Non-Transgenic CRISPR-Mediated Knockout of Entire Ergot Alkaloid Gene Clusters in Slow-Growing Asexual Polyploid Fungi

The Epichloë species of fungi include seed-borne symbionts (endophytes) of cool-season grasses that enhance plant fitness, although some also produce alkaloids that are toxic to livestock. Selected or mutated toxin-free endophytes can be introduced into forage cultivars for improved livestock performance. Long-read genome sequencing revealed clusters of ergot alkaloid biosynthesis (EAS) genes in Epichloë coenophiala strain e19 from tall fescue (Lolium arundinaceum) and Epichloë hybrida Lp1 from perennial ryegrass (Lolium perenne). The two homeologous clusters in E. coenophiala—a triploid hybrid species—were 196 kb (EAS1) and 75 kb (EAS2), and the E. hybrida EAS cluster was 83 kb. As a CRISPR-based approach to target these clusters, the fungi were transformed with ribonucleoprotein (RNP) complexes of modified Cas9 nuclease (Cas9-2NLS) and pairs of single guide RNAs (sgRNAs), plus a transiently selected plasmid. In E. coenophiala, the procedure generated deletions of EAS1 and EAS2 separately, as well as both clusters simultaneously. The technique also gave deletions of the EAS cluster in E. hybrida and of individual alkaloid biosynthesis genes (dmaW and lolC) that had previously proved difficult to delete in E. coenophiala. Thus, this facile CRISPR RNP approach readily generates non-transgenic endophytes without toxin genes for use in research and forage cultivar improvement.

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Ergot Alkaloids of the Family Clavicipitaceae

Phytopathology ◽

10.1094/phyto-12-16-0435-rvw ◽

2017 ◽

Vol 107 (5) ◽

pp. 504-518 ◽

Cited By ~ 28

Author(s):

Simona Florea ◽

Daniel G. Panaccione ◽

Christopher L. Schardl

Keyword(s):

Ergot Alkaloid ◽

Gene Loss ◽

Ergot Alkaloids ◽

Gene Clusters ◽

Lysergic Acid ◽

Forage Grasses ◽

Alkaloid Biosynthesis ◽

The Family ◽

Gene Recruitment ◽

Alkaloid Profiles

Ergot alkaloids are highly diverse in structure, exhibit diverse effects on animals, and are produced by diverse fungi in the phylum Ascomycota, including pathogens and mutualistic symbionts of plants. These mycotoxins are best known from the fungal family Clavicipitaceae and are named for the ergot fungi that, through millennia, have contaminated grains and caused mass poisonings, with effects ranging from dry gangrene to convulsions and death. However, they are also useful sources of pharmaceuticals for a variety of medical purposes. More than a half-century of research has brought us extensive knowledge of ergot-alkaloid biosynthetic pathways from common early steps to several taxon-specific branches. Furthermore, a recent flurry of genome sequencing has revealed the genomic processes underlying ergot-alkaloid diversification. In this review, we discuss the evolution of ergot-alkaloid biosynthesis genes and gene clusters, including roles of gene recruitment, duplication and neofunctionalization, as well as gene loss, in diversifying structures of clavines, lysergic acid amides, and complex ergopeptines. Also reviewed are prospects for manipulating ergot-alkaloid profiles to enhance suitability of endophytes for forage grasses.

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Faculty Opinions recommendation of Ergot alkaloid biosynthesis in Aspergillus fumigatus: conversion of chanoclavine-I to chanoclavine-I aldehyde catalyzed by a short-chain alcohol dehydrogenase FgaDH.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1466956.946054 ◽

2010 ◽

Author(s):

Sarah O'Connor

Keyword(s):

Alcohol Dehydrogenase ◽

Aspergillus Fumigatus ◽

Ergot Alkaloid ◽

Short Chain ◽

Alkaloid Biosynthesis ◽

Chain Alcohol ◽

Short Chain Alcohol

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Biosynthetic potential of uncultured Antarctic soil bacteria revealed through long-read metagenomic sequencing

The ISME Journal ◽

10.1038/s41396-021-01052-3 ◽

2021 ◽

Author(s):

Valentin Waschulin ◽

Chiara Borsetto ◽

Robert James ◽

Kevin K. Newsham ◽

Stefano Donadio ◽

...

Keyword(s):

