FungiDB: An Integrated Bioinformatic Resource for Fungi and Oomycetes

Basenko, Evelina Y.; Pulman, Jane A.; Shanmugasundram, Achchuthan; Harb, Omar S.; Crouch, Kathryn; Starns, David; Warrenfeltz, Susanne; Aurrecoechea, Cristina; Stoeckert, Christian J.; Kissinger, Jessica C.; Roos, David S.; Hertz‐Fowler, Christiane

doi:10.3390/jof4010039

Cited by 286 publications

(273 citation statements)

References 48 publications

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“…Protein homolog lists were assembled with OrthoDB (47) and PANTHERdb (48), with reference to FungiDB (107). The list of cytoplasmic ribosomal proteins was assembled in 5. cerevisiae based on (108) with help from SGD (106), extended to other fungi with PANTHERdb (47), and manually curated.…”

Section: Methodsmentioning

confidence: 99%

Start codon context controls translation initiation in the fungal kingdom

Wallace

Maufrais

Sales-Lee

et al. 2019

Preprint

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Eukaryotic protein synthesis initiates at a start codon defined by an AUG and its surrounding Kozak sequence context, but studies of S. cerevisiae suggest this context is of little importance in fungi. We tested this concept in two pathogenic Cryptococcus species by genome-wide mapping of translation and of mRNA 5’ and 3’ ends. We observed that upstream open reading frames (uORFs) are a major contributor to translation repression, that uORF use depends on the Kozak sequence context of its start codon, and that uORFs with strong contexts promote nonsense-mediated mRNA decay. Numerous Cryptococcus mRNAs encode predicted dual-localized proteins, including many aminoacyl-tRNA synthetases, in which a leaky AUG start codon is followed by a strong Kozak context in-frame AUG, separated by mitochondrial-targeting sequence. Further analysis shows that such dual-localization is also predicted to be common in Neurospora crassa. Kozak-controlled regulation is correlated with insertions in translational initiation factors in fidelity-determining regions that contact the initiator tRNA. Thus, start codon context is a signal that programs the expression and structures of proteins in fungi.

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Section: Methodsmentioning

confidence: 99%

Start codon context controls translation initiation in the fungal kingdom

Wallace

Maufrais

Sales-Lee

et al. 2019

Preprint

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“…To begin assembling a set of recommendations for reference genome database construction, we assessed the concordance and usability of available reference databases for microbial genomics. Our study considered the concordance of microbial species and genera across four fungal reference databases (Ensembl [3], RefSeq [4], JGI's 1000 fungal genomes project (JGI 1 K) [5], and FungiDB [6]) and three bacterial reference databases (Ensembl [3], RefSeq [4], and PATRIC [7]). We compared the microbial taxa in each of the databases using NCBI's universal taxonomic identifiers (hereafter referred to as taxIDs) at the ranks of species and genus (Additional file 1).…”

Section: Main Textmentioning

confidence: 99%

Improving the usability and comprehensiveness of microbial databases

et al. 2020

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Metagenomics studies leverage genomic reference databases to generate discoveries in basic science and translational research. However, current microbial studies use disparate reference databases that lack consistent standards of specimen inclusion, data preparation, taxon labelling and accessibility, hindering their quality and comprehensiveness, and calling for the establishment of recommendations for reference genome database assembly. Here, we analyze existing fungal and bacterial databases and discuss guidelines for the development of a master reference database that promises to improve the quality and quantity of omics research.

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“…Based on available A. nidulans TSS annotation 32,33 , we followed previously devised guidelines for CRISPRa in eukaryotes 34 and targeted a region 119–303 bp upstream of the TSS with a four-crRNA array named MU ( micA Upstream TSS) (Fig. 2b).…”

Section: Resultsmentioning

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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FungiDB: An Integrated Bioinformatic Resource for Fungi and Oomycetes

Cited by 286 publications

References 48 publications

Start codon context controls translation initiation in the fungal kingdom

Start codon context controls translation initiation in the fungal kingdom

Improving the usability and comprehensiveness of microbial databases

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

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