## 4 Common Strategies for Optimizing Sesquietpenol Biosynthesis
### Subcellular Organelle Engineering  
The biosynthesis of terpenoids in model microbial chassis or plant cell factories encounters inherent limitations, including cofactor imbalance, substrate competition, product toxicity, and multi-enzymatic pathway complexity [123]. Cellular compartmentalization has emerged as a strategic solution to these challenges through subcellular spatial organization (Figure 5c, Table 2). Most cellular metabolic processes are naturally compartmentalized, with distinct organelles providing specialized microenvironments. These organelles optimize biochemical reactions by spatially regulating enzymatic cascades and metabolite channeling [124]. Eukaryotic organelles enclosed by phospholipid bilayers offer unique biochemical potentials through compartment-specific resources, such as hydrogen gradients, cofactor pools, and enzymatic systems [125]. In yeast, the most frequently employed compartments for sesquiterpenol synthesis are peroxisomes and mitochondria. Peroxisomes contain acetyl-CoA generated from fatty acid \(\beta\)-oxidation, supporting mevalonate (MVA) pathway operation. [PERSON] et al. introduced the trans-nerolidol synthesis pathway genes into peroxisomes in two steps using an enhanced peroxisome targeting sequence (ePTS1). The resultant strain achieved a trans-nerolidol yield of 930 mg/L, which was 31% higher than that of the previous strain [66]. Similarly, [PERSON] et al. introduced the \(\alpha\)-bisabolol synthesis pathway into _Y. lipolytica_ peroxisomes, achieving a yield of 140.3 mg/L \(\alpha\)-bisabolol [72]. Mitochondria provide high levels of ATP/NAD(P)H, and serve as reservoirs of acetyl-CoA.  
[PERSON] et al. increased patchoul production 1.7-fold (to 109 mg/L) in _P. pastoris_ by incorporating a mitochondrial targeting signal peptide into the biosynthetic pathway [79]. [PERSON] et al. integrated the MTS-PT gene into the genome of _S. cerevisiae_ to compare patchoul production in the cytoplasm and mitochondria. The engineered strain produced 1.79 \(\pm\) 0.06 mg/L patchoul, which was 2.71-fold higher than the reference strain [78]. These results indicate that mitochondria-engineered yeast cells have significant potential for enhancing patchoul biosynthesis. In prokaryotic systems, protein-based structures such as bacterial microcompartments (BMCs) and membraneless organelles (MLOs) are employed for metabolic regulation [126]. BMCs facilitate substrate channeling and cofactor recycling, making them valuable for encapsulating terpenoid pathways. MLOs, which are liquid droplets formed through liquid-liquid phase separation, enable the dynamic spatial organization of enzymatic complexes. Both BMCs and MLOs hold promise for improving sesquiterpenal production yields in _E. coli_ -based biosynthesis.  
These compartmentalization strategies are effective across various microbial hosts. Utilizing synthetic biology tools, such as targeting peptides and scaffold proteins in combination with native organelle biochemistry, establishes a robust framework. This approach helps overcome bottlenecks in terpenoid biosynthesis, particularly in sesquiterpenal production. Future developments in programmable compartment engineering and dynamic pathway regulation are expected to enhance spatial control of metabolic flux.