Production of L-Malic Acid by Metabolically Engineered Aspergillus nidulans Based on Efficient CRISPR–Cas9 and Cre-loxP Systems
Abstract
1. Introduction
2. Materials and Methods
2.1. Strains, Media, and Culture Conditions
2.2. Construction of Plasmids
2.3. Gene Editing
2.4. Western Blotting
2.5. RNA Extraction for qRT–PCR
2.6. Cre-LoxP System
2.7. Quantification of L-Malic Acid
3. Results and Discussion
3.1. Expression of Cas9 in A. nidulans
3.2. Establishment of a CRISPR-Mediated Simultaneous Double-Gene-Editing System in A. nidulans
3.3. The Cre-loxP-Mediated Marker Recycling System for Multiple Gene Editing
3.4. Deleting the Putative Citric Acid Transporter Gene (cexA) and Overexpressing Malate Dehydrogenase Gene (mdhC) in A. nidulans
3.5. Overexpressing a Native Putative C4-dicarboxylate Transporter-DctA in A. nidulans
3.6. A. nidulans Has Good Potential to Produce L-malic Acid
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Strain | Genotype | Source |
---|---|---|
TN02A7/WT | pyrG89; pyroA4; nkuA::argB2; riboB2; veA1 | FGSC |
ZQ01/TN02A7cas9 | pyrG89; pyroA4; nkuA::argB2; riboB2; veA1; pyr4::cas9 | This study |
ZQ02/ΔoahA Pgpd-pyc | ZQ01, ΔoahA::Pgpd-pyc-loxP-Pxylp-Cre-pyroA-loxP | This study |
ZQ03/ΔoahA Ptef-pyc | ZQ01, ΔoahA::Ptef-pyc-loxP-Pxylp-Cre-pyroA-loxP | This study |
ZQ04/ΔoahA ΔpyroA Ptef-pyc | ZQ01, ΔoahA::Ptef-pyc, | This study |
ZQ05/ΔoahA ΔcexA Ptef-pyc Ptef-mdhC | ZQ04, ΔcexA::Ptef-mdhC-loxP-Pxylp-Cre-pyroA-loxP | This study |
ZQ06/ΔoahA ΔcexA ΔpyroA Ptef-pyc Ptef-mdhC | ZQ04, ΔcexA::Ptef-mdhC, | This study |
ZQ07/ΔoahA ΔcexA Ptef-pyc Ptef-mdhC Ptef-dctA | ZQ06, pryoA::Ptef-dctA | This study |
Target Gene | The Target Regions of sgRNA | PAM Sequence |
---|---|---|
oahA | 5′-GGCGGAGTTTGGAGGCAG-3′ | 5′-CGG-3′ |
cexA | 5′-GGACCTAGGATGTGGAAC-3′ | 5′-TGG-3′ |
dctA promoter | 5′- GGGATTCGAAGCTGAGGC-3′ | 5′-AGG-3′ |
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Chen, Z.; Zhang, C.; Pei, L.; Qian, Q.; Lu, L. Production of L-Malic Acid by Metabolically Engineered Aspergillus nidulans Based on Efficient CRISPR–Cas9 and Cre-loxP Systems. J. Fungi 2023, 9, 719. https://doi.org/10.3390/jof9070719
Chen Z, Zhang C, Pei L, Qian Q, Lu L. Production of L-Malic Acid by Metabolically Engineered Aspergillus nidulans Based on Efficient CRISPR–Cas9 and Cre-loxP Systems. Journal of Fungi. 2023; 9(7):719. https://doi.org/10.3390/jof9070719
Chicago/Turabian StyleChen, Ziqing, Chi Zhang, Lingling Pei, Qi Qian, and Ling Lu. 2023. "Production of L-Malic Acid by Metabolically Engineered Aspergillus nidulans Based on Efficient CRISPR–Cas9 and Cre-loxP Systems" Journal of Fungi 9, no. 7: 719. https://doi.org/10.3390/jof9070719
APA StyleChen, Z., Zhang, C., Pei, L., Qian, Q., & Lu, L. (2023). Production of L-Malic Acid by Metabolically Engineered Aspergillus nidulans Based on Efficient CRISPR–Cas9 and Cre-loxP Systems. Journal of Fungi, 9(7), 719. https://doi.org/10.3390/jof9070719