Medicinal Plant Research 2024, Vol.14, No.4, 223-233 http://hortherbpublisher.com/index.php/mpr 231 extracellular polysaccharide (EPS) production, demonstrating the potential of targeted genetic modifications to enhance metabolite yields under stress conditions (Ma et al., 2018). Furthermore, the CRISPR/Cas9 system has emerged as a powerful tool for precise genome editing, enabling the modification of specific genes involved in stress responses and secondary metabolite biosynthesis with high accuracy and efficiency (Devi et al., 2023). 9.2 Application of synthetic biology for optimized biosynthetic pathways Synthetic biology offers innovative approaches to optimize biosynthetic pathways in Ganoderma lucidum. By reconstructing and analyzing genome-scale metabolic models, researchers can identify key enzymes and regulatory nodes that can be targeted for pathway optimization. For example, the GSMM of G. lucidum has identified eight key genes for EPS overproduction, providing a roadmap for synthetic biology interventions (Ma et al., 2018). These strategies can be further refined using synthetic biology tools to construct optimized biosynthetic pathways that enhance the production of valuable secondary metabolites while maintaining cellular homeostasis under environmental stress. 9.3 The potential of CRISPR-based editing for regulating stress response genes CRISPR-based genome editing holds significant potential for regulating stress response genes in Ganoderma lucidum. The CRISPR/Cas9 system allows for precise and efficient modification of genes involved in stress responses, enabling the development of strains with enhanced resilience to environmental stressors. This technology has been widely applied in plant metabolic engineering, facilitating the discovery and functional analysis of genes involved in secondary metabolite pathways (Devi et al., 2023). By leveraging CRISPR-based editing, researchers can systematically dissect the genetic basis of stress responses and engineer G. lucidumstrains with improved stress tolerance and secondary metabolite production capabilities. 10 Conclusion In this study, we explored the regulation of secondary metabolite pathways in Ganoderma lucidumunder various environmental stress conditions. Findings reveal that different types of stress, such as water stress, heat stress, and copper stress, significantly influence the biosynthesis of ganoderic acid (GA), a key secondary metabolite with medicinal properties. Water stress was found to increase intracellular reactive oxygen species (ROS) levels, ganoderic acid content, and NADPH oxidase (NOX) activity. The cross-talk between the G. lucidum aquaporin (GlAQP) gene and NOX positively regulated GA biosynthesis via ROS at the early stage of water stress but became negative at the late stage. Heat stress (HS) also induced the production of ROS, which in turn regulated the expression of heat shock proteins (HSPs), hyphal branching, and GA biosynthesis. The AMP-activated protein kinase (AMPK)/Sucrose-nonfermenting serine-threonine protein kinase 1 (Snf1) was found to mediate metabolic rearrangement under HS, negatively regulating GA biosynthesis by removing ROS. Copper stress was shown to decrease the distance between hyphal branches and increase GA content, with ROS and Ca2+ signaling playing crucial roles in this regulation. Increased cytosolic Ca2+ levels could reduce ROS by activating antioxidases, thereby modulating hyphal growth and GA biosynthesis. Additionally, the mitogen-activated protein kinase GlSlt2 was found to regulate fungal growth, fruiting body development, cell wall integrity, oxidative stress, and GA biosynthesis. The insights gained from this study underscore the importance of managing environmental stress to optimize the cultivation of Ganoderma lucidum for therapeutic purposes. By understanding the molecular mechanisms underlying stress-induced regulation of secondary metabolite pathways, we can develop strategies to enhance the production of valuable compounds like ganoderic acid. For instance, managing water stress through the modulation of GlAQP and NOX activity can optimize GA production at different stages of fungal growth. Similarly, controlling heat stress by targeting the AMPK/Snf1 pathway and ROS levels can improve GA yield while maintaining fungal health. Copper stress management through the regulation of ROS and Ca2+ signaling can also be leveraged to enhance GA biosynthesis.
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