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Metabolic Engineering of Aspergillus via CRISPR-Based Systems
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Metabolic Engineering of Aspergillus via CRISPR-Based Systems

A special issue of Journal of Fungi (ISSN 2309-608X). This special issue belongs to the section "Fungal Genomics, Genetics and Molecular Biology".

Deadline for manuscript submissions: closed (30 November 2022) | Viewed by 18915

Special Issue Editors

Dr. Peter Stephensen Lübeck

E-Mail Website
Guest Editor
Department of Chemistry and Bioscience, Aalborg University, Aalborg, Denmark
Interests: fungal biotechnology; development of cell factories in Aspergillus fungi producing biochemicals; proteins or peptides using molecular tools as CRISPR-Cas9; biorefinery concepts
Dr. Lei Yang

E-Mail Website
Guest Editor
Department of Chemistry and Bioscience, Aalborg University, Aalborg, Denmark
Interests: microbial biotechnology; development of microbial cell factories for production of chemicals and fuels using synthetic biology approach; exploitation of novel genome editing tools; protein engineering
Dr. Mette Lubeck

E-Mail Website
Guest Editor
Department of Chemistry and Bioscience, Aalborg University, Aalborg, Denmark
Interests: fungal biotechnology; development of cell factories in Aspergillus fungi producing enzymes and biochemicals; biorefinery concepts
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Aspergillus is a widely distributed fungal genus with great medical and commercial influence and with a high degree of metabolic diversity and complexity. Some species are known to cause infections in humans and animals, others produce harmful mycotoxins in food products, and many species are used industrially in food fermentation or for production of enzymes and biochemicals. In recent years, the newly developed CRISPR-Cas9 system has been recognized as a novel potent approach for precise genome editing in Aspergillus, which now powers fundamental research in physiology and genetics of aspergilli and greatly facilitates strain engineering for industrial applications. This Special Issue will collect original research and review papers covering the application of CRISPR-based systems in metabolic engineering of Aspergillus within all areas ranging from fundamental physiology and genetics studies to applied aspects, as well as optimization of the CRISPR-based systems.

Dr. Peter Stephensen Lübeck
Dr. Lei Yang
Dr. Mette Lubeck
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Journal of Fungi is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2600 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • Aspergillus
  • CRISPR
  • genome editing
  • gene targeting
  • metabolic engineering
  • protein engineering
  • cell factories
  • fungal metabolites
  • biochemicals
  • enzymes

Published Papers (6 papers)

