## 4 Production of Plant Secondary Metabolites Using Microbial Chassis
### Pathway Establishment and Optimization  
Microbial metabolic networks have developed tight regulation to maintain metabolic homeostasis, and introducing exogenous synthetic pathways may lead to disruptions in endogenous metabolism, increase cellular metabolic load, and the introduction of new substances that are toxic to cells. Therefore, it is essential to carefully design synthetic pathways to balance the overall metabolic flux and cofactor levels [219]. Before constructing a pathway, it is important to consider the form in which exogenous DNA exists in microorganisms. While integrating exogenous DNA into the genome is ideal due to the instability of plasmids and variations in copy number, many laboratory-scale studies still use plasmids due to their convenience. Traditional homologous recombination systems using counter-selection markers are time-consuming, so site-specific recombination and transposon-mediated random insertion are often used. The former is widely used in yeast, utilizing sites such as nutrient and amino acid synthesis gene loci, delta sites, and rDNA sites. The CRISPR/Cas system can induce site-specific breaks, allowing for knockouts, in-del mutations, and the insertion of exogenous DNA. Coupling specific sites with CRISPR/Cas9 can enable a higher insertion copy number, potentially increasing production further [220]. Manipulating individual or combinations of genes remains labor-intensive and iterative. Each change that leads to a change in phenotype needs to be considered in the next step. The ideal approach involves multiplex genome engineering, such as oligo-based, recombinase-based, and CRISPR-based methods, along with biosensors and high-throughput screening tools [221]. However, this also increases uncertainty and operational complexity, so it is not widely applied in the successful cases we have investigated. Similar to plant metabolic engineering, microbial strategies mainly include the regulation of pathway enzyme gene expression, inhibition of branches, and control of transport. The expression levels of pathway enzyme genes are one of the crucial factors influencing product synthesis. Many studies choose to overexpress the enzyme genes responsible for precursor supply pathways to increase the capacity of precursor pools. For example, to increase upstream products of terpenoids, IDII and HMGR are often overexpressed [159; 160]. However, it is worth noting that, during the early stages of cultivation, the overaccumulation of precursors or end products may have adverse effects on cell growth, such as precursors of terpenoids and polyphenols [160; 222]. At the same time, the early expression of exogenous pathways can slow down cell growth due to increased metabolic burden. In such cases, the dynamic control of gene expression can be chosen. In the study of [PERSON] et al. [222], the expression of genes involved in the MVA pathway and squalene synthesis pathway initially remained at normal levels. It was only after the cells reached high density that these genes were overexpressed, achieving a form of sequential control. Dynamic control is often achieved through condition- or inducible-type promoters that regulate gene expression, such as GAL promoters [223] or hexose transporter promoters [162]. Dealing with nonlinear diverging-converging metabolic pathways often involves a more precise control of the supply of two or more precursors, and adjusting the metabolic flux in one branch may affect the others. This requires laborious work to optimize gene copy numbers, expression levels, ribosome binding sites, and other factors. Separating diverging pathways and building them separately in different strains can reduce metabolic stress, create distinct cellular environments, and minimize interference from unwanted pathway modules. For example, the synthesis of rosmarinic acid was divided into three _E. coli_ strains to provide two precursors and downstream pathways.