Improved natural food colorant production in the filamentous fungus Monascus ruber using CRISPR-based engineering

Yoon, Hye Ree; Han, Suk Jung; Shin, Seung Chul; Yeom, Su Cheong; Kim, Hyo Jin

doi:10.1016/j.foodres.2023.112651

Cited by 6 publications

(6 citation statements)

References 41 publications

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“…For example, in M. purpureus , one citrinin-free transformant without integrating screening marker has been obtained by knocking out the gene responsible for citrinin biosynthesis and further applied to produce MPs (Liu et al 2022 ). Ree et al ( 2023 ) also applied CGES method to M. ruber for knocking out negative regulatory factors responsible for biosynthesizing MPs, and the yields of MPs were significantly enhanced. All these examples suggested that CGES is feasible for site-specific gene editing in industrial strains of Monascus species.…”

Section: Discussionmentioning

confidence: 99%

“…For instance, CGES combined with donor DNA fragments has been applied to knock out multiple genes in Aspergillus niger , and the GRF of this system was as high as 90% (Leeuwe et al 2019 ). Although CGES has been successfully applied to Monascus purpureus (Liu et al 2022 ) and M. ruber (Ree et al 2023 ), there are more than 10 species recognized internationally in genus Monascus , and they are interspecific diversities with different characteristics (He et al 2018 ), so the GRF of CGES in other Monascus species remains to be verified.…”

Section: Introductionmentioning

confidence: 99%

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CRISPR/Cas9 system is a suitable gene targeting editing tool to filamentous fungus Monascus pilosus

Gong,

Li,

Liu

et al. 2024

Appl Microbiol Biotechnol

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Monascus pilosus has been used to produce lipid-lowering drugs rich in monacolin K (MK) for a long period. Genome mining reveals there are still many potential genes worth to be explored in this fungus. Thereby, efficient genetic manipulation tools will greatly accelerate this progress. In this study, we firstly developed the protocol to prepare protoplasts for recipient of CRISPR/Cas9 system. Subsequently, the vector and donor DNA were co-transformed into recipients (106 protoplasts/mL) to produce 60–80 transformants for one test. Three genes (mpclr4, mpdot1, and mplig4) related to DNA damage response (DDR) were selected to compare the gene replacement frequencies (GRFs) of Agrobacterium tumefaciens-mediated transformation (ATMT) and CRISPR/Cas9 gene editing system (CGES) in M. pilosus MS-1. The results revealed that GRF of CGES was approximately five times greater than that of ATMT, suggesting that CGES was superior to ATMT as a targeting gene editing tool in M. pilosus MS-1. The inactivation of mpclr4 promoted DDR via the non-homologous end-joining (NHEJ) and increased the tolerances to DNA damaging agents. The inactivation of mpdot1 blocked DDR and led to the reduced tolerances to DNA damaging agents. The inactivation of mplig4 mainly blocked the NHEJ pathway and led to obviously reduced tolerances to DNA damaging agents. The submerged fermentation showed that the ability to produce MK in strain Δmpclr4 was improved by 52.6% compared to the wild type. This study provides an idea for more effective exploration of gene functions in Monascus strains. Key points • A protocol of high-quality protoplasts for CGES has been developed in M. pilosus. • The GRF of CGES was about five times that of ATMT in M. pilosus. • The yield of MK for Δmpclr4 was enhanced by 52.6% compared with the wild type.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

CRISPR/Cas9 system is a suitable gene targeting editing tool to filamentous fungus Monascus pilosus

Gong,

Li,

Liu

et al. 2024

Appl Microbiol Biotechnol

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“…It is noteworthy that, as of now, RNA-mediated gene silencing has not been applied to investigate azaphilones BGCs. In the case of CRISPR-Cas9, this methodology was employed in M. ruber to inactivate two negative regulatory genes within the azaphilone BGC, resulting in an enhanced pigment production capacity [55]. To the best of our knowledge, no other examples of CRISPR/Cas9 application for studying azaphilone BGCs have been described.…”

