Evaluation of <i>Aspergillus niger</i> Six Constitutive Strong Promoters by Fluorescent-Auxotrophic Selection Coupled with Flow Cytometry: A Case for Citric Acid Production
<i>Aspergillus niger</i> 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 <i>A. niger</i> are l...
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| Format: | Article |
| Language: | English |
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MDPI AG
2022-05-01
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| Series: | Journal of Fungi |
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| Online Access: | https://www.mdpi.com/2309-608X/8/6/568 |
| _version_ | 1797485706587668480 |
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| author | Yudan Lu Xiaomei Zheng Yu Wang Lihui Zhang Lixian Wang Yu Lei Tongcun Zhang Ping Zheng Jibin Sun |
| author_facet | Yudan Lu Xiaomei Zheng Yu Wang Lihui Zhang Lixian Wang Yu Lei Tongcun Zhang Ping Zheng Jibin Sun |
| author_sort | Yudan Lu |
| collection | DOAJ |
| description | <i>Aspergillus niger</i> 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 <i>A. niger</i> are limited. Herein, to efficiently assess promoters, we developed an accurate and intuitive fluorescent-auxotrophic selection workflow based on <i>mCherry</i>, <i>pyrG</i>, CRISPR/Cas9 system, and flow cytometry. With this workflow, we characterized six endogenous constitutive promoters in <i>A. niger</i>. The endogenous glyceraldehyde-3-phosphate dehydrogenase promoter P<i>gpdAg</i> showed a 2.28-fold increase in promoter activity compared with the most frequently used strong promoter P<i>gpdAd</i> from <i>A. nidulans</i>. Six predicted conserved motifs, including the <i>gpdA</i>-box, were verified to be essential for the P<i>gpdAg</i> activity. To demonstrate its application, the promoter P<i>gpdAg</i> was used for enhancing the expression of citrate exporter <i>cexA</i> in a citric acid-producing isolate D353.8. Compared with the <i>cexA</i> controlled by P<i>gpdAd</i>, the transcription level of the <i>cexA</i> gene driven by P<i>gpdAg</i> increased by 2.19-fold, which is consistent with the promoter activity assessment. Moreover, following <i>cexA</i> 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 <i>A. niger</i> promoters and strong constitutive promoters for fungal cell factory construction and strain engineering. |
| first_indexed | 2024-03-09T23:22:44Z |
| format | Article |
| id | doaj.art-af4f7a838ae04e88b4554bb2c01b456c |
| institution | Directory Open Access Journal |
| issn | 2309-608X |
| language | English |
| last_indexed | 2024-03-09T23:22:44Z |
| publishDate | 2022-05-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Journal of Fungi |
| spelling | doaj.art-af4f7a838ae04e88b4554bb2c01b456c2023-11-23T17:24:15ZengMDPI AGJournal of Fungi2309-608X2022-05-018656810.3390/jof8060568Evaluation of <i>Aspergillus niger</i> Six Constitutive Strong Promoters by Fluorescent-Auxotrophic Selection Coupled with Flow Cytometry: A Case for Citric Acid ProductionYudan Lu0Xiaomei Zheng1Yu Wang2Lihui Zhang3Lixian Wang4Yu Lei5Tongcun Zhang6Ping Zheng7Jibin Sun8College of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, ChinaTianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, ChinaTianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, ChinaTianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, ChinaTianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, ChinaTianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, ChinaCollege of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, ChinaTianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, ChinaTianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China<i>Aspergillus niger</i> 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 <i>A. niger</i> are limited. Herein, to efficiently assess promoters, we developed an accurate and intuitive fluorescent-auxotrophic selection workflow based on <i>mCherry</i>, <i>pyrG</i>, CRISPR/Cas9 system, and flow cytometry. With this workflow, we characterized six endogenous constitutive promoters in <i>A. niger</i>. The endogenous glyceraldehyde-3-phosphate dehydrogenase promoter P<i>gpdAg</i> showed a 2.28-fold increase in promoter activity compared with the most frequently used strong promoter P<i>gpdAd</i> from <i>A. nidulans</i>. Six predicted conserved motifs, including the <i>gpdA</i>-box, were verified to be essential for the P<i>gpdAg</i> activity. To demonstrate its application, the promoter P<i>gpdAg</i> was used for enhancing the expression of citrate exporter <i>cexA</i> in a citric acid-producing isolate D353.8. Compared with the <i>cexA</i> controlled by P<i>gpdAd</i>, the transcription level of the <i>cexA</i> gene driven by P<i>gpdAg</i> increased by 2.19-fold, which is consistent with the promoter activity assessment. Moreover, following <i>cexA</i> 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 <i>A. niger</i> promoters and strong constitutive promoters for fungal cell factory construction and strain engineering.https://www.mdpi.com/2309-608X/8/6/568<i>Aspergillus niger</i>promoterfluorescence proteinflow cytometrycitric acidCRISPR/Cas9 |
| spellingShingle | Yudan Lu Xiaomei Zheng Yu Wang Lihui Zhang Lixian Wang Yu Lei Tongcun Zhang Ping Zheng Jibin Sun Evaluation of <i>Aspergillus niger</i> Six Constitutive Strong Promoters by Fluorescent-Auxotrophic Selection Coupled with Flow Cytometry: A Case for Citric Acid Production Journal of Fungi <i>Aspergillus niger</i> promoter fluorescence protein flow cytometry citric acid CRISPR/Cas9 |
| title | Evaluation of <i>Aspergillus niger</i> Six Constitutive Strong Promoters by Fluorescent-Auxotrophic Selection Coupled with Flow Cytometry: A Case for Citric Acid Production |
| title_full | Evaluation of <i>Aspergillus niger</i> Six Constitutive Strong Promoters by Fluorescent-Auxotrophic Selection Coupled with Flow Cytometry: A Case for Citric Acid Production |
| title_fullStr | Evaluation of <i>Aspergillus niger</i> Six Constitutive Strong Promoters by Fluorescent-Auxotrophic Selection Coupled with Flow Cytometry: A Case for Citric Acid Production |
| title_full_unstemmed | Evaluation of <i>Aspergillus niger</i> Six Constitutive Strong Promoters by Fluorescent-Auxotrophic Selection Coupled with Flow Cytometry: A Case for Citric Acid Production |
| title_short | Evaluation of <i>Aspergillus niger</i> Six Constitutive Strong Promoters by Fluorescent-Auxotrophic Selection Coupled with Flow Cytometry: A Case for Citric Acid Production |
| title_sort | evaluation of i aspergillus niger i six constitutive strong promoters by fluorescent auxotrophic selection coupled with flow cytometry a case for citric acid production |
| topic | <i>Aspergillus niger</i> promoter fluorescence protein flow cytometry citric acid CRISPR/Cas9 |
| url | https://www.mdpi.com/2309-608X/8/6/568 |
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