## 4 Common Strategies for Optimizing Sesquietpenol Biosynthesis
### Enzyme Engineering
#### 4.1.3 Enzyme Colocalization  
Enzyme colocalization represents a pivotal strategy for enhancing metabolic efficiency and product yield in biosynthetic pathways (Figure 4c, Table 2). This approach fuses key enzymes into multi-enzyme complexes, increasing local concentrations of pathway enzymes and metabolites while establishing substrate channeling [101]. In sesquiterpenol biosynthesis, enzyme fusion strategies have proven effective for metabolic engineering, with farnesyl pyrophosphate (FPP) serving as the critical precursor [102]. The inherent metabolic competition for FPP and product feedback inhibition often limits sesquiterpenol production. Recent advances in constructing fusion proteins of FPP synthase (FPPS) with sesquiterpenol synthases have demonstrated remarkable improvements in yield. Previous studies have shown that the composition and length of peptide linkers in enzyme fusions can affect terpene production. Consequently, [PERSON] et al. designed FPPS-NES fusion constructs incorporating four distinct peptide linkers. Each of these four fusion constructs achieved a remarkable >110-fold increase in nerolidol production, yielding titers between 3.51 and 4.20 g/L in culture [56]. [PERSON] et al. found that fusing PTS to the C-terminus of ERG20, rather than the N-terminus, greatly increased patchoul yield [80]. To understand why, they generated protein 3D models using the AlphaFold2 Multimer. The simulations revealed that the active-site distance between ERG20 and PTS was shorter in the ERG20-PTS fusion than in the PTS-ERG20 fusion. Similarly, [PERSON] et al. [44] and [PERSON] et al. [74] also utilized the ERG20-PTS fusion protein for patchoul synthesis. These studies together prove that the fusion-protein strategy has been used in various microbial chassis with significant outcomes. Biomolecular scaffolding systems also facilitate enzyme colocalization. Scaffold-mediated enzyme organization, which can be categorized into protein-based and nucleic acid-based systems, offers an alternative approach to spatial control [103]. [PERSON] et al. engineered an affibody scaffold in _S. cerevisiae_ to colocalize FPPS and farresene synthase, increasing farresene yield by 135% under glucose-fed conditions [104]. Additionally, [PERSON] et al. utilized Tya, a component of the Ty1 retrotransposon, to assemble VLPs by incorporating three farnesol biosynthetic enzymes in _S. cerevisiae_, thereby quadrupling farnesol production [99].  
These colocalization strategies synergistically optimize enzyme proximity, substrate channeling, and metabolic flux partitioning. The fusion protein approach is particularly effective in redirecting FPP flux toward target sesquiterpenols. Scaffold systems enable modular control of enzyme stoichiometry. The success of these strategies across various microbial chassis underscores their universal applicability in sesquiterpenol biosynthesis, and provides crucial technical support for industrial-scale production. Further breakthroughs in metabolic pathway optimization are anticipated as programmable scaffolding systems are developed and AI-driven fusion protein design advances.