Recent advances in reconstructing microbial secondary metabolites biosynthesis in Aspergillus spp.

He, Yi; Wang, Bin; Chen, Wanping; Cox, Russell J.; He, Jingren; Chen, Fusheng

doi:10.1016/j.biotechadv.2018.02.001

Cited by 66 publications

(48 citation statements)

References 300 publications

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“…Genome sequencing efforts have identified that filamentous fungi encodes a significant number of PKS‐NRPS containing gene clusters, many of which have unknown function or are not transcriptionally active under standard cultivation conditions . Consequently, there is of significant interest to mine these clusters for the production of encoded natural products, using either genetic methods in the natural host or heterologous expression techniques in model hosts . Here, we demonstrate a cryptic PKS‐NRPS pathway in the plant endophyte Penicillium thymicola can produce pyranonigrin A when reconstituted in a heterologous host.…”

Section: Introductionmentioning

confidence: 95%

Identification of the pyranonigrin A biosynthetic gene cluster by genome mining in Penicillium thymicola IBT 5891

et al. 2018

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Significance A polyketide synthase and nonribosomal peptide synthetase hybrid gene cluster from the genome of Penicillium thymicola was identified through genome mining. Heterologous expression of this cluster leads to the production of pyranonigrin A. A series of experiments established that only four genes are sufficient to biosynthesize pyranonigrin A. Based on the results from the current study, a biosynthetic pathway of pyranonigrin A is proposed. © 2018 American Institute of Chemical Engineers AIChE J, 64: 4182–4186, 2018

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Section: Introductionmentioning

confidence: 95%

Identification of the pyranonigrin A biosynthetic gene cluster by genome mining in Penicillium thymicola IBT 5891

et al. 2018

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“…A recent review has provided more information about reconstructing fungal biosynthetic gene clusters in the Aspergillus spp. 82…”

Section: Host Organism: Aspergillus Nidulans and Aspergillus Oryzaementioning

confidence: 99%

Advances in targeting and heterologous expression of genes involved in the synthesis of fungal secondary metabolites

Qiao

Zhu

2019

RSC Adv.

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“…BGC from fungi that are genetically intractable require expression in a heterologous host 5 . Filamentous fungi are the most compatible heterologous hosts for expression of fungal BGCs, not requiring intron removal or codon optimization 11 . For example, Aspergillus nidulans has been successfully utilised as a heterologous host by several groups 12–14 , including ours 15,16 .…”

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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Recent advances in reconstructing microbial secondary metabolites biosynthesis in Aspergillus spp.

Cited by 66 publications

References 300 publications

Identification of the pyranonigrin A biosynthetic gene cluster by genome mining in Penicillium thymicola IBT 5891

Identification of the pyranonigrin A biosynthetic gene cluster by genome mining in Penicillium thymicola IBT 5891

Advances in targeting and heterologous expression of genes involved in the synthesis of fungal secondary metabolites

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

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