## 3 Production of Plant Secondary Metabolites Using Natural Hosts
### Plant Metabolic Engineering  
The complexity of plant secondary metabolite biosynthetic pathways results in a diverse range of strategies for manipulating metabolic pathways [120], including utilizing gene editing tools, such as Clustered, Regularly Interspaced, Short Palindromic Repeats (CRISPR)/dead CRISPR associated protein 9 (dCAS9), to activate single or multiple-target genes; reducing the expression of genes in competitive pathways through techniques such as RNA interference (RNAi) and CRISPR/CAS9; modifying the expression of transcription factors that control multiple biosynthetic genes; and enhancing the compartmentalization and/or release of the desired product by transporter engineering. Enhancing the activity of key enzymes controlling the biosynthesis of a specific secondary metabolite through genetic engineering can increase the content of the target secondary metabolite in transgenic plants. For example, overexpressing FPP synthase, squalene synthase, or the upstream 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) in the mevalonate pathway (MVA), for example, can increase the production of downstream terpenoid end-products. However, due to the presence of downstream branch pathways, increasing the expression of upstream pathway enzyme genes alone may not achieve the accumulation of the target compound. For instance, overexpressing the first two genes, tabersonine 16-hydroxylase (T16H) and 16-O-methyl transferase (16 OMT), in _C. roseus_ hairy roots failed to produce the target vindoline [121]. Using RNAi to disrupt undesired metabolic branches can redirect synthetic metabolism toward the target. [PERSON] et al. [122] used this technique to silence the gene family of codeinone reductase (COR) in poppies, blocking the synthesis of opioid substances while leading to the significant accumulation of the precursor to codeine, (S)-reticuline. Overexpression limonene synthase while inhibiting the expression of by-product synthetic gene limonene-3-hydroxylase substantially increased the content of limonene and its proportion in the total essential oil (up to 79%) [28]. The activity and expression of these biosynthetic enzymes are regulated by corresponding transcription factors and other regulatory genes, making metabolic engineering of transcription factors potentially highly effective [123]. Currently, transcription factors involved in plant secondary metabolite biosynthetic pathways mainly include WRKY, myeloblasts (MYB), basic helix-loop-helix (bHLH), APETALA2/Ethylene-Responsive-Factor (AP2/ERF), jasmonate-responsive ERF (JRE), basic Leucine Zipper (bZIP), SQUAMOSA promoter-binding protein-like (SPL), and YABBY classes, among others [11; 124]. Takingthe indole alkaloid biosynthetic pathway as an example, the overexpression of ORCA3 together with the addition of the terpene precursor [88] or overexpression of downstream key enzyme genes [89] can increase the final yield of the target compound. The excessive accumulation of secondary metabolites within cells can have two main effects: on one hand, it can increase cellular toxicity and hinder cell growth; on the other hand, it can result in the feedback inhibition of synthetic enzyme activity, reducing overall biosynthesis [125; 126]. Metabolic flux balance analysis indicates that the extracellular secretion and transfer of secondary metabolites are among the key limiting steps for improving yield [127]. Additionally, due to the simplicity of the culture medium, the economic aspects of purifying secondary metabolites through medium separation are favorable. Avoiding cell disruption can further save on cultivation and production costs [128]. Therefore, enhancing the extracellular secretion of secondary metabolites through genetic engineering is of significant importance in plant or microbial biosynthesis research [127; 129]. Two main types of proteins have been identified to be involved in the transport of plant secondary metabolites: ABC (ATP-binding cassette) and MATE (Multidrug and toxic compound extrusion) [130; 131].