scholarly journals Highly efficient gene targeting in Aspergillus oryzae industrial strains under ligD mutation introduced by genome editing: Strain-specific differences in the effects of deleting EcdR, the negative regulator of sclerotia formation

2017 ◽  
Vol 63 (3) ◽  
pp. 172-178 ◽  
Author(s):  
Hidetoshi Nakamura ◽  
Takuya Katayama ◽  
Tomoya Okabe ◽  
Kazuhiro Iwashita ◽  
Wataru Fujii ◽  
...  
Keyword(s):  
Genome Editing ◽  
Gene Targeting ◽  
Aspergillus Oryzae ◽  
Negative Regulator ◽  
Highly Efficient ◽  
Efficient Gene ◽  
Industrial Strains

Highly efficient gene targeting in the Aspergillus niger kusA mutant

2007 ◽  
Vol 128 (4) ◽  
pp. 770-775 ◽  
Author(s):  
Vera Meyer ◽  
Mark Arentshorst ◽  
Aymen El-Ghezal ◽  
Ann-Christin Drews ◽  
Rolf Kooistra ◽  
...  
Keyword(s):  
Aspergillus Niger ◽  
Gene Targeting ◽  
Highly Efficient ◽  
Efficient Gene

Highly efficient gene targeting in Penicillium chrysogenum using the bi-partite approach in Δlig4 or Δku70 mutants

2010 ◽  
Vol 47 (10) ◽  
pp. 839-846 ◽  
Author(s):  
Paulo de Boer ◽  
Jeroen Bastiaans ◽  
Hesselien Touw ◽  
Richard Kerkman ◽  
Jurian Bronkhof ◽  
...  
Keyword(s):  
Gene Targeting ◽  
Penicillium Chrysogenum ◽  
Highly Efficient ◽  
Efficient Gene

scholarly journals Forced Recycling of an AMA1-Based Genome-Editing Plasmid Allows for Efficient Multiple Gene Deletion/Integration in the Industrial Filamentous Fungus Aspergillus oryzae

2018 ◽  
Vol 85 (3) ◽  
Author(s):  
Takuya Katayama ◽  
Hidetoshi Nakamura ◽  
Yue Zhang ◽  
Arnaud Pascal ◽  
Wataru Fujii ◽  
...  
Keyword(s):  
Secondary Metabolites ◽  
Genetic Engineering ◽  
Genome Editing ◽  
Aspergillus Oryzae ◽  
Filamentous Fungus ◽  
Gene Deletion ◽  
Multiple Gene ◽  
Content Type ◽  
Industrial Strains ◽  
Marker Free

ABSTRACT Filamentous fungi are used for food fermentation and industrial production of recombinant proteins. They also serve as a source of secondary metabolites and are recently expected as hosts for heterologous production of useful secondary metabolites. Multiple-step genetic engineering is required to enhance industrial production involving these fungi, but traditional sequential modification of multiple genes using a limited number of selection markers is laborious. Moreover, efficient genetic engineering techniques for industrial strains have not yet been established. We have previously developed a clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas9-based mutagenesis technique for the industrial filamentous fungus Aspergillus oryzae, enabling mutation efficiency of 10 to 20%. Here, we improved the CRISPR/Cas9 approach by including an AMA1-based autonomously replicating plasmid harboring the drug resistance marker ptrA. By using the improved mutagenesis technique, we successfully modified A. oryzae wild and industrial strains, with a mutation efficiency of 50 to 100%. Conditional expression of the Aoace2 gene from the AMA1-based plasmid severely inhibited fungal growth. This enabled forced recycling of the plasmid, allowing repeated genome editing. Further, double mutant strains were successfully obtained with high efficiency by expressing two guide RNA molecules from the genome-editing plasmid. Cotransformation of fungal cells with the genome-editing plasmid together with a circular donor DNA enabled marker-free multiplex gene deletion/integration in A. oryzae. The presented repeatable marker-free genetic engineering approach for mutagenesis and gene deletion/integration will allow for efficient modification of multiple genes in industrial fungal strains, increasing their applicability. IMPORTANCE Multiple gene modifications of specific fungal strains are required for achieving industrial-scale production of enzymes and secondary metabolites. In the present study, we developed an efficient multiple genetic engineering technique for the filamentous fungus Aspergillus oryzae. The approach is based on a clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas9 system and recycling of an AMA1-based autonomous replicating plasmid. Because the plasmid harbors a drug resistance marker (ptrA), the approach does not require the construction of auxotrophic industrial strains prior to genome editing and allows for forced recycling of the gene-editing plasmid. The established plasmid-recycling technique involves an Aoace2-conditional expression cassette, whose induction severely impairs fungal growth. We used the developed genetic engineering techniques for highly efficient marker-free multiple gene deletion/integration in A. oryzae. The genome-editing approaches established in the present study, which enable unlimited repeatable genetic engineering, will facilitate multiple gene modification of industrially important fungal strains.

