## 2 Botanical Biopesticides
### Botanical Biopsticides and Synthetic Biology
#### 2.2.1 Selection and Modification of Highly Adaptable Chassis  
The synthetic processes of botanical natural products are usually complex. To achieve their efficient synthesis in microbial chassis, the selection of the chassis is particularly important and should consider ease of handling, ease of cultivation, and adaptation to exogenous enzymes and products. _Saccharomyces cerevisiae_ is a suitable chassis for the expression of natural products of plant origin. Using brewer's yeast to express some pathways that require transmembrane proteins eliminates the need to modify transmembrane proteins. Moreover, these proteins contain microstructures such as mitochondria that mimic the subcellular localization of plant natural product synthesis [45]. For example, botanical P450 is a transmembrane protein that requires suitable membrane structures, such as the endoplasmic reticulum, for proper localization and folding. [PERSON] et al. fused the key capsaicin gene sesquiterpene synthase EAS with cytochrome P450 EAH for _capsaicin_ and expressed the gene in yeast (EPY300) [66]. Since acidification during fermentation alters the structure of the product, the heterologous expression of _capsaicin_ was successfully achieved (250 mg/mL) after adding the buffer HEPES to adjust the pH to 7.0 during the configuration of the medium YPA [67]. [PERSON] et al. heterologously expressed the oxidized squalene cyclase (OSC) genes AiOSC1 and MaOSC1 from _azadirachta indica_ in the mevalonate pathway (MVA) optimized _S cerevisiae_ and co-integrated AiOSC1 with AtAQS2 and PgtHMGR into the _delta_ locus of the _S cerevisiae_ chromosome to construct a recombinant strain with a high yield of the azadirachtin precursor substance irtucalla-7,24-dien-3\(\beta\)-ol, thereby providing \begin{table}
\begin{tabular}{c c c c} \hline
**Item** & **Purpose** & **Technique** & **Application** \\ \hline \multirow{3}{*}{Recombinant DNA Technique} & Gene testing and analysis; & PCR & Eg. Limonene biosynthesis: \\  & Site-directed mutagenesis & Gibson assembly & synthetic gene screening [64] \\ \hline \multirow{7}{*}{Modular Metabolic Engineering} & \multirow{7}{*}{Optimize metabolic pathways} & \multirow{7}{*}{Modular metabolic engineering} & Eg. Pyrethrin biosynthesis: \\  & & & Reconstructed the \\  & & & chrysanthine acid \\  & & & biosynthetic pathway in \\  & & & tomato fruit [62]; Rhodopsin \\  & & & biosynthesis: co-fermentation \\  & & & of C. tigrinum with strain T-34, \\  & & & optimizing the fermentation \\  & & & and extraction conditions [50] \\ \hline \multirow{7}{*}{Genome Editing} & \multirow{7}{*}{Edit Genes} & Zinc-finger nuclease, ZFN & \multirow{7}{*}{Eg. Pyrethrin biosynthesis: \\  & & Transcription activator-like & \\  & & effector nuclease, TALEN & \\  & & Clustered regulatory & overexpressed the gene \\  & & interspaced short palindence & encoding the synthase of \\  & & repeat, CRISPR & \\  & & multiplex automated genome & \\  & & engineering, & \\  & & MAGE/conjugative assembly & \\  & & genome engineering, CAGE & \\ \hline \end{tabular}
\end{table} 
Table 3: Synthetic biotechniques in the biosynthesis of botanical biopesticides. a large number of substrates for the identification of important genes in the _azadirachttin_ biosynthetic pathway [57]. Lipolytic yeast is another suitable chassis. [PERSON] et al. applied four different metabolic engineering strategies using lipolytic yeast to increase the formation of an acetylcholinesterase enzyme A precursor, an important precursor substance for the production of natural products such as terpenoids [68]. It was found that the enhancement of pyruvate branched pathways (pyruvate decarboxylase PDC, acetaldehyde dehydrogenase ALD, and acetyl-coenzyme A synthase ACS) and \(\beta\)-oxidation pathways yielded the most significant increase in cytoplasmic acetyl coenzyme A synthesis, while the synthesis of triacetate lactone (TAL) was promoted by the overexpression of the above pathways, resulting in a TAL yield of 35.9 g [68]. [PERSON] et al.