## 3 Discussion
We hence postulated that the compound-2 (eluted from the HPLC column at 22.3 min), obtained from the negative- and positive-ion mode analyses, is presumably the tautomeric form of ferulenol (2). The obtained MS/MS spectra (Figures 3G and 7D) were further elucidated to gain insight into the structural information of this preynylated product. In the negative ion mode analyses, the three chemical losses explained the daughter ions at _m_/_z_ 214.06, 228.08, 282.12, signifying the prenyl side chain was partially removed from the 2-hydroxy-4-chromenone (c), the core structure of this preynylated product. A similar trend was observed in the positive ion mode analyses, where the partial loss of prenyl side chain attaching to the 2-hydroxy-4-chromenone could be characterized via the signals with _m_/_z_ 177.08, 215.06, 229.08, and 243.10, respectively. The existence of product ions at _m_/_z_ 189.05 [M \(+\) H]\({}^{+}\) in the MS/MS spectrum of both preynylated products (Figure 7C,D) were likely to be the outcome of non-enantioselective epoxidation of the double bond present in the farnesyl side chain of ferulenol (2), as recently described by [PERSON] et al. [59]. Based on the obtained evidence, the compound-2 (3) was interpretedas the farnesylated derivative of 2-hydroxy-4-chromenone, however further elucidation (e.g., NMR) is required to fully support a chemical point of view. The availability of prenyl precursors influences the patterns of isoprenoid-derived natural products produced by the bacterial systems [60]. Here, ferulenol (2) and its proposed isomeric structure (3) were exclusively detected as the predominant products produced by _E. coli_-carried pCDFDuet-AtaPT-CstDXS. Although AtaPT exhibits remarkable substrate promiscuity towards a variety of prenyl-donors such as DMAPP, GPP, FPP, and GGPP [46], the presence of two farnesylated-4 HC analogs (compounds-1 and -2) clearly supports our hypothesis that the FPP accumulated inside the bacterial cells is more easily accessible for synthesizing the two prenylated products rather than the others (DMAPP, GPP, and GGPP). Our result was also consistent with the previous finding, where the farnesylated-menadione was the major product of the whole-cell catalysis by _P. pastoris_, harboring the gene encoding for aromatic prenyltransferase (NovQ) [37]. Studies have shown that overexpression of multiple rate-limiting enzymes in the DXP pathway, including 1-deoxy-D-xylulose 5-phosphate reductio isomerase (DXR), isopentenyl pyrophosphate isomerase (IDI), along with FPP synthase (e.g., IspA), causes the substantially increased production of terpen in their engineered microbes [23; 24; 25]. Furthermore, 4 HC (1) as the fed precursor is unlikely and unsustainable from an economic standpoint, as a considerable amount of this expensive substrate must be supplied to the bacterial culture to make its desired prenylated analogs. This strategy might be impracticable for industrially scaled applications, where the newly designed strains capable of de novo manufacture of the two farnesylated-4 HC analogs or the usage of the lower cost precursors (e.g., sodium salicate) should be established. Previous research has shown that _E. coli_ can be engineered to produce 4 HC (1) via the inherent chorismate pathway, which is accomplished through a series of reactions catalyzed by isochorismate synthase (ICS), isochorismate pyruvate-lyase (IPL), salicate-CoA ligase (SCL), and biphenyl synthase (BIS) [61]. Based on the broad substrate specificity of benzoate-CoA ligase (BadA) and benzophenone synthase (GmBPS), our group demonstrated that _E. coli_ BL21(DE3)pLysS carrying pETDuet-BadA-GmBPS was able to synthesize 4 HC (1) from the fed salicate (sodium salt) as well (data unpublished). By considering these advantages, further establishments of _E.