Construction of single, double, or triple mutants within kojic acid synthesis genes kojA, kojR, and kojT by the CRISPR/Cas9 tool in Aspergillus oryzae

Li, Yuzhen; Zhang, Huanxin; Chen, Ziming; Fan, Junxia; Chen, YangQuan; Zeng, Bin

doi:10.1007/s12223-022-00949-6

Cited by 8 publications

(5 citation statements)

References 25 publications

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“…The AMA plasmid can easily be recycled after several rounds of subculturing under nonselective conditions, allowing the reuse of the dominant selection marker(s) during transformation [ 94 ]. CRISPR/Cas9-based approaches involving an autonomously replicating AMA1-plasmid have been successfully established for the editing of single or multiple genes in industrial strains of Aspergillus niger [ 95 ], the edible fungus Cordyceps militaris [ 96 ], A. terreus [ 97 ], and A. oryzae [ 98 ]. For instance, an AMA1-based CRISPR/Cas9 genome-editing system was used in Paecilomyces variotii and Penicillium roquefortii for creating melanin-deficient strains by knocking out the associated PKS genes and investigating the effect of melanin on the heat and UV-C radiation resistance of conidia from these food-associated fungi [ 99 ].…”

Section: Application Of Crispr/cas Systems In Fungal Genetic Engineeringmentioning

confidence: 99%

Advances and Challenges in CRISPR/Cas-Based Fungal Genome Engineering for Secondary Metabolite Production: A Review

Wang

Jin

et al. 2023

JoF

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Fungi represent an important source of bioactive secondary metabolites (SMs), which have wide applications in many fields, including medicine, agriculture, human health, and many other industries. The genes involved in SM biosynthesis are usually clustered adjacent to each other into a region known as a biosynthetic gene cluster (BGC). The recent advent of a diversity of genetic and genomic technologies has facilitated the identification of many cryptic or uncharacterized BGCs and their associated SMs. However, there are still many challenges that hamper the broader exploration of industrially important secondary metabolites. The recent advanced CRISPR/Cas system has revolutionized fungal genetic engineering and enabled the discovery of novel bioactive compounds. In this review, we firstly introduce fungal BGCs and their relationships with associated SMs, followed by a brief summary of the conventional strategies for fungal genetic engineering. Next, we introduce a range of state-of-the-art CRISPR/Cas-based tools that have been developed and review recent applications of these methods in fungi for research on the biosynthesis of SMs. Finally, the challenges and limitations of these CRISPR/Cas-based systems are discussed and directions for future research are proposed in order to expand their applications and improve efficiency for fungal genetic engineering.

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Section: Application Of Crispr/cas Systems In Fungal Genetic Engineeringmentioning

confidence: 99%

Advances and Challenges in CRISPR/Cas-Based Fungal Genome Engineering for Secondary Metabolite Production: A Review

Wang

Jin

et al. 2023

JoF

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“…oryzae houses kojR (AO090113000137), which encodes a Zn(II) 2 Cys 6 transcriptional activator that is integral to KA biosynthesis [5,6]. kojR deletion significantly downregulates KA synthesis [6,7]. kojR is located between kojA (AO090113000136) and kojT (AO090113000138) and plays a pivotal role in regulating their expression [5][6][7].…”

Section: Introductionmentioning

confidence: 99%

“…kojR deletion significantly downregulates KA synthesis [6,7]. kojR is located between kojA (AO090113000136) and kojT (AO090113000138) and plays a pivotal role in regulating their expression [5][6][7]. Chang et al identified an 11-bp palindromic sequence within the kojA and kojT promoter regions in 23 Aspergillus species including A. oryzae and A. flavus [8].…”

Section: Introductionmentioning

confidence: 99%

The Identification of a Target Gene of the Transcription Factor KojR and Elucidation of Its Role in Carbon Metabolism for Kojic Acid Biosynthesis in Aspergillus oryzae

