Bioscience Methods 2024, Vol.15, No.3, 114-123 http://bioscipublisher.com/index.php/bm 118 4.3 Overexpression and silencing of key genes Overexpression and silencing of key genes are fundamental strategies in metabolic engineering to modulate the biosynthetic pathways of bioactive compounds. Overexpression involves the introduction of additional copies of a gene or the use of strong promoters to increase the expression levels of target genes. Conversely, gene silencing can be achieved through RNA interference (RNAi) or CRISPR interference (CRISPRi), which reduce or eliminate the expression of specific genes. These approaches have been used to enhance the production of various bioactive compounds in plants. For example, the overexpression of genes in the methylerythritol-phosphate (MEP) pathway has been shown to increase the production of β-carotene in Escherichia coli (Li et al., 2015). Similarly, CRISPRi has been employed to temporarily control gene expression without altering the genomic sequence, providing a flexible tool for metabolic pathway regulation (Nishida and Kondo, 2020; Zhao et al., 2020). 4.4 Pathway redirection for enhanced compound production Pathway redirection involves the reconfiguration of metabolic pathways to channel intermediates towards the desired end products. This can be achieved through the overexpression of key enzymes, the knockout of competing pathways, or the introduction of novel biosynthetic routes. CRISPR-based metabolic pathway engineering has been particularly effective in this regard, enabling the simultaneous modulation of multiple genes to optimize pathway flux. For instance, the CRISPR/Cas9-facilitated multiplex pathway optimization (CFPO) technique has been developed to modulate the expression of multiple genes in the xylose utilization pathway of E. coli, resulting in a significant improvement in xylose utilization (Zhu et al., 2017). Additionally, combinatorial metabolic engineering strategies using CRISPR systems have been employed to enhance the production of β-carotene and other valuable compounds in yeast (Lian et al., 2017). These approaches can be adapted for the metabolic engineering of tea to enhance the production of bioactive compounds by redirecting metabolic fluxes towards the desired pathways. 5 Case Study 5.1 Introduction to the case study This case study focuses on a metabolic engineering project aimed at enhancing the production of bioactive compounds in tea (Camellia sinensis). Tea is renowned for its health benefits, primarily due to its rich content of bioactive compounds such as catechins, theaflavins, and caffeine. However, the natural levels of these compounds can vary significantly, and there is a growing interest in optimizing their production through metabolic engineering. 5.2 Description of the metabolic engineering project The project involved the genetic modification of tea plants to either overexpress or silence specific genes involved in the biosynthesis of key bioactive compounds. One approach was to enhance the production of catechins and other flavonoids by targeting genes associated with flavonoid metabolic biosynthesis (Figure 2). This was achieved by leveraging the findings from the tea tree genome, which revealed lineage-specific expansions of these genes (Xia et al., 2017). Another strategy focused on reducing or eliminating caffeine content by manipulating the caffeine biosynthesis pathway. This involved either overexpressing caffeine degradative pathway genes or silencing caffeine biosynthesis pathway genes (Yadav and Ahuja, 2007). 5.3 Results and impact on bioactive compound production The genetic modifications led to significant changes in the levels of bioactive compounds in the engineered tea plants. For instance, the overexpression of flavonoid biosynthesis genes resulted in increased production of catechins, which are crucial for tea flavor and health benefits (Xia et al., 2017). On the other hand, the silencing of caffeine biosynthesis genes successfully reduced caffeine levels, making the tea more suitable for individuals sensitive to caffeine (Yadav and Ahuja, 2007). Additionally, the biotransformation of catechins through enzymatic treatments further enhanced the antioxidant capacity of the tea extracts (Baik et al., 2015). These modifications not only improved the health benefits of the tea but also enhanced its processing suitability and quality (Xia et al., 2017).
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