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Research

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12 pages, 2208 KiB  
Article
Production of L-Malic Acid by Metabolically Engineered Aspergillus nidulans Based on Efficient CRISPR–Cas9 and Cre-loxP Systems
by Ziqing Chen, Chi Zhang, Lingling Pei, Qi Qian and Ling Lu
J. Fungi 2023, 9(7), 719; https://doi.org/10.3390/jof9070719 - 30 Jun 2023
Cited by 1 | Viewed by 1357
Abstract
Aspergillus nidulans has been more extensively characterized than other Aspergillus species considering its morphology, physiology, metabolic pathways, and genetic regulation. As it has a rapid growth rate accompanied by simple nutritional requirements and a high tolerance to extreme cultural conditions, A. nidulans is [...] Read more.
Aspergillus nidulans has been more extensively characterized than other Aspergillus species considering its morphology, physiology, metabolic pathways, and genetic regulation. As it has a rapid growth rate accompanied by simple nutritional requirements and a high tolerance to extreme cultural conditions, A. nidulans is a promising microbial cell factory to biosynthesize various products in industry. However, it remains unclear for whether it is also a suitable host for synthesizing abundant L-malic acid. In this study, we developed a convenient and efficient double-gene-editing system in A. nidulans strain TN02A7 based on the CRISPR–Cas9 and Cre-loxP systems. Using this gene-editing system, we made a L-malic acid-producing strain, ZQ07, derived from TN02A7, by deleting or overexpressing five genes (encoding Pyc, pyruvate carboxylase; OahA, oxaloacetate acetylhydrolase; MdhC, malate dehydrogenase; DctA, C4-dicarboxylic acid transporter; and CexA, citric acid transporter). The L-malic acid yield in ZQ07 increased to approximately 9.6 times higher (up to 30.7 g/L titer) than that of the original unedited strain TN02A7, in which the production of L-malic acid was originally very low. The findings in this study not only demonstrate that A. nidulans could be used as a potential host for biosynthesizing organic acids, but also provide a highly efficient gene-editing strategy in filamentous fungi. Full article
(This article belongs to the Special Issue Metabolic Engineering of Aspergillus via CRISPR-Based Systems)
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12 pages, 2094 KiB  
Article
CRISPR/Cas9-Mediated Multiplexed Genome Editing in Aspergillus oryzae
by Qinghua Li, Jinchang Lu, Guoqiang Zhang, Jingwen Zhou, Jianghua Li, Guocheng Du and Jian Chen
J. Fungi 2023, 9(1), 109; https://doi.org/10.3390/jof9010109 - 13 Jan 2023
Cited by 8 | Viewed by 3343
Abstract
Aspergillus oryzae has great potential and competitive advantages to be developed as an excellent expression system, owing to its powerful protein secretion ability, complex post-translational modification, and safety characteristics. However, the low efficiency of genetic modification and gene function analysis is an urgent [...] Read more.
Aspergillus oryzae has great potential and competitive advantages to be developed as an excellent expression system, owing to its powerful protein secretion ability, complex post-translational modification, and safety characteristics. However, the low efficiency of genetic modification and gene function analysis is an urgent problem to be solved in A. oryzae and other filamentous fungal systems. Therefore, establishing efficient genetic transformation and multiplexed genome editing tools is significant for developing A. oryzae expression systems, and revealing its intrinsic mechanisms. In this study, the high-efficiency transformation of A. oryzae was achieved by optimizing the preparation conditions of protoplasts, and the random editing efficiency of the CRISPR/Cas9 system in A. oryzae for single and double genes reached 37.6% and 19.8%, respectively. With the aid of the selection marker, such as color or resistance, the editing efficiency of single and double genes can reach 100%. Based on the developed CRISPR/Cas9 genome editing method, the heterologous lipase gene (TLL) achieves precise integration at different genetic loci in one step. The efficient and accurate acquisition of positive transformants indicated that the morphological gene yA could be used as a helpful selection marker for genome editing in A. oryzae. In conclusion, the developed system improves the efficiency of transformation and multiplexed genome editing for A. oryzae. It provides a practical method for developing the A. oryzae high-efficiency expression system for heterologous proteins. Full article
(This article belongs to the Special Issue Metabolic Engineering of Aspergillus via CRISPR-Based Systems)
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13 pages, 3100 KiB  
Article
A Mad7 System for Genetic Engineering of Filamentous Fungi
by Katherina Garcia Vanegas, Jakob Kræmmer Haar Rendsvig, Zofia Dorota Jarczynska, Marcio Vinicius de Carvalho Barros Cortes, Abel Peter van Esch, Martí Morera-Gómez, Fabiano Jares Contesini and Uffe Hasbro Mortensen
J. Fungi 2023, 9(1), 16; https://doi.org/10.3390/jof9010016 - 22 Dec 2022
Cited by 8 | Viewed by 4597
Abstract
The introduction of CRISPR technologies has revolutionized strain engineering in filamentous fungi. However, its use in commercial applications has been hampered by concerns over intellectual property (IP) ownership, and there is a need for implementing Cas nucleases that are not limited by complex [...] Read more.
