## 2 Botanical Biopesticides
### Botanical Biopsticides and Synthetic Biology
#### 2.2.3 Optimization and Integration of Synthetic Biology Components  
Although the biosynthetic pathways of some plant natural products can be resolved by intensive mining and functional validation in vitro and in vivo, it is not easy to take a broadly well-defined pathway, functionally express it in a heterologous microbial host, and produce the target product [82; 83]. Some enzymes cannot be expressed correctly or achieve their functions in heterologous hosts due to incorrect folding, post-transcriptional modifications, mislocalization, a lack of cofactors, non-optimal pH, non-natural substrates, product feedback inhibition, etc. Some of these problems can be solved or improved by means of enzyme engineering. [PERSON] et al. cloned five GGPPS genes, key enzymes for terpene biosynthesis, from _rayed_ and introduced the cloned GGPPS and SmCPS/KSL genes into _E. coli_. By analyzing the expression of TwGGPPS in MeJA-induced suspension cells, the authors found that the accumulation of ragweed methicin was enhanced with an increase in TwGGPPS1 and TwGGPPS4 [84]. [PERSON] et al. overexpressed and purified an enzyme homologous to GDSL lipase in _E. coli_ and termed it TcGLIP. By expressing all the variants of TcGLIP, variants JN418993 and JN418996 were found to have similar acyltransferase activity, with JN418990 showing significantly higher activity. These findings enhanced our understanding of the structural basis of TcGLIP in exerting _pyrethrin_ biosynthetic activity [85]. During attempts to synthesize _morphine_ and its semisynthetic derivatives in yeast, researchers found that a considerable amount of the by-product neomorphine accumulates in the cells [85], mainly due to codeine ketone reductase (COR) activity, which can be isolated from the substrate after localizing COR to the endoplasmic reticulum using a tag, thereby allowing the spontaneous rearrangement of neomorphine to form codeine ketones. This method ultimately resulted in a seven-fold increase in the yield of the target product, _morphine_. [PERSON] et al. used recombinant _E. coli_ BL21 (DE3) p ET28a-agl A to efficiently express the glucosyltransferase gene agl A derived from _Xanthomonas campestris_ and obtained the recombinant enzyme via isolation and purification, which catalyzed the glycosylation of _eugenol_ with a product conversion rate of 75% and effectively increased the yield of _eugenol_ synthase \(\alpha\)-EG [86]. [PERSON] et al. further clarified the pathway of chimeric OMT in _berberine_ biosynthesis by expressing recombinant chimeric methyltransferase in _E. coli_. The authors found that only the cell lysates of recombinant _E. coli_ with pET-64'OMT possessed the methylation activity of demethylated dodecanol; i.e., the N-terminal end of 6-OMT and the C-terminal end of 4'-OMT were functionally complementary, thus allowing the chimeric OMT to play a role in the biosynthesis of _berberine_[87]. In _E. coli_, [PERSON] expressed two recombinant methyltransferases, recombinant S-adenosyl I L methionine scoulerine 9-O-methyltransferasease and S-adenosyl-L-methionine. Coclaurine N methyltransferase, (S)-THC, and (R,S)-N-methyl tetrahydroroberberine were prepared, and the accumulation of _berberine_ was successfully induced via the introduction of the enzymes, suggesting that the relatively high ectopic expression of a novel branching enzyme is one of the key factors behind the diversification of _berberine_ metabolism [88]. For many synthetic pathways in which enzymes are involved, some intermediate metabolites are not directly available. Consequently, the activity of some enzymes cannot be verified with certainty, which can also make the heterologous synthesis of plant natural products difficult. In this case, the longer synthetic pathways can be broken down into shorter modules, ideally with each module yielding the desired intermediate. [PERSON] et al.