Genetic Transformation of Filamentous Fungi: Achievements and Challenges

Lichius, Alexander; Ruiz, Dubraska Moreno; Zeilinger, Susanne

doi:10.1007/978-3-030-29541-7_5

Cited by 21 publications

(14 citation statements)

References 213 publications

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“…Since genetical modification of filamentous fungi involves a number of challenges and different transformation techniques are required for different fungal species ( Li et al, 2017 ; Lichius et al, 2020 ), the current focus is on heterologous protein expression in model organisms to bring versatile UPOs closer to large-scale use and industrial application. First successful attempts of heterologous UPO expression were published almost a decade after the first UPO characterization in 2013 using the mold A. oryzae followed by the yeast organism S. cerevisiae in 2014 ( Babot et al, 2013 ; Molina-Espeja et al, 2014 ).…”

Section: Upo Production Approachesmentioning

confidence: 99%

Identification and Expression of New Unspecific Peroxygenases – Recent Advances, Challenges and Opportunities

Kinner

Rosenthal

Lütz

2021

Front. Bioeng. Biotechnol.

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In 2004, the fungal heme-thiolate enzyme subfamily of unspecific peroxygenases (UPOs) was first described in the basidiomycete Agrocybe aegerita. As UPOs naturally catalyze a broad range of oxidative transformations by using hydrogen peroxide as electron acceptor and thus possess a great application potential, they have been extensively studied in recent years. However, despite their versatility to catalyze challenging selective oxyfunctionalizations, the availability of UPOs for potential biotechnological applications is restricted. Particularly limiting are the identification of novel natural biocatalysts, their production, and the description of their properties. It is hence of great interest to further characterize the enzyme subfamily as well as to identify promising new candidates. Therefore, this review provides an overview of the state of the art in identification, expression, and screening approaches of fungal UPOs, challenges associated with current protein production and screening strategies, as well as potential solutions and opportunities.

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Section: Upo Production Approachesmentioning

confidence: 99%

Identification and Expression of New Unspecific Peroxygenases – Recent Advances, Challenges and Opportunities

Kinner

Rosenthal

Lütz

2021

Front. Bioeng. Biotechnol.

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“…PMT is highly recommended for a wide number of fungi, including the filamentous fungal pathogen Stagonospora nodorum and a newly explored fungus Pseudogymnoascus verrucosu from Antarctic region [21, 22, 37]. Moreover, high transformation efficiency of PMT has been detected in a special class of fungi known as Dothideomycetes [20, 38]. Recently, this method has been successfully applied to another medically important species Shiraia bambusicola to obtain transformants [39].…”

Section: Traditional Genetic Transformation Techniques In Filamentousmentioning

confidence: 99%

“…AMT is highly efficient in a wide number of filamentous fungi, including Botryosphaeria dothidea [62] and Mortierella alpine [63]. However, the efficiency of AMT in particular strains is highly dependent on certain parameters, like the biological properties of the fungus, coculturing conditions, and the fungus‐to‐ Agrobacterium ratio, which need to be optimized accordingly [38, 64]. Additional examples of filamentous fungi in which this method has successfully applied include Fusarium proliferatum [65] , B. dothidea [62] , Penicillium chrysogenum [66] A. oryzae [67], and many others [24].…”

Section: Traditional Genetic Transformation Techniques In Filamentousmentioning

confidence: 99%

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CRISPR/Cas9‐based genome engineering: A new breakthrough in the genetic manipulation of filamentous fungi

Ullah

Lin

Xie

et al. 2020

Biotech and App Biochem

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Filamentous fungi have several industrial, environmental, and medical applications. However, they are rarely utilized owing to the limited availability of full‐genome sequences and genetic manipulation tools. Since the recent discovery of the full‐genome sequences for certain industrially important filamentous fungi, CRISPR/Cas9 technology has drawn attention for the efficient development of engineered strains of filamentous fungi. CRISPR/Cas9 genome editing has been successfully applied to diverse filamentous fungi. In this review, we briefly discuss the use of common genetic transformation techniques as well as CRISPR/Cas9‐based systems in filamentous fungi. Furthermore, we describe potential limitations and challenges in the practical application of genome engineering of filamentous fungi. Finally, we provide suggestions and highlight future research prospects in the area.

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“…HDR-mediated recombination allows for precise gene knockouts/knock-ins and is generally more predictable, and thus better suited, for targeted genome engineering. However, the frequency of HDR events is critically low in filamentous fungi leaving the process of targeted integration of DNA to be very inefficient [8]. Deletion of genes involved in NHEJ pathways, such as the ku70/ku80 gene, has been shown to result in high HDR frequency in both filamentous ascomycete and basidiomycete fungi [5,[9][10][11][12].…”

Section: Introductionmentioning

confidence: 99%

Application of CRISPR/Cas9 Tools for Genome Editing in the White-Rot Fungus Dichomitus squalens

2021

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Dichomitus squalens is an emerging reference species that can be used to investigate white-rot fungal plant biomass degradation, as it has flexible physiology to utilize different types of biomass as sources of carbon and energy. Recent comparative (post-) genomic studies on D. squalens resulted in an increasingly detailed knowledge of the genes and enzymes involved in the lignocellulose breakdown in this fungus and showed a complex transcriptional response in the presence of lignocellulose-derived compounds. To fully utilize this increasing amount of data, efficient and reliable genetic manipulation tools are needed, e.g., to characterize the function of certain proteins in vivo and facilitate the construction of strains with enhanced lignocellulolytic capabilities. However, precise genome alterations are often very difficult in wild-type basidiomycetes partially due to extremely low frequencies of homology directed recombination (HDR) and limited availability of selectable markers. To overcome these obstacles, we assessed various Cas9-single guide RNA (sgRNA) ribonucleoprotein (RNP) -based strategies for selectable homology and non-homologous end joining (NHEJ) -based gene editing in D. squalens. We also showed an induction of HDR-based genetic modifications by using single-stranded oligodeoxynucleotides (ssODNs) in a basidiomycete fungus for the first time. This paper provides directions for the application of targeted CRISPR/Cas9-based genome editing in D. squalens and other wild-type (basidiomycete) fungi.

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Genetic Transformation of Filamentous Fungi: Achievements and Challenges

Cited by 21 publications

References 213 publications

Identification and Expression of New Unspecific Peroxygenases – Recent Advances, Challenges and Opportunities

Identification and Expression of New Unspecific Peroxygenases – Recent Advances, Challenges and Opportunities

CRISPR/Cas9‐based genome engineering: A new breakthrough in the genetic manipulation of filamentous fungi

Application of CRISPR/Cas9 Tools for Genome Editing in the White-Rot Fungus Dichomitus squalens

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