Identification and functional characterization of glycerol dehydrogenase reveal the role in kojic acid synthesis in Aspergillus oryzae

Fan, Junxia; Zhang, Huanxin; Long, Chuannan; He, Bin; Hú, Zhìhóng; Jiang, Chunmiao; Zeng, Bin

doi:10.1007/s11274-020-02912-4

Cited by 22 publications

(7 citation statements)

References 41 publications

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“…The U6 promoter with the target sequence for Aokap4 was amplified using the plasmid pPTRII-Cas9-AoGld3 [ 26 ] as a template with the primers PU6-F and PU6-Aokap4-R, generating the fragment PU6-Aokap4. The U6 terminator and sgRNA sequence were amplified from the vector pPTRII-Cas9-AoGld3 with the primers TU6-Aokap4-F and TU6-R, yielding the fragment Aokap4-TU6.…”

Section: Methodsmentioning

confidence: 99%

A Novel Major Facilitator Superfamily Transporter Gene Aokap4 near the Kojic Acid Gene Cluster Is Involved in Growth and Kojic Acid Production in Aspergillus oryzae

Chen

et al. 2022

JoF

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Kojic acid is an important secondary metabolite of industrial importance produced by Aspergillus oryzae. The kojic acid gene cluster plays an essential role in kojic acid production, and harbors kojA, kojR and kojT. The deletion of kojT, encoding a major facilitator superfamily (MFS) transporter, did not completely abolish kojic acid production, implying that other transporters are required for the transport of kojic acid. The aim of this study is to look for the transporters involved in kojic acid production. Here, Aokap4 encoding a novel MFS transporter was identified, which was adjacent to kojT in the kojic acid gene cluster. The deletion of Aokap4 contributed to the hyphal growth, conidial production and biomass of A. oryzae. Moreover, Aokap4 is required for heat- and cell-wall-stress tolerance but not oxidative and osmotic stress. The disruption of Aokap4 impaired kojic acid production with the reduced expression of kojA, kojR and kojT. Furthermore, when kojT was deleted in the Aokap4-disrupted strain, the yield of kojic acid declined to the same level as that of the kojT-deletion mutant, whereas the production of kojic acid was recovered when kojT was overexpressed in the Aokap4 knockout strain, suggesting that kojT acts downstream of Aokap4. AoKap4 was the second identified MSF transporter involved in kojic acid production after kojT was found a decade ago. This study contributes to a better understanding of the biological roles of the MFS transporter and lays a foundation for future studies on kojic acid synthesis in A. oryzae.

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Section: Methodsmentioning

confidence: 99%

A Novel Major Facilitator Superfamily Transporter Gene Aokap4 near the Kojic Acid Gene Cluster Is Involved in Growth and Kojic Acid Production in Aspergillus oryzae

Chen

et al. 2022

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“…Then, CRISPR/Cas9-based genome editing was used to delete the Aooch1 encoding a key enzyme of hyper-mannosylation process, to assess the recombinant antibody’s capacity to bind to FcγRIIIa [ 90 ]. This genome editing system was used to investigate the functional characterization of glycerol dehydrogenase, revealing that AoGld3, a glycerol dehydrogenase, is involved in the production of the secondary metabolite kojic acid by influencing the expression of kojA (an enzyme gene) and kojR (a transcription factor gene) involved in the kojic acid biosynthesis [ 91 ]. Moreover, using the CRISPR/Cas9 technology, single and double gene disruption of two intracellular triacylglycerol lipases, AoTgla and AoTglb, revealed that disfunction of either AoTgla or AoTglb improved total lipid contents, particularly in the triacylglycerol (TAG) fraction [ 92 ].…”

Section: Development and Application Of Crispr/cas9-based Genome Edit...mentioning

confidence: 99%

CRISPR/Cas9-Based Genome Editing and Its Application in Aspergillus Species

Jin

Wang

et al. 2022

JoF

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Aspergillus, a genus of filamentous fungi, is extensively distributed in nature and plays crucial roles in the decomposition of organic materials as an important environmental microorganism as well as in the traditional fermentation and food processing industries. Furthermore, due to their strong potential to secrete a large variety of hydrolytic enzymes and other natural products by manipulating gene expression and/or introducing new biosynthetic pathways, several Aspergillus species have been widely exploited as microbial cell factories. In recent years, with the development of next-generation genome sequencing technology and genetic engineering methods, the production and utilization of various homo-/heterologous-proteins and natural products in Aspergillus species have been well studied. As a newly developed genome editing technology, the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system has been used to edit and modify genes in Aspergilli. So far, the CRISPR/Cas9-based approach has been widely employed to improve the efficiency of gene modification in the strain type Aspergillus nidulans and other industrially important and pathogenic Aspergillus species, including Aspergillus oryzae, Aspergillus niger, and Aspergillus fumigatus. This review highlights the current development of CRISPR/Cas9-based genome editing technology and its application in basic research and the production of recombination proteins and natural products in the Aspergillus species.

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“…In addition, the CRISPR/Cas9 system was also used to delete AoGld3, which is one of 45 glycerol dehydrogenase genes in A. oryzae, to investigate its effect on the production of the secondary metabolite kojic acid. 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).…”

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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Identification and functional characterization of glycerol dehydrogenase reveal the role in kojic acid synthesis in Aspergillus oryzae

Cited by 22 publications

References 41 publications

A Novel Major Facilitator Superfamily Transporter Gene Aokap4 near the Kojic Acid Gene Cluster Is Involved in Growth and Kojic Acid Production in Aspergillus oryzae

A Novel Major Facilitator Superfamily Transporter Gene Aokap4 near the Kojic Acid Gene Cluster Is Involved in Growth and Kojic Acid Production in Aspergillus oryzae

CRISPR/Cas9-Based Genome Editing and Its Application in Aspergillus Species

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

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