully identify the causative mutations in 12 of the 17 isolates that we sequenced.…”

Section: Fig 2 Gene Deletions Of Novel Regulators Inmentioning

confidence: 99%

Revitalization of a Forward Genetic Screen Identifies Three New Regulators of Fungal Secondary Metabolism in the Genus Aspergillus

Pfannenstiel

Zhao

Wortman

et al. 2017

mBio

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The study of aflatoxin in Aspergillus spp. has garnered the attention of many researchers due to aflatoxin's carcinogenic properties and frequency as a food and feed contaminant. Significant progress has been made by utilizing the model organism Aspergillus nidulans to characterize the regulation of sterigmatocystin (ST), the penultimate precursor of aflatoxin. A previous forward genetic screen identified 23 A. nidulans mutants involved in regulating ST production. Six mutants were characterized from this screen using classical mapping (five mutations in mcsA) and complementation with a cosmid library (one mutation in laeA). The remaining mutants were backcrossed and sequenced using Illumina and Ion Torrent sequencing platforms. All but one mutant contained one or more sequence variants in predicted open reading frames. Deletion of these genes resulted in identification of mutant alleles responsible for the loss of ST production in 12 of the 17 remaining mutants. Eight of these mutations were in genes already known to affect ST synthesis (laeA, mcsA, fluG, and stcA), while the remaining four mutations (in laeB, sntB, and hamI) were in previously uncharacterized genes not known to be involved in ST production. Deletion of laeB, sntB, and hamI in A. flavus results in loss of aflatoxin production, confirming that these regulators are conserved in the aflatoxigenic aspergilli. This report highlights the multifaceted regulatory mechanisms governing secondary metabolism in Aspergillus. Additionally, these data contribute to the increasing number of studies showing that forward genetic screens of fungi coupled with wholegenome resequencing is a robust and cost-effective technique.IMPORTANCE In a postgenomic world, reverse genetic approaches have displaced their forward genetic counterparts. The techniques used in forward genetics to identify loci of interest were typically very cumbersome and time-consuming, relying on Mendelian traits in model organisms. The current work was pursued not only to identify alleles involved in regulation of secondary metabolism but also to demonstrate a return to forward genetics to track phenotypes and to discover genetic pathways that could not be predicted through a reverse genetics approach. While identification of mutant alleles from whole-genome sequencing has been done before, here we illustrate the possibility of coupling this strategy with a genetic screen

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“…Due to increased polarity, 2 was only extractable from the culture medium with acidified ethyl acetate or adsorbent resin (Supplementary Fig. 8b), which might explain why this compound has not been observed in previous studies where ethyl acetate extraction was used for metabolite profiling of A. nidulans 31,38 .…”

Section: Resultsmentioning

confidence: 97%

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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Discovery of McrA, a master regulator of Aspergillus secondary metabolism

Cited by 66 publications

References 90 publications

Biosynthesis of alkylcitric acids inAspergillus nigerinvolves both co-localized and unlinked genes

Biosynthesis of alkylcitric acids inAspergillus nigerinvolves both co-localized and unlinked genes

Revitalization of a Forward Genetic Screen Identifies Three New Regulators of Fungal Secondary Metabolism in the Genus Aspergillus

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

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