scholarly journals Efficient CRISPR/Cas-mediated homologous recombination in the model diatom Thalassiosira pseudonana

2017 ◽  
Author(s):  
Nigel Belshaw ◽  
Irina Grouneva ◽  
Lior Aram ◽  
Assaf Gal ◽  
Amanda Hopes ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Cell Size ◽  
Gene Targeting ◽  
Algal Species ◽  
Thalassiosira Pseudonana ◽  
Cycle Phase ◽  
Metabolic Potential ◽  
Highly Efficient ◽  
Efficient Gene ◽  
Photosynthetic Organism

AbstractCRISPR/Cas enables targeted genome editing in many different plant and algal species including the model diatom Thalassiosira pseudonana. However, efficient gene targeting by homologous recombination (HR) to date is only reported for photosynthetic organisms in their haploid life-cycle phase and there are no examples of efficient nuclease-meditated HR in any photosynthetic organism. Here, a CRISPR/Cas construct, assembled using Golden Gate cloning, enabled highly efficient HR for the first time in a diploid photosynthetic organism. HR was induced in T. pseudonana by means of sequence specific CRISPR/Cas, paired with a donor matrix, generating substitution of the silacidin gene by a resistance cassette (FCP:NAT). Approximately 85% of NAT resistant T. pseudonana colonies screened positive for HR using a nested PCR approach and confirmed by sequencing of the PCR products. The knockout of the silacidin gene in T. pseudonana caused a significant increase in cell size, confirming the role of this gene for cell-size regulation in centric diatoms. Highly efficient gene targeting by HR makes T. pseudonana as genetically tractable as Nannochloropsis and Physcomitrella, hence rapidly advancing functional diatom biology, bionanotechnology and any biotechnological application targeted on harnessing the metabolic potential of diatoms.

scholarly journals Genome Editing Technology and Its Application Potentials in the Industrial Filamentous Fungus Aspergillus oryzae

2021 ◽  
Vol 7 (8) ◽  
pp. 638
Author(s):  
Jun-ichi Maruyama
Keyword(s):  
Genome Editing ◽  
Aspergillus Oryzae ◽  
Filamentous Fungus ◽  
Genetic Manipulation ◽  
Wild Strain ◽  
Soy Sauce ◽  
Transcription Activator ◽  
Gene Modification ◽  
A Genome ◽  
Industrial Strains

Aspergillus oryzae is a filamentous fungus that has been used in traditional Japanese brewing industries, such as the sake, soy sauce, and miso production. In addition, A. oryzae has been used in heterologous protein production, and the fungus has been recently used in biosynthetic research due to its ability to produce a large amount of heterologous natural products by introducing foreign biosynthetic genes. Genetic manipulation, which is important in the functional development of A. oryzae, has mostly been limited to the wild strain RIB40, a genome reference suitable for laboratory analysis. However, there are numerous industrial brewing strains of A. oryzae with various specialized characteristics, and they are used selectively according to the properties required for various purposes such as sake, soy sauce, and miso production. Since the early 2000s, genome editing technologies have been developed; among these technologies, transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) have been applied to gene modification in A. oryzae. Notably, the CRISPR/Cas9 system has dramatically improved the efficiency of gene modification in industrial strains of A. oryzae. In this review, the development of genome editing technology and its application potentials in A. oryzae are summarized.

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