Mizutani,

Oka,

Goto

et al. 2024

JoF

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DNA-binding transcription factors are broadly characterized as proteins that bind to specific sequences within genomic DNA and modulate the expression of downstream genes. This study focused on KojR, a transcription factor involved in the metabolism of kojic acid, which is an organic acid synthesized in Aspergillus oryzae and is known for its tyrosinase-inhibitory properties. However, the regulatory mechanism underlying KojR-mediated kojic acid synthesis remains unclear. Hence, we aimed to obtain a comprehensive identification of KojR-associated genes using genomic systematic evolution of ligands by exponential enrichment with high-throughput DNA sequencing (gSELEX-Seq) and RNA-Seq. During the genome-wide exploration of KojR-binding sites via gSELEX-Seq and identification of KojR-dependent differentially expressed genes (DEGs) using RNA-Seq, we confirmed that KojR preferentially binds to 5′-CGGCTAATGCGG-3′, and KojR directly regulates kojT, as was previously reported. We also observed that kojA expression, which may be controlled by KojR, was significantly reduced in a ΔkojR strain. Notably, no binding of KojR to the kojA promoter region was detected. Furthermore, certain KojR-dependent DEGs identified in the present study were associated with enzymes implicated in the carbon metabolic pathway of A. oryzae. This strongly indicates that KojR plays a central role in carbon metabolism in A. oryzae.

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“…A. oryzae houses kojR (AO090113000137), which encodes a Zn(II)2Cys6 transcriptional activator that is integral to the KA biosynthesis [5,6]. kojR deletion significantly downregulates KA synthesis [6,7]. kojR is located between kojA (AO090113000136) and kojT (AO090113000138) and plays a pivotal role in regulating their expression [5][6][7].…”

Section: Introductionmentioning

confidence: 99%

Elucidating the role of KojR in transcriptional regulation of carbon metabolism for kojic acid biosynthesis in Aspergillus oryzae

Mizutani,

Oka,

Goto

et al. 2024

Preprint

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DNA-binding transcription factors are broadly characterized as proteins that bind to specific sequences within genomic DNA and modulate the expression of downstream genes. This study focused on KojR—a transcription factor involved in the metabolism of kojic acid, which is an organic acid synthesized in Aspergillus oryzae and is known for its tyrosinase-inhibitory properties. However, the regulatory mechanism underlying KojR-mediated kojic acid synthesis in remains unclear. Hence, we aimed to obtain a comprehensive identification of KojR-associated genes using genomic Systematic Evolution of Ligands by Exponential enrichment (gSELEX-Seq) and RNA-Seq. During the genome-wide exploration of KojR-binding sites via gSELEX-Seq and identification of KojR-dependent differentially expressed genes (DEGs) using RNA-Seq, we confirmed that KojR directly regulates kojT, as was previously reported. We also observed that kojA expression, which may be controlled by KojR, was significantly reduced in a ΔkojR strain. Notably, no binding of KojR to the kojA promoter region was detected. Furthermore, certain KojR-dependent DEGs identified in the present study were associated with enzymes implicated in the carbon metabolic pathway of A. oryzae. This strongly indicates that KojR plays a central role in carbon metabolism in A. oryzae.

show abstract

Construction of single, double, or triple mutants within kojic acid synthesis genes kojA, kojR, and kojT by the CRISPR/Cas9 tool in Aspergillus oryzae

Cited by 8 publications

References 25 publications

Advances and Challenges in CRISPR/Cas-Based Fungal Genome Engineering for Secondary Metabolite Production: A Review

Advances and Challenges in CRISPR/Cas-Based Fungal Genome Engineering for Secondary Metabolite Production: A Review

The Identification of a Target Gene of the Transcription Factor KojR and Elucidation of Its Role in Carbon Metabolism for Kojic Acid Biosynthesis in Aspergillus oryzae

Elucidating the role of KojR in transcriptional regulation of carbon metabolism for kojic acid biosynthesis in Aspergillus oryzae

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