## 4 Common Strategies for Optimizing Sesquietpenol Biosynthesis
### The Strategies for the Regulation of the Synthesis Pathway
#### 4.2.1 The Engineering of Host Metabolism
While both IUP and IPA pathways show significant potential, their industrial implementation  
Figure 5: The regulation of the synthesis pathway, subcellular compartmentalization, and toxicity tolerance engineering: (**a**) engineering the MVA pathway; (**b**) enhancing the central carbon flux; (**c**) subcellular compartmentalization; (**d**) strategies of improving cell toxicity tolerance in terpenoid production: secretion; adaptive laboratory evolution (ALE); bio-derivatization. requires further mechanistic optimization, particularly in phosphokinase screening and pathway regulation. These heterologous pathways are expected to be employed in the biosynthesis of sesquiterpenols to enhance production yields. Downregulating the activity of metabolic branches can reduce metabolic flux loss, thereby enhancing the efficiency of the target product synthesis (Figure 4a, Table 2). The biosynthesis pathways of squalene and ergosterol are major competing pathways for sesquiterpenol production in yeast. Farnesyl diphosphate (FPP) is predominantly utilized for squalene synthesis through the high expression of squalene synthase (ERG9) rather than for sesquiterpenol synthases, diverting metabolic flux toward these competing pathways [114]. Downregulating squalene biosynthesis enables redirection of FPP precursors toward sesquiterpenol synthesis. However, completely knocking out the squalene and ergosterol pathways remains unfeasible due to their essential roles in cellular physiology. Current suppression strategies often involve promoter engineering, such as replacing the native ERG9 promoter with regulated ones, like PMET3 (methionine-repressible) or PHXT1 (glucose-responsive). The PHXT1 promoter provides better dynamic control [115]. [PERSON] et al. achieved a record farnesol titer of 393.13 mg/L under flask culture conditions in _S. cerevisiae_ by implementing PHXT1-driven ERG9 regulation [55]. Additionally, [PERSON] et al. introduced the MrBBS gene for (-)-\(\alpha\)-bisabolol biosynthesis into the _S. cerevisiae_ genome. Initially, no (-)-\(\alpha\)-bisabolol was detected. To resolve this, the native promoter of the squalene synthase gene ERG9 was replaced with the HXT1 promoter. This strategy led to (-)-\(\alpha\)-bisabolol production reaching 75.83 mg/L under flask fermentation conditions [73]. These findings demonstrate that downregulating ERG9 expression effectively enhances sesquiterpenol biosynthesis by redirecting metabolic flux toward target pathways. Complementary approaches involve blocking sesquiterpenol degradation by knocking out _DPP1_ and _LPP1_, thereby conserving FPP flux for target compounds. [PERSON] et al. demonstrated this by disrupting three extraneous pathways, including the farnesol synthase genes _DPP1_ and _LPP1_, which led to physiological adaptations and 1.63 g/L patchoul production [77]. Additionally, research has shown that the alkaline phosphatase _PhoA_ from _E. coli_ can hydrolyze geranylgeranyl pyrophosphate (GGPP) to produce geraniol. Drawing on this finding, [PERSON] et al. hypothesized that the endogenous phospholipase in _S. marcescens_ might recognize farnesyl pyrophosphate (FPP) and catalyze its hydrolysis to produce farnesol. To test this hypothesis, they used the CRISPR-Cas9 system to knockout the phospholipase gene _phoA_ in _S. marcescens_, resulting in a 70% reduction in farnesol titers and an increase in (-)-\(\alpha\)-bisabololol production to 3.21 g/L [5]. These findings establish that attenuating branch pathways is an effective strategy for enhancing metabolic flux partitioning toward desired. While these strategies significantly enhance sesquiterpenol production, excessive gene overexpression might impose metabolic burdens that affect cellular fitness. Emerging solutions include AI/ML-assisted dynamic regulation systems [116]. These systems integrate multi-omics data to achieve orthogonal control between growth and product synthesis, enabling real-time pathway optimization in response to environmental and metabolic fluctuations.