Detailed analysis of targeted gene mutations caused by the Platinum-Fungal TALENs in Aspergillus oryzae RIB40 strain and a ligD disruptant

Mizutani, Osamu; Arazoe, Takayuki; Toshida, Kenji; Hayashi, Risa; Ohsato, Shuichi; Sakuma, Tetsushi; Yamamoto, Takashi; Kuwata, Shigeru

doi:10.1016/j.jbiosc.2016.09.014

Cited by 28 publications

(12 citation statements)

References 44 publications

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“…A potato plant cultivar, Sassy, was cultured in vitro on Murashige and Skoog (1962) medium, without phytohormones (pH 5.7) or grown in soil, with a photoperiod of 16 h at 23°C.…”

Section: Plant Materials and Bacterial Strainmentioning

confidence: 99%

“…Highly active TALENs with variable TALE repeats harboring non-repeat-variable di-residue (non-RVD) variations called Platinum TALENs have been developed (Sakuma et al 2013a). Platinum TALENs have been applied to introduce targeted mutagenesis in cultured human cell lines (Sakuma et al 2013a), frogs (Sakane et al 2014;Sakuma et al 2013a), rats (Sakuma et al 2013a), fungi (Mizutani et al 2016), and plant tissues (Kusano et al 2016). However, there have been no reports regarding the application of Platinum TALEN for genome editing in whole regenerated plants.…”

Section: Introductionmentioning

confidence: 99%

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Efficient genome engineering using Platinum TALEN in potato

Yasumoto

Umemoto

Lee

et al. 2019

Plant Biotechnology

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Potato (Solanum tuberosum) is one of the most important crops in the world. However, it is generally difficult to breed a new variety of potato crops because they are highly heterozygous tetraploid. Steroidal glycoalkaloids (SGAs) such as α-solanine and α-chaconine found in potato are antinutritional specialized metabolites. Because of their toxicity following intake, controlling the SGA levels in potato varieties is critical in breeding programs. Recently, genome-editing technologies using artificial site-specific nucleases such as TALEN and CRISPR-Cas9 have been developed and used in plant sciences. In the present study, we developed a highly active Platinum TALEN expression vector construction system, and applied to reduce the SGA contents in potato. Using Agrobacterium-mediated transformation, we obtained three independent transgenic potatoes harboring the TALEN expression cassette targeting SSR2 gene, which encodes a key enzyme for SGA biosynthesis. Sequencing analysis of the target sequence indicated that all the transformants could be SSR2-knockout mutants. Reduced SGA phenotype in the mutants was confirmed by metabolic analysis using LC-MS. In vitro grown SSR2knockout mutants exhibited no differences in morphological phenotype or yields when compared with control plants, indicating that the genome editing of SGA biosynthetic genes such as SSR2 could be a suitable strategy for controlling the levels of toxic metabolites in potato. Our simple and powerful plant genome-editing system, developed in the present study, provides an important step for future study in plant science.

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“…A potato plant cultivar, Sassy, was cultured in vitro on Murashige and Skoog (1962) medium, without phytohormones (pH 5.7) or grown in soil, with a photoperiod of 16 h at 23°C.…”

Section: Plant Materials and Bacterial Strainmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Efficient genome engineering using Platinum TALEN in potato

Yasumoto

Umemoto

Lee

et al. 2019

Plant Biotechnology

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“…Targeted gene inactivation can correlate a BGC with a specific NP or halt the biosynthesis of a pathway at a specific point, revealing intermediate metabolites not yet characterized. Traditionally, targeted gene disruption in fungi has been difficult to achieve due to the high rates of nonhomologous end joining (NHEJ); however, several methods have been developed to provide more efficient gene-targeting options [24][25][26]. Using the Cre/loxP marker recycling system combined with heterologous gene expression in Ustilaginoidea virens, the first fungal ribosomally synthesized and post-translationally modified peptide (RiPP) BGC, encoding ustiloxin B, was elucidated [27,28].…”