The introduction of CRISPR technologies has revolutionized strain engineering in filamentous fungi. However, its use in commercial applications has been hampered by concerns over intellectual property (IP) ownership, and there is a need for implementing Cas nucleases that are not limited by complex IP constraints. One promising candidate in this context is the Mad7 enzyme, and we here present a versatile Mad7-CRISPR vector-set that can be efficiently used for the genetic engineering of four different Aspergillus species: Aspergillus nidulans, A. niger, A. oryzae and A. campestris, the latter being a species that has never previously been genetically engineered. We successfully used Mad7 to introduce unspecific as well as specific template-directed mutations including gene disruptions, gene insertions and gene deletions. Moreover, we demonstrate that both single-stranded oligonucleotides and PCR fragments equipped with short and long targeting sequences can be used for efficient marker-free gene editing. Importantly, our CRISPR/Mad7 system was functional in both non-homologous end-joining (NHEJ) proficient and deficient strains. Therefore, the newly implemented CRISPR/Mad7 was efficient to promote gene deletions and integrations using different types of DNA repair in four different Aspergillus species, resulting in the expansion of CRISPR toolboxes in fungal cell factories. Full article
(This article belongs to the Special Issue Metabolic Engineering of Aspergillus via CRISPR-Based Systems)
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14 pages, 2534 KiB  
Article
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
by Tianming Chen, Ziming Chen, Yuzhen Li, Bin Zeng and Zhe Zhang
J. Fungi 2022, 8(8), 885; https://doi.org/10.3390/jof8080885 - 22 Aug 2022
Cited by 6 | Viewed by 1614
Abstract
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 [...] Read more.
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. Full article
(This article belongs to the Special Issue Metabolic Engineering of Aspergillus via CRISPR-Based Systems)
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17 pages, 2716 KiB  
Article
Evaluation of Aspergillus niger Six Constitutive Strong Promoters by Fluorescent-Auxotrophic Selection Coupled with Flow Cytometry: A Case for Citric Acid Production
by Yudan Lu, Xiaomei Zheng, Yu Wang, Lihui Zhang, Lixian Wang, Yu Lei, Tongcun Zhang, Ping Zheng and Jibin Sun
J. Fungi 2022, 8(6), 568; https://doi.org/10.3390/jof8060568 - 26 May 2022
Cited by 13 | Viewed by 2721
Abstract
Aspergillus niger is an important industrial workhorse for the biomanufacturing of organic acids, proteins, etc. Well-controlled genetic regulatory elements, including promoters, are vital for strain engineering, but available strong promoters for A. niger are limited. Herein, to efficiently assess promoters, we developed an [...] Read more.
Aspergillus niger is an important industrial workhorse for the biomanufacturing of organic acids, proteins, etc. Well-controlled genetic regulatory elements, including promoters, are vital for strain engineering, but available strong promoters for A. niger are limited. Herein, to efficiently assess promoters, we developed an accurate and intuitive fluorescent-auxotrophic selection workflow based on mCherry, pyrG, CRISPR/Cas9 system, and flow cytometry. With this workflow, we characterized six endogenous constitutive promoters in A. niger. The endogenous glyceraldehyde-3-phosphate dehydrogenase promoter PgpdAg showed a 2.28-fold increase in promoter activity compared with the most frequently used strong promoter PgpdAd from A. nidulans. Six predicted conserved motifs, including the gpdA-box, were verified to be essential for the PgpdAg activity. To demonstrate its application, the promoter PgpdAg was used for enhancing the expression of citrate exporter cexA in a citric acid-producing isolate D353.8. Compared with the cexA controlled by PgpdAd, the transcription level of the cexA gene driven by PgpdAg increased by 2.19-fold, which is consistent with the promoter activity assessment. Moreover, following cexA overexpression, several genes involved in carbohydrate transport and metabolism were synergically upregulated, resulting in up to a 2.48-fold increase in citric acid titer compared with that of the parent strain. This study provides an intuitive workflow to speed up the quantitative evaluation of A. niger promoters and strong constitutive promoters for fungal cell factory construction and strain engineering. Full article
(This article belongs to the Special Issue Metabolic Engineering of Aspergillus via CRISPR-Based Systems)
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Review

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16 pages, 340 KiB  
Review
CRISPR/Cas9-Based Genome Editing and Its Application in Aspergillus Species
by Feng-Jie Jin, Bao-Teng Wang, Zhen-Dong Wang, Long Jin and Pei Han
J. Fungi 2022, 8(5), 467; https://doi.org/10.3390/jof8050467 - 30 Apr 2022
Cited by 19 | Viewed by 4198
Abstract
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 [...] Read more.
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. Full article
(This article belongs to the Special Issue Metabolic Engineering of Aspergillus via CRISPR-Based Systems)
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