## 2 Results
### Identification of Farnesylated-4 HC Derivatives by Using LC-QTOF-MS/MS in Positive ESI Mode  
The high-resolution LC-QTOF-MS/MS in positive ion mode was thus implemented to corroborate both prenylated products based on monitoring of the molecular ion _m_/z 367.227320 [M + H]\({}^{+}\) computed by the web tool ([[https://www.sisweb.com/referenc/tools/exactmass.htm?formula=C24H31O3](https://www.sisweb.com/referenc/tools/exactmass.htm?formula=C24H31O3)]([https://www.sisweb.com/referenc/tools/exactmass.htm?formula=C24H31O3](https://www.sisweb.com/referenc/tools/exactmass.htm?formula=C24H31O3)) (accessed on 7 August 2021)). In the extracted ion chromatogram (EIC) of the medium extract of clones carrying pCDFDuet-AtaPT-CstDXS, there were two extracted ions (namely compound-1 and compound-2) eluted at the retention times of 21.3 and 22.3 min that exhibited identical _m_/z 367.22 [M + H]\({}^{+}\), where they presumably farnesylated derivatives of 4 HC (Figure 7A). The resulting MS/MS spectrum of compound-1 (_m_/z 367.22; Rt = 21.3 min) was clearly identical to those belonging to the authentic feruelenol (2) established under the same LC-MS/MS condition (Figure 7B). In the MS/MS spectrum of this prenylated product, the presence of proposed substructures  
Figure 6: Structural annotation and classifications for the query feruelenol (2) (_m_/z 365.21 [M - H]\({}^{-}\)) synthesized by the recombinant _E. coli_ carrying pCDFDuet-AtaPT-CstDXS based on CSI:FingerID and CANOPUS integrated in the SIRIUS (version 4.8.2). (**A**) In all included databases, ferulenol (2) as the target metabolite was ranked as the tenth potential candidate (highlighted in yellow) for the prenylated 4 HC analog _m_/z 365.21 [M - H]\({}^{-}\), meanwhile, the rank of this product significantly increased towards the fifth- and top-ranked candidates when structural identification was performed against the biological databases included Natural Products, COCONUTS, and NORMAN. (**B**) The examples of substructures (molecular fingerprints) predicted to exist in the query subject, including those belonging to the 4 HC (1) core structure (No. 1284, 1222, and 1234), and the farnesyl moiety (No. 435, 411, and 502). (**C**) The examples of substructures belonging to feruelenol (2) found to exist in the training structures of the SIRIUS tool. (**D**) Based on the experimentally observed MS/MS data, CANOPUS showing the query compound (C\({}_{24}\)H\({}_{30}\)O\({}_{3}\) (SIRIUS score 100%), _m_/z 365.21 [M - H]\({}^{-}\)) was categorized as coumarins and derivatives, describing the polycyclic aromatic compounds that contains a 1-benzopyran moiety with a ketone group at the C2 carbon atom (1-benzopyran-2-one). at _m/z_ 175.04 (base peak), 217.08, 231.10, and 243.10 illustrated the partial losses of the premyl side chain specifically attached to a C3 position of the 4 HC core structure (1) (Figure 7C). The obtained evidence leds us to suggest that the compound-1 (_m/z_ 367.22 [M + H]\({}^{+}\); Rt 21.3 min) was indeed ferulenol (2), a product derived from C3-farnesylation of 4 HC (1). Hence, the compound-2 (_m/z_ 367.22 [M + H]\({}^{+}\); Rt = 22.3 min) was presumably the tautomeric structure of ferulenol (2), where the 2-hydroxy-4-chromenone (c) served as the core structure (Figure 3E). In the MS/MS spectra along with proposed structures of this product, the existing product ions at _m/z_ 177.08 (base peak), 215.06, 229.08, and 243.10 signify the partial elimination of premyl moiety that incorporated the C3 region of 2-hydroxy-4-chromenone (Figure 7D). The existing of signals at _m/z_ 189.05, which were presented in the MS/MS spectrum of both products as shown in Figure 7C,D, were presumably obtained from the premyl moieties which were adjacent to the core structure of ferulenol (2) and its isomeric structure (3). Based on the acquired evidence, compound-2 was tentatively defined as the isomeric structure of ferulenol (2) which was formed via \"tautomerization\". Further elucidation (e.g., NMR) is required to verify the tentative confirmation of this premylated product.