CRISPRi-Mediated Down-Regulation of the Cinnamate-4-Hydroxylase (C4H) Gene Enhances the Flavonoid Biosynthesis in Nicotiana tabacum

Abstract

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 NtC4H and cloned them into a CRISPRi construct. After being introduced into Nicotiana tabacum 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 NtC4H 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 Nt4CL and NtCHS, in the C4H-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.

Molecular Cloning, Characterization and Expression Analysis of Cinnamate 4-Hydroxylase (C4H) from Ferula pseudalliacea

“…Additionally, the flavonoid content at different stages in the tea plant Camellia sinensis was correlated with the expression pattern of C4Hb [54]. In addition, downregulation of the C4H gene in Nicotiana tabacum via the use of CRISPRi could increase flavonoid biosynthesis [55]. According to these results, it is hypothesized that the C4H gene is an important element in the biosynthesis of secondary metabolites and that this gene affects the improvement of plant resistance to adverse environmental conditions.…”

Advances in Plant Epigenome Editing Research and Its Application in Plants

“…In addition, these three TRDs, D144, DLS, and MIX, were also successfully verified in monocotyledonous plant wheat to inhibit transcriptional activity and produce an albino phenotype [ 33 ]. In the regulation pathway of secondary metabolites, CRISPRi successfully mediated the downregulation of the Cinnamate-4-Hydroxylase (C4H) gene and enhanced flavonoid biosynthesis in Nicotiana tabacum [ 39 ].…”

Plant-based expression platforms to produce high-value metabolites and proteins

“…The transgenic plants produced a significantly higher hyoscyamine content but without anisodamine and scopolamine alkaloids (Zeng et al, 2021). In another study, Karlson et al (2022) used CRISPR/Cas9 to silence cinnamate-4-hydroxylase (C4H) for enhanced flavonoid production into N. tabacum cell suspension. C4Hsilenced cells produced a significantly higher concentration of cinnamic acid, chlorogenic acid, pinostrobin, and naringenin than wild-type cells (Karlson et al, 2022).…”

“…The transgenic plants produced a significantly higher hyoscyamine content but without anisodamine and scopolamine alkaloids ( Zeng et al., 2021 ). In another study, Karlson et al. (2022) used CRISPR/Cas9 to silence cinnamate-4-hydroxylase (C4H) for enhanced flavonoid production into N. tabacum cell suspension.…”

“…(2022) used CRISPR/Cas9 to silence cinnamate-4-hydroxylase (C4H) for enhanced flavonoid production into N. tabacum cell suspension. C4H-silenced cells produced a significantly higher concentration of cinnamic acid, chlorogenic acid, pinostrobin, and naringenin than wild-type cells ( Karlson et al., 2022 ).…”