CRISPRi-Mediated Down-Regulation of the Cinnamate-4-Hydroxylase (C4H) Gene Enhances the Flavonoid Biosynthesis in <i>Nicotiana tabacum</i>
Flavonoids are an important class of natural compounds present in plants. However, despite various known biological activities and therapeutic potential, the low abundance of flavonoids in nature limits their development for industrial applications. In this study, we aimed to enhance flavonoid produ...
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MDPI AG
2022-07-01
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| author | Chou Khai Soong Karlson Siti Nurfadhlina Mohd Noor Norzulaani Khalid Boon Chin Tan |
| author_facet | Chou Khai Soong Karlson Siti Nurfadhlina Mohd Noor Norzulaani Khalid Boon Chin Tan |
| author_sort | Chou Khai Soong Karlson |
| collection | DOAJ |
| description | Flavonoids are an important class of natural compounds present in plants. However, despite various known biological activities and therapeutic potential, the low abundance of flavonoids in nature limits their development for industrial applications. In this study, we aimed to enhance flavonoid production by silencing cinnamate-4-hydroxylase (C4H), an enzyme involved in the branch point of the flavonoid biosynthetic pathway, using the clustered regularly interspaced short palindromic repeats interference (CRISPRi) approach. We designed three sgRNAs targeting the promoter region of <i>NtC4H</i> and cloned them into a CRISPRi construct. After being introduced into <i>Nicotiana tabacum</i> cell suspension culture, the transformed cells were sampled for qPCR and liquid chromatography-mass spectrometry analyses. Sixteen of 21 cell lines showed PCR-positive, confirming the presence of the CRISPRi transgene. The <i>Nt</i><i>C4H</i> transcript in the transgenic cells was 0.44-fold lower than in the wild-type. In contrast, the flavonoid-related genes in the other branching pathways, such as <i>Nt</i><i>4CL</i> and <i>Nt</i><i>CHS</i>, in the <i>C4H</i>-silenced cells showed higher expression than wild-type. The upregulation of these genes increased their respective products, including pinostrobin, naringenin, and chlorogenic acid. This study provides valuable insight into the future development of CRISPRi-based metabolic engineering to suppress target genes in plants. |
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| language | English |
| last_indexed | 2024-03-09T10:01:39Z |
| publishDate | 2022-07-01 |
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| spelling | doaj.art-90bbc01242094a53b681fab8469931442023-12-01T23:25:57ZengMDPI AGBiology2079-77372022-07-01118112710.3390/biology11081127CRISPRi-Mediated Down-Regulation of the Cinnamate-4-Hydroxylase (C4H) Gene Enhances the Flavonoid Biosynthesis in <i>Nicotiana tabacum</i>Chou Khai Soong Karlson0Siti Nurfadhlina Mohd Noor1Norzulaani Khalid2Boon Chin Tan3Center for Research in Biotechnology for Agriculture (CEBAR), Universiti Malaya, Kuala Lumpur 50603, MalaysiaInstitute of Microengineering and Nanoelectronics (IMEN), Universiti Kebangsaan Malaysia, Bangi 43600, MalaysiaCenter for Research in Biotechnology for Agriculture (CEBAR), Universiti Malaya, Kuala Lumpur 50603, MalaysiaCenter for Research in Biotechnology for Agriculture (CEBAR), Universiti Malaya, Kuala Lumpur 50603, MalaysiaFlavonoids are an important class of natural compounds present in plants. However, despite various known biological activities and therapeutic potential, the low abundance of flavonoids in nature limits their development for industrial applications. In this study, we aimed to enhance flavonoid production by silencing cinnamate-4-hydroxylase (C4H), an enzyme involved in the branch point of the flavonoid biosynthetic pathway, using the clustered regularly interspaced short palindromic repeats interference (CRISPRi) approach. We designed three sgRNAs targeting the promoter region of <i>NtC4H</i> and cloned them into a CRISPRi construct. After being introduced into <i>Nicotiana tabacum</i> cell suspension culture, the transformed cells were sampled for qPCR and liquid chromatography-mass spectrometry analyses. Sixteen of 21 cell lines showed PCR-positive, confirming the presence of the CRISPRi transgene. The <i>Nt</i><i>C4H</i> transcript in the transgenic cells was 0.44-fold lower than in the wild-type. In contrast, the flavonoid-related genes in the other branching pathways, such as <i>Nt</i><i>4CL</i> and <i>Nt</i><i>CHS</i>, in the <i>C4H</i>-silenced cells showed higher expression than wild-type. The upregulation of these genes increased their respective products, including pinostrobin, naringenin, and chlorogenic acid. This study provides valuable insight into the future development of CRISPRi-based metabolic engineering to suppress target genes in plants.https://www.mdpi.com/2079-7737/11/8/1127metabolic engineeringcell suspension cultureCRISPRiflavonoidsgene silencing |
| spellingShingle | Chou Khai Soong Karlson Siti Nurfadhlina Mohd Noor Norzulaani Khalid Boon Chin Tan CRISPRi-Mediated Down-Regulation of the Cinnamate-4-Hydroxylase (C4H) Gene Enhances the Flavonoid Biosynthesis in <i>Nicotiana tabacum</i> Biology metabolic engineering cell suspension culture CRISPRi flavonoids gene silencing |
| title | CRISPRi-Mediated Down-Regulation of the Cinnamate-4-Hydroxylase (C4H) Gene Enhances the Flavonoid Biosynthesis in <i>Nicotiana tabacum</i> |
| title_full | CRISPRi-Mediated Down-Regulation of the Cinnamate-4-Hydroxylase (C4H) Gene Enhances the Flavonoid Biosynthesis in <i>Nicotiana tabacum</i> |
| title_fullStr | CRISPRi-Mediated Down-Regulation of the Cinnamate-4-Hydroxylase (C4H) Gene Enhances the Flavonoid Biosynthesis in <i>Nicotiana tabacum</i> |
| title_full_unstemmed | CRISPRi-Mediated Down-Regulation of the Cinnamate-4-Hydroxylase (C4H) Gene Enhances the Flavonoid Biosynthesis in <i>Nicotiana tabacum</i> |
| title_short | CRISPRi-Mediated Down-Regulation of the Cinnamate-4-Hydroxylase (C4H) Gene Enhances the Flavonoid Biosynthesis in <i>Nicotiana tabacum</i> |
| title_sort | crispri mediated down regulation of the cinnamate 4 hydroxylase c4h gene enhances the flavonoid biosynthesis in i nicotiana tabacum i |
| topic | metabolic engineering cell suspension culture CRISPRi flavonoids gene silencing |
| url | https://www.mdpi.com/2079-7737/11/8/1127 |
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