Genome Mining ◽

Gene Clusters ◽

Biosynthetic Gene Cluster ◽

Full Length ◽

Metagenomic Sequencing ◽

Short Read ◽

Short Read Sequencing ◽

Rich Diversity ◽

Long Read ◽

The Rich

AbstractThe growing problem of antibiotic resistance has led to the exploration of uncultured bacteria as potential sources of new antimicrobials. PCR amplicon analyses and short-read sequencing studies of samples from different environments have reported evidence of high biosynthetic gene cluster (BGC) diversity in metagenomes, indicating their potential for producing novel and useful compounds. However, recovering full-length BGC sequences from uncultivated bacteria remains a challenge due to the technological restraints of short-read sequencing, thus making assessment of BGC diversity difficult. Here, long-read sequencing and genome mining were used to recover >1400 mostly full-length BGCs that demonstrate the rich diversity of BGCs from uncultivated lineages present in soil from Mars Oasis, Antarctica. A large number of highly divergent BGCs were not only found in the phyla Acidobacteriota, Verrucomicrobiota and Gemmatimonadota but also in the actinobacterial classes Acidimicrobiia and Thermoleophilia and the gammaproteobacterial order UBA7966. The latter furthermore contained a potential novel family of RiPPs. Our findings underline the biosynthetic potential of underexplored phyla as well as unexplored lineages within seemingly well-studied producer phyla. They also showcase long-read metagenomic sequencing as a promising way to access the untapped genetic reservoir of specialised metabolite gene clusters of the uncultured majority of microbes.

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An improved method for precise genome editing in zebrafish using CRISPR-Cas9 technique

Molecular Biology Reports ◽

10.1007/s11033-020-06125-8 ◽

2021 ◽

Author(s):

Eugene V. Gasanov ◽

Justyna Jędrychowska ◽

Michal Pastor ◽

Malgorzata Wiweger ◽

Axel Methner ◽

...

Keyword(s):

Genome Editing ◽

High Efficiency ◽

Target Site ◽

Proof Of Concept ◽

Intervention Site ◽

End Joining ◽

Guide Rna ◽

Guide Rnas ◽

Cas9 Nuclease ◽

Non Homologous End Joining

AbstractCurrent methods of CRISPR-Cas9-mediated site-specific mutagenesis create deletions and small insertions at the target site which are repaired by imprecise non-homologous end-joining. Targeting of the Cas9 nuclease relies on a short guide RNA (gRNA) corresponding to the genome sequence approximately at the intended site of intervention. We here propose an improved version of CRISPR-Cas9 genome editing that relies on two complementary guide RNAs instead of one. Two guide RNAs delimit the intervention site and allow the precise deletion of several nucleotides at the target site. As proof of concept, we generated heterozygous deletion mutants of the kcng4b, gdap1, and ghitm genes in the zebrafish Danio rerio using this method. A further analysis by high-resolution DNA melting demonstrated a high efficiency and a low background of unpredicted mutations. The use of two complementary gRNAs improves CRISPR-Cas9 specificity and allows the creation of predictable and precise mutations in the genome of D. rerio.

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Genetically Encoded CRISPR components Yield Efficient Gene Editing in the Invasive Pest,Drosophila suzukii

10.1101/2021.03.15.435483 ◽

2021 ◽

Author(s):

Nikolay P. Kandul ◽

Esther J. Belikoff ◽

Junru Liu ◽

Anna Buchman ◽

Fang Li ◽

...

Keyword(s):

Population Control ◽

Invasive Pest ◽

Drosophila Suzukii ◽

Guide Rnas ◽

Cas9 Nuclease ◽

Efficient Gene ◽

Transgenic Lines ◽

Repeated Applications ◽

Very High ◽

Genetic Strategies

AbstractOriginally from Asia,Drosophila suzukii(Matsumura, 1931, Diptera:Drosophilidae) is presently a global pest of economically important soft-skinned fruits. Also commonly known as spotted wingDrosophila(SWD), it is largely controlled through repeated applications of broad-spectrum insecticides. There is a pressing need for a better understanding of SWD biology and for developing alternative environmentally-friendly methods of control. The RNA-guided Cas9 nuclease has revolutionized functional genomics and is an integral component of several recently developed genetic strategies for population control of insects. Here we have developed transgenic strains that encode three different terminators and four different promoters to express Cas9 in both the soma and/or germline of SWD. The Cas9 lines were evaluated through genetic crossing to transgenic lines that encode single guide RNAs targeting the conserved X-linkedyellowbody andwhiteeye genes. We find that several Cas9/gRNA lines display very high editing capacity. Going forward, these tools will be instrumental for evaluating gene function in SWD and may provide tools useful for the development of new genetic strategies for control of this invasive species.

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Metagenomic exploration of the marine sponge mycale hentscheli uncovers multiple polyketide-producing bacterial symbionts

10.26686/wgtn.12444086 ◽

2020 ◽

Author(s):

Mathew Storey ◽

SK Andreassend ◽

Joe Bracegirdle ◽

Alistair Brown ◽

Robert Keyzers ◽

...

