## 1 Introduction
However, an attempt at synthesizing ferulenol (2), the product of C-3 farnesylation of 4 HC (1), through the genetic manipulation of the DXP pathway in _E. coli_ has not been reported yet. Therefore, this gives rise to our interest in establishing a new synthesis pathway for producing this product by using the engineered microbe based on feeding of 4 HC (1) to minimize chemical consumption. Aromatic prenyltransferase (aPTs) are the enzymes that catalyze the region-selective prepnylation of the prenyl groups (so-called prenyl donors), incorporating the aromatic compounds (known as aromatic acceptors) that contain the electron-rich regions through a mechanism comparable to Friedel-Crafts aromatic electrophilic substitution [39; 40; 41; 42]. Nowadays, the genes encoded for aPTs have been isolated from plants, fungi, and bacteria and have been found to exhibit broad substrates with specificity both in terms of aromatic acceptors and prenyl donors, creating a diverse range of prenylated products [1; 3; 43; 44; 45]. Of those enzymes characterized, _Aspergillus terreus_ aromatic prenyltransferase (AtaPT) possesses the broad range substrate specificity towards various types of aromatic acceptors (e.g., coumarins, resveratrol, and naringenin) and prenyl donors, i.e., DMAPP, GPP, FPP, and GGPP, yielding prenylated products with structural diversity [46]. This enzyme also differs from formerly characterized aPTs, as it can generate mono-, di-, and/or triprenylated products via C-C- and/or C-O-bonded prenylation. Since 4-hydroxycoumarin (4 HC) (1) contains the two promising regions for electrophilic alkylation, including the C3 and the oxygen atom at C4 positions [15; 47], it was proposed to be utilized by AtaPT due to the unique catalytic activity of this enzyme. In wild-type _E. coli_, its native isoprenoid precursors are always insufficient for pathway engineering purposes, therefore driving metabolic fluxes via overexpression of the rate-limiting step enzymes in the DXP pathway, e.g., DXS, 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), and isopentenyl pyrophosphate isomerase (IDI) are required [19; 20; 21; 22; 23; 24]. According to the previous findings, transcriptional profiling analysis revealed a positive correlation between CsDXS gene expression and plaunotol (acyclic diterpene alcohol) content in the young leaves of _Croton stellatopilosus_, and this enzyme has been suggested to control the metabolic flux of isoprenoid precursorsin that plant [48]. Therefore, the truncated CsDXS (namely CstDXS) was chosen to co-express with the AtaPT in _E. coli_ BL21(DE3)pLysS to establish a new synthetic route for the in vivo formation ferulenol (2), in which 4 HC (1) was only a fed precursor. In this study, _E. coli_ was used as the bioconversion system of the newly designed pathway leading to the formation of farnesylated-4 HC analogs, which includes two steps (Figure 1). The CstDXS was overexpressed for driving metabolic flux of isoprenoid precursors (e.g., GPP, FPP) in the DXP pathway [47]. Second, prenylation of those prenyls to the core structure of 4 HC (1) through the catalytic function of AtaPT produced its corresponded products (2 and 3) [46]. This process requires magnesium ion (Mg\({}^{2+}\)) as a cofactor to facilitate the formation of an electrophilic phenyl carbocation, which subsequently binds to 4 HC (1) via the Friedel-Crafts reaction [46]. The structure elucidation of the target compounds by NMR techniques has been limited to the study of metabolic engineering and metabolomics, which deals with trace-level metabolites [49]. Therefore, high-resolution LC-ESI-QTOF-MS/MS has played a crucial role instead through its high sensitivity and can distinguish between the closed atomic mass numbers [49; 50; 51]. The experimental mass spectra can be interpreted into the chemical structures based on the in silico prediction tools by matching against the in silico -fragmented spectra of compounds in databases (i.e., PubChem, MetaCyc) [51; 52; 53].