## 5 Conclusions  
Sesquiterpenals, with their structural diversity and significant pharmacological potential, represent a key class of bioactive compounds. Compared to conventional plant extraction and chemical synthesis methods, engineering microbial cell factories for sesquiterpenal production offers a promising alternative. Despite notable advances in microbial engineering for sesquiterpenal production, several challenges hinder large-scale industrial applications. First, metabolic pathway regulation requires more comprehensive understanding. While the native MVA and MEP pathways are tightly regulated, their control networks, particularly in the MEP pathway, remain incompletely understood. This complexity limits the efficient redirection of metabolic flux toward target products. Therefore, deeper insights into these regulatory mechanisms are essential for precise control of the MVA and MEP pathways, providing a theoretical basis for enhancing sesquiterpenal yield and efficacy. Second, the structure-function relationships of sesquiterpenal synthases warrant immediate investigation. While protein engineering can enhance the catalytic efficiency of sesquiterpenal synthases, our understanding of their tertiary structures and catalytic mechanisms remains limited. Elucidating the spatial structures and comprehending the catalytic mechanisms of these synthases is crucial for advancing sesquiterpenal metabolic engineering. Third, high-throughput screening platforms are urgently needed. A significant limitation in terpenoid metabolic engineering is the absence of high-throughput screening methods, which hampers the efficiency of metabolic engineering strategies. New screening technologies, such as fluorescence-based or colorimetric assays, could accelerate the identification of high-producing strains and the optimization of biosynthetic pathways. Fourth, carbon conversion efficiency must be significantly improved. The existence of competing metabolic pathways often results in relatively low carbon conversion efficiency for sesquiterpenals. These pathways consume valuable carbon sources and release CO2 during decarboxylation reactions, thereby reducing overall production efficiency. Thus, enhancing carbon source conversion efficiency through pathway design and metabolic engineering is imperative for cost-effective sesquiterpenal production. Finally, by-product formation during production requires mitigation. In sesquiterpenal production, by-product formation not only reduces the yield of the target product but increases downstream purification costs. Therefore, employing metabolic engineering strategies to minimize or eliminate these by-products is a vital approach to improving production efficiency and reducing costs.  
In summary, the microbial production of sesquiterpenals faces multiple challenges, ranging from elucidating the fundamental mechanisms of metabolic pathway regulation to addressing practical aspects such as improving enzyme catalytic efficiency, developing high-throughput screening technologies, and optimizing carbon source conversion efficiency. Overcoming these challenges requires interdisciplinary research efforts, including close collaboration across fields such as molecular biology, biochemistry, computational biology, and chemical engineering. With the continued advancements in synthetic biology and systems biology, future research in these areas will provide new strategies and tools for the efficient production of sesquiterpenals.  
Conceptualization, M.L. and R.C.; investigation, M.L. and R.C.; writing--original draft preparation, M.L. and R.C.; writing--review and editing, M.L. and R.C.; supervision, W.L., H.Z. and J.Q. All authors have read and agreed to the published version of the manuscript.  
This work was supported by National Key Research and Development Project of China (2020 YFA0907900) and Zhejiang Provincial Natural Science Foundation of China under Grant No. LQN25C010006.  
Not applicable.  
The authors declare no conflict of interest.