Keyword(s):

Marine Sponge ◽

Bacterial Species ◽

Gene Clusters ◽

Taxonomic Diversity ◽

Marine Sponges ◽

Metagenomic Sequencing ◽

Defensive Symbiosis ◽

Long Read ◽

Comprehensive Picture ◽

Mycale Hentscheli

© 2020 Storey et al. Marine sponges have been a prolific source of unique bioactive compounds that are presumed to act as a deterrent to predation. Many of these compounds have potential therapeutic applications; however, the lack of efficient and sustainable synthetic routes frequently limits clinical development. Here, we describe a metag-enomic investigation of Mycale hentscheli, a chemically gifted marine sponge that pos-sesses multiple distinct chemotypes. We applied shotgun metagenomic sequencing, hybrid assembly of short-and long-read data, and metagenomic binning to obtain a comprehensive picture of the microbiome of five specimens, spanning three chemo-types. Our data revealed multiple producing species, each having relatively modest secondary metabolomes, that contribute collectively to the chemical arsenal of the holo-biont. We assembled complete genomes for multiple new genera, including two species that produce the cytotoxic polyketides pateamine and mycalamide, as well as a third high-abundance symbiont harboring a proteusin-type biosynthetic pathway that appears to encode a new polytheonamide-like compound. We also identified an additional 188 biosynthetic gene clusters, including a pathway for biosynthesis of peloruside. These re-sults suggest that multiple species cooperatively contribute to defensive symbiosis in M. hentscheli and reveal that the taxonomic diversity of secondary-metabolite-producing sponge symbionts is larger and richer than previously recognized. IMPORTANCE Mycale hentscheli is a marine sponge that is rich in bioactive small mol-ecules. Here, we use direct metagenomic sequencing to elucidate highly complete and contiguous genomes for the major symbiotic bacteria of this sponge. We identify complete biosynthetic pathways for the three potent cytotoxic polyketides which have previously been isolated from M. hentscheli. Remarkably, and in contrast to previous studies of marine sponges, we attribute each of these metabolites to a different producing mi-crobe. We also find that the microbiome of M. hentscheli is stably maintained among in-dividuals, even over long periods of time. Collectively, our data suggest a cooperative mode of defensive symbiosis in which multiple symbiotic bacterial species cooperatively contribute to the defensive chemical arsenal of the holobiont.

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Probing ergot alkaloid biosynthesis: synthesis and feeding of a proposed intermediate along the biosynthetic pathway. A new amidomalonate for tryptophan elaboration

The Journal of Organic Chemistry ◽

10.1021/jo00239a034 ◽

1988 ◽

Vol 53 (4) ◽

pp. 863-869 ◽

Cited By ~ 18

Author(s):

Alan P. Kozikowski ◽

Makoto Okita ◽

Motomasa Kobayashi ◽

Heinz G. Floss

Keyword(s):

Ergot Alkaloid ◽

Biosynthetic Pathway ◽

Alkaloid Biosynthesis

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Probing ergot alkaloid biosynthesis: identification of advanced intermediates along the biosynthetic pathway

Journal of the American Chemical Society ◽

10.1021/ja00214a054 ◽

1988 ◽

Vol 110 (6) ◽

pp. 1970-1971 ◽

Cited By ~ 10

Author(s):

Alan P. Kozikowski ◽

Jiang Ping. Wu ◽

Masaaki. Shibuya ◽

Heinz G. Floss

Keyword(s):

Ergot Alkaloid ◽

Biosynthetic Pathway ◽

Alkaloid Biosynthesis

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Dynamics in Secondary Metabolite Gene Clusters in Otherwise Highly Syntenic and Stable Genomes in the Fungal Genus Botrytis

Genome Biology and Evolution ◽

10.1093/gbe/evaa218 ◽

2020 ◽

Vol 12 (12) ◽

pp. 2491-2507

Author(s):

Claudio A Valero-Jiménez ◽

Maikel B F Steentjes ◽

Jason C Slot ◽

Xiaoqian Shi-Kunne ◽

Olga E Scholten ◽

...

Keyword(s):

Secondary Metabolite ◽

Gene Clusters ◽

Single Species ◽

White Rot ◽

White Rot Fungus ◽

Broad Host Range ◽

Allium Species ◽

Sclerotium Cepivorum ◽

Single Host ◽

Long Read

Abstract Fungi of the genus Botrytis infect >1,400 plant species and cause losses in many crops. Besides the broad host range pathogen Botrytis cinerea, most other species are restricted to a single host. Long-read technology was used to sequence genomes of eight Botrytis species, mostly pathogenic on Allium species, and the related onion white rot fungus, Sclerotium cepivorum. Most assemblies contained <100 contigs, with the Botrytis aclada genome assembled in 16 gapless chromosomes. The core genome and pan-genome of 16 Botrytis species were defined and the secretome, effector, and secondary metabolite repertoires analyzed. Among those genes, none is shared among all Allium pathogens and absent from non-Allium pathogens. The genome of each of the Allium pathogens contains 8–39 predicted effector genes that are unique for that single species, none stood out as potential determinant for host specificity. Chromosome configurations of common ancestors of the genus Botrytis and family Sclerotiniaceae were reconstructed. The genomes of B. cinerea and B. aclada were highly syntenic with only 19 rearrangements between them. Genomes of Allium pathogens were compared with ten other Botrytis species (nonpathogenic on Allium) and with 25 Leotiomycetes for their repertoire of secondary metabolite gene clusters. The pattern was complex, with several clusters displaying patchy distribution. Two clusters involved in the synthesis of phytotoxic metabolites are at distinct genomic locations in different Botrytis species. We provide evidence that the clusters for botcinic acid production in B. cinerea and Botrytis sinoallii were acquired by horizontal transfer from taxa within the same genus.

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