Heterologous biosynthesis of elsinochrome A sheds light on the formation of the photosensitive perylenequinone system

Hu, Jinyu; Sarrami, Farzaneh; Li, Hang; Zhang, Guozhi; Stubbs, Keith A.; Lacey, Ernest; Stewart, Scott G.; Karton, Amir; Piggott, Andrew M.; Chooi, Yit‐Heng

doi:10.1039/c8sc02870b

Cited by 74 publications

(129 citation statements)

References 37 publications

(63 reference statements)

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“…AMA1-bearing vectors are found at multiple copies per nucleus although their genetic stability has been reported to be limited under non-selective conditions. We built on the triple auxotrophic mutant A. nidulans LO8030 25 , which can maintain AMA1 vectors by complementation with the selectable markers pyrG , riboB , pyroA 15 . The modular nature of the AMA1 vector set allowed rapid building, testing and exchange of the different CRISPRa components.…”

Section: Resultsmentioning

confidence: 99%

“…This led to the rediscovery of dehydromicroperfuranone, in which the molecule was associated with the mic cluster for the first time. Additionally, we demonstrated CRISPRa-mediated upregulation of genes in episomal constructs, which could be a valuable tool to activate BGCs from diverse fungi captured on vectors and heterologously expressed in A. nidulans as a chassis 12,15 .…”

Section: Discussionmentioning

confidence: 99%

“…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 . Whether in the native fungus or in a heterologous host, promoter exchange can be cumbersome due to the need for marker recycling 13 for chromosomal manipulations, or the challenging episomal cloning of multiple and often large biosynthetic genes 15 .…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Roux

Woodcraft

et al. 2020

Preprint

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“…However, the enzyme responsible for the aldol reaction to construct the unique tricyclo[6.2.2.0]dodecane scaffold in 1 and the related betaenone A ( 5 ) from P. betae remained enigmatic. Herein, to further shed light into the biosynthesis of 1 , we employed a similar heterologous pathway reconstruction approach in A. nidulans LO7890 by using a hybrid yeast‐fungal artificial chromosome (pYFAC) expression system . Co‐expression of the polyketide synthase gene sthA and the partnering trans ‐ER gene sthE in A. nidulans led to the production of dehydroprobetaenone I 2 (Figure ), which corresponds to co‐expression of bet1 and bet3 (homologous to sthA and E , respectively) in A. oryzae .…”

Section: Figurementioning

confidence: 99%

Biosynthesis of a Tricyclo[6.2.2.0^2,7]dodecane System by a Berberine Bridge Enzyme‐Like Aldolase

Wei

et al. 2019

Chemistry A European J

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The aldol reaction is one of the most fundamental stereocontrolled carbon–carbon bond‐forming reactions and is mainly catalyzed by aldolases in nature. Despite the fact that the aldol reaction has been widely proposed to be involved in fungal secondary metabolite biosynthesis, a dedicated aldolase that catalyzes stereoselective aldol reactions has only rarely been reported in fungi. Herein, we activated a cryptic polyketide biosynthetic gene cluster that was upregulated in the fungal wheat pathogen Parastagonospora nodorum during plant infection; this resulted in the production of the phytotoxic stemphyloxin II (1). Through heterologous reconstruction of the biosynthetic pathway and in vitro assay by using cell‐free lysate from Aspergillus nidulans, we demonstrated that a berberine bridge enzyme (BBE)‐like protein SthB catalyzes an intramolecular aldol reaction to establish the bridged tricyclo[6.2.2.02,7]dodecane skeleton in the post‐assembly tailoring step. The characterization of SthB as an aldolase enriches the catalytic toolbox of classic reactions and the functional diversities of the BBE superfamily of enzymes.

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“…[5,6] In these examples,the polyketide monomers possess several activated positions,o f which only one is selected for coupling by adistinct laccase.In contrast to inherent regioselectivity,e nantioselectivity has only been found when laccases were combined with other proteins. [7][8][9] Specifically,so-called dirigent proteins are able to mediate enantioselective coupling of radicals produced by alaccase. [10] In this study,w ef ocused on ag roup of laccases that catalyzes coupling reactions in the biosynthesis of ustilaginoidins,w hich are dimeric g-naphthopyrones isolated from different fungal species.…”

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Enantioselective Phenol Coupling by Laccases in the Biosynthesis of Fungal Dimeric Naphthopyrones

et al. 2019

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Biaryl compounds are ubiquitous metabolites that are often formed by dimerization through oxidative phenol coupling.Hindered rotation around the biaryl bond can cause axial chirality.I nn ature,d imerizations are catalyzed by oxidative enzymes such as laccases.T his class of enzymes is knownf or non-specific oxidase reactions while inherent enantioselectivity is hitherto unknown. Here,w ed escribe four related fungal laccases that catalyze g-naphthopyrone dimerization in ar egio-and atropselective manner.I nvitro assays revealed that three enzymes were highly P-selective (ee > 95 %), while one enzyme showed remarkable flexibility.I ts selectivity for M-or P-configured dimers varied depending on the reaction conditions.F or example,alower enzyme concentration yielded primarily (P)-ustilaginoidin A, whereas the Matropisomer was favored at higher concentration. These results demonstrate inherent enantioselectivity in an enzyme class that was previously thought to comprise only nonselective oxidases.

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Heterologous biosynthesis of elsinochrome A sheds light on the formation of the photosensitive perylenequinone system

Abstract: Formation of the three C–C bridges between the two naphthol monomers for elsinochrome (1) involves three distinct classes of oxidases.

Cited by 74 publications

References 37 publications

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

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

Biosynthesis of a Tricyclo[6.2.2.0^2,7]dodecane System by a Berberine Bridge Enzyme‐Like Aldolase

Enantioselective Phenol Coupling by Laccases in the Biosynthesis of Fungal Dimeric Naphthopyrones

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Heterologous biosynthesis of elsinochrome A sheds light on the formation of the photosensitive perylenequinone system

Abstract: Formation of the three C–C bridges between the two naphthol monomers for elsinochrome (1) involves three distinct classes of oxidases.

Cited by 74 publications

References 37 publications

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

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

Biosynthesis of a Tricyclo[6.2.2.02,7]dodecane System by a Berberine Bridge Enzyme‐Like Aldolase

Enantioselective Phenol Coupling by Laccases in the Biosynthesis of Fungal Dimeric Naphthopyrones

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Biosynthesis of a Tricyclo[6.2.2.0^2,7]dodecane System by a Berberine Bridge Enzyme‐Like Aldolase