## 3 Discussion  
_E. coli_ has the capability to supply various isoprenoid precursors through the native DXP pathway [19; 20; 21; 22; 23; 24]. In the past few decades, this microorganism has been engineered to produce various bioactive terpenoids and valuable precursors such as limonene, taxadiene (taxol precursor), amorphadiene (artemisinin precursor), and carotenoids (e.g., lycopene, astaxanthin, carotenoids) by the heterologous expression of terpene synthases with the rate-limiting step enzymes in the DXP pathway [29; 30; 31; 32; 33; 34]. However, few studies have focused on the use of this microorganism as the in vivo prenylation system of aromatic natural products. In wild-type _E. coli_, PPP is involved in the biosynthesis of various molecules (e.g., ubiquinone and peptidoglycan) and its level is tightly maintained, as it is essential for bacterial growth and viability [25; 26; 27; 28]. A previous study also demonstrated that the deletion of genes encoded for FPP synthase (IspA) resulted in the observed growth retardation in the mutant strains of _E. coli_[25]. We hence speculated that FPP might become more readily available for the in vivo prenylation of 4 HC (1) for ferulenol (2) than others (e.g., DMAPP, GPP, and GGPP). However, the native isoprenoid precursors of _E. coli_ are always insufficient for metabolic engineering purposes, and the enhanced metabolic fluxes via overexpression of the rate-limiting step enzymes in the DXP pathway (e.g., DXS, DXR, and IDI) are required [7; 19; 20; 21; 22; 23; 24]. The transcriptional profile analysis in the young leaves of _C. stellatopilosus_ revealed a positive association between CsDXS gene expression and plaunotol (acyclic diterpene alcohol) content, and this enzyme was hence suggested as one of the rate-limiting steps in the DXP pathway controlling the flux of isoprenoid precursors in the plant [48]. Since the active form of CsDXS could be achieved after the removal of the signaling peptide region [30; 48], the truncated form of this enzyme, CstDXS, was used in this study to drive the metabolic flux of the DXP pathway in the _E. coli_ BL21(DE3)pLysS strain. We demonstrated that _E. coli_ BL21(DE3)pLysS harboring pCDFUet-AtaPT-CstDXS is capable of producing the putative mass peak corresponding to ferulenol (2) from the fed 4 HC (1) without relying on the external supply of prenyl-donors (e.g., DMAPP, GPP, and FPP). The synthesized product was found to be excreted into the culture medium. This means the step of breaking the cell during the downstream processes is not necessary. Furthermore, this expression system did not require the use of high-priced precursors. These reflected the potential economic system of the prenylated 4 HC derivatives production by using recombinant _E. coli_. Besides, our results extend the findings of [PERSON] et al. [46] in that AtaPT can utilize various substrates by showing that 4 HC (1) could also be recognized as the aromatic acceptor, even though it has never been reported that this compound acted as the substrate of AtaPT. Although NMR elucidation is required to verify the structures of the resulting prenylated product (compound-1), this technique is restricted to researchers in the fields of metabolic engineering and metabolomics, which have to deal with trace-level metabolites [49; 50; 51]. The structural elucidation of compound-1 was thus based on the use of in silico tools designed to annotate the structures of the query metabolites using the experimental mass spectra (MS2) (Figure S4). Having been confirmed to be highly consistent with the unique MS/MS fragmentation profile of ferulenol (2) from the previous work [56], MetFrag analysis clearly showed that the compound-1 was best annotated as \"ferulenol (2)\" out of the 2266 candidates presented from PubChem database. However, information regarding the molecular formula, chemical structure, substructures, and classifications of this product are still needed.