Section: Discussionmentioning

confidence: 99%

Expanding the Toolbox for Genetic Manipulation in Pseudogymnoascus: RNAi-Mediated Silencing and CRISPR/Cas9-Mediated Disruption of a Polyketide Synthase Gene Involved in Red Pigment Production in P. verrucosus

Palma,

Oliva,

Montanares

et al. 2024

JoF

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Fungi belonging to the genus Pseudogymnoascus have garnered increasing attention in recent years. One of the members of the genus, P. destructans, has been identified as the causal agent of a severe bat disease. Simultaneously, the knowledge of Pseudogymnoascus species has expanded, in parallel with the increased availability of genome sequences. Moreover, Pseudogymnoascus exhibits great potential as a producer of specialized metabolites, displaying a diverse array of biological activities. Despite these significant advancements, the genetic landscape of Pseudogymnoascus remains largely unexplored due to the scarcity of suitable molecular tools for genetic manipulation. In this study, we successfully implemented RNAi-mediated gene silencing and CRISPR/Cas9-mediated disruption in Pseudogymnoascus, using an Antarctic strain of Pseudogymnoascus verrucosus as a model. Both methods were applied to target azpA, a gene involved in red pigment biosynthesis. Silencing of the azpA gene to levels of 90% or higher eliminated red pigment production, resulting in transformants exhibiting a white phenotype. On the other hand, the CRISPR/Cas9 system led to a high percentage (73%) of transformants with a one-nucleotide insertion, thereby inactivating azpA and abolishing red pigment production, resulting in a white phenotype. The successful application of RNAi-mediated gene silencing and CRISPR/Cas9-mediated disruption represents a significant advancement in Pseudogymnoascus research, opening avenues for comprehensive functional genetic investigations within this underexplored fungal genus.

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“…However, it is essential to recognize that the research landscape is dynamic and subject to change over time. Recent years have seen rapid developments in gene editing tools and techniques, potentially altering the trajectory of research on Monascus species [21,[42][43][44][45], underscoring the improved understanding and exploration of genetic manipulation in Monascus.…”

Section: Discussionmentioning

confidence: 99%

Effect of Exogenous and Endogenous Ectoine on Monascus Development, Metabolism, and Pigment Stability

Gong,

Shi,

Tang

et al. 2023

Foods

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Monascus, a key player in fermented food production, is known for generating Monascus pigments (MPs) and monacolin K (MK), possessing bioactive properties. However, the limited stability of MPs and mycotoxin citrinin (CTN) constrain the Monascus industry. Extremolytes like ectoine, derived from bacteria, exhibit cytoprotective potential. Here, we investigated the impact of ectoine on Monascus purpureus ATCC 16365, emphasizing development and secondary metabolism. Exogenous 5 mM ectoine supplementation substantially increased the yields of MPs and MK (105%–150%) and reduced CTN production. Ectoine influenced mycelial growth, spore development, and gene expression in Monascus. Remarkably, ectoine biosynthesis was achieved in Monascus, showing comparable effects to exogenous addition. Notably, endogenous ectoine effectively enhanced the stability of MPs under diverse stress conditions. Our findings propose an innovative strategy for augmenting the production and stability of bioactive compounds while reducing CTN levels, advancing the Monascus industry.

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Improved natural food colorant production in the filamentous fungus Monascus ruber using CRISPR-based engineering

Cited by 6 publications

References 41 publications

CRISPR/Cas9 system is a suitable gene targeting editing tool to filamentous fungus Monascus pilosus

CRISPR/Cas9 system is a suitable gene targeting editing tool to filamentous fungus Monascus pilosus

Expanding the Toolbox for Genetic Manipulation in Pseudogymnoascus: RNAi-Mediated Silencing and CRISPR/Cas9-Mediated Disruption of a Polyketide Synthase Gene Involved in Red Pigment Production in P. verrucosus

Effect of Exogenous and Endogenous Ectoine on Monascus Development, Metabolism, and Pigment Stability

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