Section: Pathway Engineeringmentioning

confidence: 99%

Strategies for Engineering Natural Product Biosynthesis in Fungi

Skellam

2019

Trends in Biotechnology

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Fungi are a prolific source of bioactive compounds, some of which have been developed as essential medicines and life-enhancing drugs. Genome sequencing has revealed that fungi have the potential to produce considerably more natural products (NPs) than are typically observed in the laboratory. Recently, there have been significant advances in the identification, understanding, and engineering of fungal biosynthetic gene clusters (BGCs). This review briefly describes examples of the engineering of fungal NP biosynthesis at the global, pathway, and enzyme level using in vivo and in vitro approaches and refers to the range and scale of heterologous expression systems available, developments in combinatorial biosynthesis, progress in understanding how fungal BGCs are regulated, and the applications of these novel biosynthetic enzymes as biocatalysts. Fungal NPs and Human Health NPs (see Glossary) or NP derivatives represented 25% of all FDA-approved drugs in the years spanning 1981 to 2014 [1]. Fungi are prolific producers of some of these important drugs, such as antibiotics (e.g., penicillin, pleuromutilin), cholesterol-lowering drugs (e.g., lovastatin, compactin), and immunosuppressants (e.g., mycophenolic acid, cyclosporine). Fungal NPs have many biological activities, ranging from carcinogens (e.g., aflatoxins), deadly toxins (e.g., a-amanitin), and industrial fungicides (e.g., strobilurin) to food colorants (e.g., azaphilones), hormones (e.g., gibberellin), and psychedelics (e.g., psilocybin) [2,3]. In nature, these NPs serve to protect fungi from predators (e.g., insects), UV radiation, and competition from other microorganisms and have evolved to secure their survival in niche habitats; they just happen to have had a profound effect on human health. The broad spectrum of biological activities reflects their structural diversity, which arises from their biosynthetic classification (e.g., polyketides, peptides, terpenes, alkaloids) (Figure 1). The complexity of fungal secondary metabolism presents numerous challenges for traditional drug discovery. Fungi often produce NPs at very low levels or in response to specific environmental cues that are difficult to reproduce in the laboratory [4]. The prominence of NPs in the pharmaceutical industry and re-emerging interest in NPs as lead structures in drug discovery [5] require methods to engineer improved NP analogs and activate silent biosynthetic pathways. In recent years there have been substantial advances in strategies for engineering fungal NP biosynthesis (Figure 2, Key Figure), which are highlighted in this review. These strategies can be divided into three broad categories: (i) inducing global transcriptional perturbations (e.g., through epigenetic modifications or the overexpression of global transcriptional regulators), which effectively 'awakens' the biosynthetic machinery and often affects multiple pathways resulting in the production of a variety of NPs, but is unpredictable and difficult to control Highlights

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“…The result showed that the deletion of AoGld3 resulted in the inhibition of kojic acid production by affecting the expression of kojA and kojR, which encode an enzyme and a transcription factor involved in the kojic acid biosynthesis (Fan et al, 2020). In addition, a platinumfungal TALEN (Transcription activator-like effector nucleases) -based genome editing technique has also been developed for targeted gene mutations in A. oryzae (Mizutani et al, 2017). The platinum-fungal TALEN technique resulted in various different mutation patterns with almost half of deletions larger than 1 kb in the target region.…”

Section: Crispr/cas9-based Genome Engineeringmentioning

confidence: 99%

Advances in Genetic Engineering Technology and Its Application in the Industrial Fungus Aspergillus oryzae

Jin

Wang

et al. 2021

Front. Microbiol.

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The filamentous fungus Aspergillus oryzae is an important strain in the traditional fermentation and food processing industries and is often used in the production of soy sauce, soybean paste, and liquor-making. In addition, A. oryzae has a strong capacity to secrete large amounts of hydrolytic enzymes; therefore, it has also been used in the enzyme industry as a cell factory for the production of numerous native and heterologous enzymes. However, the production and secretion of foreign proteins by A. oryzae are often limited by numerous bottlenecks that occur during transcription, translation, protein folding, translocation, degradation, transport, secretion, etc. The existence of these problems makes it difficult to achieve the desired target in the production of foreign proteins by A. oryzae. In recent years, with the decipherment of the whole genome sequence, basic research and genetic engineering technologies related to the production and utilization of A. oryzae have been well developed, such as the improvement of homologous recombination efficiency, application of selectable marker genes, development of large chromosome deletion technology, utilization of hyphal fusion techniques, and application of CRISPR/Cas9 genome editing systems. The development and establishment of these genetic engineering technologies provided a great deal of technical support for the industrial production and application of A. oryzae. This paper reviews the advances in basic research and genetic engineering technologies of the fermentation strain A. oryzae mentioned above to open up more effective ways and research space for the breeding of A. oryzae production strains in the future.

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Detailed analysis of targeted gene mutations caused by the Platinum-Fungal TALENs in Aspergillus oryzae RIB40 strain and a ligD disruptant

Cited by 28 publications

References 44 publications

Efficient genome engineering using Platinum TALEN in potato

Efficient genome engineering using Platinum TALEN in potato

Strategies for Engineering Natural Product Biosynthesis in Fungi

Advances in Genetic Engineering Technology and Its Application in the Industrial Fungus Aspergillus oryzae

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