Bioscience Methods 2024, Vol.15, No.3, 114-123 http://bioscipublisher.com/index.php/bm 117 Figure 1 Functional analysis of key genes in theanine biosynthesis (Adapted from Wei et al., 2018) Image Caption: A: Theanine biosynthesis pathway, involving key genes such as glutamine synthetase (GS), glutamate synthase (GOGAT), glutamate dehydrogenase (GDH), arginine decarboxylase (ADC), and theanine synthetase (TS); B: A phylogenetic tree was constructed by comparing glutamine synthetase genes known in other organisms; C: The theanine synthesis activity of CsTSI was detected by overexpressing the tea plant TS gene (CsTSI) in Arabidopsis, with or without feeding ethylamine (Adapted from Wei et al., 2018) Wei et al. (2018) enhanced the understanding of amino acid metabolism in tea plants by revealing the key pathways and genes involved in the biosynthesis of theanine. The experimental results indicated that the TS candidate gene (CS) in tea plants shows high similarity to known GSI-type genes, and the CsTSI gene plays a crucial role in theanine synthesis. Moreover, treatment with ethylamine significantly increased theanine content. This provides an important molecular foundation for future genetic engineering efforts to improve tea quality. 4.2 CRISPR-Cas9 and genome editing approaches The advent of CRISPR-Cas9 technology has revolutionized the field of genetic engineering, providing a more precise and efficient method for genome editing. CRISPR-Cas9 allows for targeted modifications at specific genomic loci, enabling the knockout, knock-in, or alteration of genes with high precision. This technology has been successfully applied in various organisms, including plants, to enhance the production of bioactive compounds. For instance, CRISPR-Cas9 has been used to knock out genes involved in the biosynthesis of benzylisoquinoline alkaloids in opium poppy, resulting in altered alkaloid profiles (Alagoz et al., 2016). Additionally, CRISPR-based tools have been employed for multiplex pathway modifications and transcriptional regulations, allowing for comprehensive metabolic engineering (Jakočiūnas et al., 2017; Nishida and Kondo, 2020). The versatility and efficiency of CRISPR-Cas9 make it an ideal tool for metabolic engineering in tea to enhance the production of valuable bioactive compounds.
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