Medicinal Plant Research 2025, Vol.15, No.2, 51-61 http://hortherbpublisher.com/index.php/mpr 56 5 Genetic Modification and Optimization Strategies of G. lucidum 5.1 Promoter selection and optimization for higher expression In the genetic improvement of G. lucidum, the selection of promoters is a key step. Whether the promoter can drive the stable expression of the target gene directly affects the synthesis efficiency of triterpenoid compounds. For example, studies have found that the endogenous u6 promoter works well in the CRISPR-Cas9 system and is very helpful in improving editing efficiency (Wang et al., 2019). If more stable and efficient promoters like this can be found, it will be helpful to promote the manipulation of G. lucidumfunctional genes. Some promoters can also respond to induction signals, such as methyl jasmonate (MeJA). Such promoters can enhance the expression of genes related to triterpene synthesis under specific induction conditions (Xu et al., 2022b). In addition, there are also studies on engineering existing promoters, such as introducing self-cleaving ribozymes HDV or performing site-directed mutagenesis to improve their activity and specificity. With the support of transcriptome data, promoters with obvious expression under different environmental conditions can also be screened, providing a basis for subsequent optimization (Xu et al., 2022b). 5.2 Gene stacking To put it simply, gene stacking is to integrate multiple target genes at one time to form a "combined force" to make the entire triterpene synthesis pathway more efficient. Now tools like CRISPR-Cas9 can already achieve this kind of precise editing of multiple genes. For example, researchers used CRISPR-Cas9 to knock out P450 genes such as cyp5150l8 and cyp505d13, and found that the types and proportions of triterpene compounds changed significantly (Wang et al., 2020). This shows that it is indeed effective to regulate triterpene synthesis by multiple genes at the same time. The advantage of this method is that it can "link" multiple links in one step, making the entire metabolism flow more smoothly. But it is not without difficulties. It is common for multiple genes to "hinder" each other in the body, and if you are not careful, it may also interfere with other metabolic pathways. But in general, gene stacking is still the most worthwhile way to increase the production of triterpenes in Ganoderma lucidum. 5.3 Use of transcription factors to boost triterpenoid production The triterpenoid synthesis pathway is coordinated by a series of genes, and many of these genes are regulated by transcription factors. Transcription factors such as GlbHLH5 have been found to positively regulate the synthesis of triterpenes. Studies have shown that when GlbHLH5 expression is enhanced in G. lucidum, it will drive the gene expression of multiple key enzymes in the synthesis pathway, thereby allowing more triterpenoid compounds to accumulate (Xu et al., 2022a). In addition, transcription factors that respond to methyl jasmonate (MeJA) are also receiving more and more attention. As regulatory points under exogenous induction, such factors themselves can also become targets for overexpression to increase triterpene production (Xu et al., 2022b). Except for the direct regulation of a single transcription factor, signaling pathways such as HO-1/CO may also indirectly promote triterpenoid synthesis by affecting the expression of related genes in the mevalonic acid (MVA) pathway (Cui et al., 2021). After all, the MVA pathway is the main route for triterpenoid synthesis, and regulating it is equivalent to regulating the basic flow of triterpenoids. At the same time, transcriptome technology can also help us screen out transcription factors whose expression levels change significantly under specific induction or stress conditions, which may become a breakthrough for subsequent gene editing (Xu et al., 2022b). 6 Case Studies 6.1 CRISPR/Cas9 for increasing triterpenoid yield The CRISPR/Cas9 system, as a third-generation gene editing tool, has been widely applied in gene editing research in fungi, plants, and animals due to its efficiency and precision. However, the genetic diversity of G. lucidum and codon preference variations among different strains limit the application of plasmid-dependent CRISPR systems (Tu et al., 2021).
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