## 2 Botanical Biopesticides
### Botanical Biopsticides and Synthetic Biology
Secondly, two molecules of IPP and one molecule of DMAPP are catalyzed by farnesyl pyrophosphatases (FPPs) to synthesize FPP, which is then cyclized into the sesquiterpene skeleton via the action of sesquiterpene synthases. Finally, the structurally diverse sesquiterpenes are formed via the post-modification of P450 enzymes, BAHD acyltransferases [57]. Through transcriptome sequencing and differential gene expression analysis, our lab identified 15 sesquiterpene synthase genes, 8 P450 genes, and 4 BAHD acyltransferase genes that may be involved in the _celangulin_ biosynthetic pathway. Through bioinformatic mining, a functional information dataset was established. The function of CaTPS2 was verified in vitro by recombinant protein, which was involved in the _celangulin_ biosynthesis pathway and could catalyze the formation of \(\gamma\)-eudsemol [57]. Then, we integrated the CaTPS2 in the yeast chromosome multicopy locus, and the production of \(\gamma\)-eudsemol with an approximately 4.6-fold enhancement was obtained [57]. This laid the foundation for the development of synthetic biology in botanical pesticides. Currently, synthetic biotechniques are widely used in the synthesis of botanical biopesticides. In general, these techniques include the recombinant DNA technique, modular metabolic engineering, and genome editing. The recombinant DNA technique mainly includes the use of PCR and a Gibson assembly, which are widely employed in the synthesis of _pyrethroids_[58]_physicon_[59], and _eugenol_[60]. This technique can detect and analyze genes and targeted mutations. Genome editing includes zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), clustered regulatory inter-spaced short palindromic repeats (CRISPR), and multiplex automated genome engineering (MAGE)/conjugative assembly genome engineering (CAGE) [61]. Table 3 shows the application of synthetic biotechniques in the biosynthesis of botanical biopesticides. Using _pyrethrin_ biosynthesis as an example. [PERSON] et al. overexpressed the gene encoding the synthase of _pyrethroid_ acid ligands in _Chrysanthemum morifolium_ and detected the release of volatile chrysanthemyl alcohol and chrysanthemyl-6-O-malonyl-\(\beta\)-D glucopyranoside accumulation, reaching 47 pmol/(h\(\cdot\)g) of chrysanthemyl alcohol and 1.1 mmol/L of chrysanthemic acid [53]. [PERSON] et al. reconstructed the chrysanthemic acid biosynthetic pathway in _Solanum lycopersicum_, overexpressing the TcCDS gene from _pyrethrum_ and the ADH gene from wild tomato with ALDH, and reaching 67.1 \(\upmu\)g/g FW of chrysanthemic acid, and 97% of the transferred DMAPP was converted [54]. [PERSON] et al. reconstituted the synthetic pathway of pyrethric acid in a tobacco transient expression system, and the total yield of pyrethric acid reached (24.0 \(\pm\) 2.7) \(\upmu\)g/g [62]. Significant progress has also been made in the biosynthesis of rhodopsin. [PERSON] et al. found that co-fermentation of _C. tigrinum_ with a \(\beta\)-glucosidase producing _Rrizobium_ strain T-34 was able to hydrolyze the glycosidic bond of rhodopsin \(\beta\)-glucoside in C. tigrinum, and after optimizing the fermentation and extraction conditions, the yield of _emodin_ could be increased to 2.25% [50]. [PERSON] found that _Aspergillus ochracous_ can produce large amounts of rhodopsin, and the content can reach 14.58 mg/mL by inducing high yield strain through ion injection and further optimizing the fermentation process [63]. There have also been major breakthroughs in the biosynthesis of some biopesticides. [PERSON] et al. designed an orthogonal _limonene_ synthesis pathway, and a yield of 917 mg/L was achieved in yeast chassis and 1.3 g/L in _E. coli_ through synthetic gene screening and the modification of the precursor metabolic pathway [64]. [PERSON] et al. reconstructed the _rhodopsin_ biosynthesis pathway in a _Saccharomyces cerevisiae_ and achieved the highest yield reported in the literature by supplemental fermentation [65].